Bcl-2 Prevents Bax Oligomerization in the Mitochondrial OuterMembrane*

Received for publication, January 24, 2001, and in revised form, February 14, 2001Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M100655200

Valery Mikhailov, Margarita Mikhailova, Donna J. Pulkrabek, Zheng Dong,Manjeri A. Venkatachalam, and Pothana Saikumar‡

From the Department of Pathology, University of Texas Health Science Center, San Antonio, Texas 78229

ATP depletion results in Bax translocation from cy-tosol to mitochondria and release of cytochrome c frommitochondria into cytosol in cultured kidney cells. Over-expression of Bcl-2 prevents cytochrome c release, with-out ameliorating ATP depletion or Bax translocation,with little or no association between Bcl-2 and Bax asdemonstrated by immunoprecipitation (Saikumar, P.,Dong, Z., Patel, Y., Hall, K., Hopfer, U., Weinberg, J. M.,and Venkatachalam, M. A. (1998) Oncogene 17, 3401–3415). Now we show that translocated Bax forms homo-oligomeric structures, stabilized as chemical adducts bybifunctional cross-linkers in ATP-depleted wild typecells, but remains monomeric in Bcl-2-overexpressingcells. The protective effects of Bcl-2 did not require Bcl-2/Bax association, at least to a degree of proximity oraffinity that was stable to conditions of immunoprecipi-tation or adduct formation by eight cross-linkers of di-verse spacer lengths and chemical reactivities. On theother hand, nonionic detergents readily induced ho-modimers and heterodimers of Bax and Bcl-2. Moreover,associations between translocated Bax and the voltage-dependent anion channel protein or the adenine nucle-otide translocator protein could not be demonstrated byimmunoprecipitation of Bax, or by using bifunctionalcross-linkers. Our data suggest that the in vivo actions ofBax are at least in part dependent on the formation ofhomo-oligomers without requiring associations withother molecules and that Bcl-2 cytoprotection involvesmechanisms that prevent Bax oligomerization.

Mitochondria are central to the apoptosis activation pathwayin many physiological and pathological conditions. Members ofthe Bcl-2 family of proteins are known to affect mitochondrialfunction and regulate the release of apoptosis-activating fac-tors (2–5). Anti-apoptotic members of Bcl-2 family (e.g. Bcl-2and Bcl-xL) act primarily to preserve mitochondrial integrity bysuppressing the release of cytochrome c (5). In contrast, pro-apoptotic members (Bax, Bid, etc.) induce the release of cyto-chrome c and cause mitochondrial dysfunction (1, 6–8). Thepro-apoptotic protein, Bax, which normally resides in the cy-tosol, translocates to mitochondria when triggered by certain

stimuli (6, 9). Translocated Bax has been shown to inducecytochrome c release both in vivo (1, 6, 10) and in vitro (11) andthis is followed by caspase activation (10, 12). The mitochon-drial permeability transition, an event that results in disrup-tion of the mitochondrial potential gradient, has been reportedto induce cytochrome c release and apoptosis (13). However,our observations and several other reports suggest that theeffects of Bax are targeted at the outer mitochondrial mem-brane and that the mitochondrial inner membrane remainsintact even after Bax-induced release of cytochrome c (6, 10,14–16).

How cytochrome c leaves mitochondria during apoptosis af-ter relocation of Bax to the mitochondrial outer membrane stillremains an unanswered puzzle. Potential mechanisms involvemitochondrial swelling caused by opening the permeabilitytransition pore in the inner membrane (17) or by mitochondrialhyperpolarization followed by swelling and membrane rupture(18). However, it has been reported that the pro-apoptotic pro-teins Bid and Bax can release cytochrome c from isolated mi-tochondria in the absence of detectable mitochondrial swelling(19). Although it was believed earlier that Bax induces therelease of cytochrome c by inhibiting Bcl-2 function throughbinding of the Bcl-2 homology domains BH1, BH2, and BH3,there is evidence to suggest that Bax and Bcl-2 function inde-pendently in regulating apoptosis (20, 21). Formation of ionchannels in synthetic lipid bilayer by members of the Bcl-2family (22) has suggested that pro-apoptotic members mayrearrange in the outer mitochondrial membranes to allow theefflux of cytochrome c by forming large channels. Even thoughboth Bcl-2 and Bax are capable of forming ion channels inartificial membranes, it is unclear how these proteins can formsimilar channels and still exert opposing actions. The datawould suggest that Bax function can be inhibited by Bcl-2/Bcl-xL, but does not require direct Bax/Bcl-2 or Bax/Bcl-xL

interaction for regulating Bax function (23, 24). For example,enforced dimerization of Bax, as a chimeric protein with FK506binding protein, resulted in its translocation to the mitochon-dria and induced cell death even in the presence of Bcl-xL (25).Likewise, Bax mutant proteins that fail to bind to Bcl-2 arecapable of inducing apoptosis (20). In addition, Youle’s group(26) have shown that nonionic detergents induce Bax homo- andheterodimerization with Bcl-2 or Bcl-xL and suggested that suchsimple dimers alone are not sufficient to regulate apoptosis.Bax has recently been reported to interact directly with VDAC1* This work was supported by National Institutes of Health Grants

DK54472 (to P. S.) and DK37139 (to M. A. V.), by a Morrison Trustgrant (to P. S.), and by a Texas Advanced Research Program grant (toZ. D.). The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

‡ To whom correspondence should be addressed: Dept. of Pathology,University of Texas Health Science Center, 7703 Floyd Curl Dr., SanAntonio, TX 78229. Tel.: 210-567-6597; Fax: 210-567-2367; E-mail:saikumar@uthscsa.edu.

1 The abbreviations used are: VDAC, voltage-dependent anion chan-nel protein; RPTC, rat proximal tubule cell; Bcl-21, human Bcl-2-over-expressing rat proximal tubule cell; ANT, adenine nucleotide translo-cator protein; DSP, dithiobis(succinimidyl propionate); DTSSP, 3,39-dithiobis(sulfosuccinimidyl propionate); DTBP, dimethyl 3,39-dithiobispropionimidate; EGS, ethylene glycol bis(succinimidylsuccinate); DPDPB, 1,4-di-[39-(29-pyridyldithio)-propionamido]butane;

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 21, Issue of May 25, pp. 18361–18374, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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on the outer membrane to release cytochrome c or with ANT onthe inner membrane to initiate the permeability transition,indirectly leading to cytochrome c release (27, 28). More re-cently, it has been reported that Bax may cause instability inartificial lipid membranes, suggesting another mechanism bywhich Bax may permeabilize the outer mitochondrial mem-brane (29). A caveat in this model is that Bcl-xL, which isknown to block cytochrome c release from intact mitochondria,did not prevent the membrane-destabilizing effects of Bax.

We have been investigating the roles played by Bax andBcl-2 in the regulation of cytochrome c release from mitochon-dria in a model of apoptotic cell death induced by cellular ATPdepletion. We have shown previously that severe ATP deple-tion of cultured rat kidney proximal tubule cells induced byhypoxia or chemical inhibitors of mitochondrial respirationtriggers the translocation of cytosolic Bax to mitochondria andcytochrome c release into the cytosol (1). In this model, mito-chondrial insertion of Bax does not compromise the integrity ofinner mitochondrial membranes (14). Thus, the outer mito-chondrial membrane would appear to be a reasonable site forthe permeabilizing actions of Bax, at least in the context ofhypoxia. Using this model, we now report that, following inser-tion into the mitochondrial outer membrane, Bax oligomerizesto form a multimeric structure that could explain the release ofcytochrome c. We also show that the protective actions of Bcl-2may stem from its ability to block Bax oligomerization in themitochondrial outer membrane without forming physical com-plexes with the Bax protein.

EXPERIMENTAL PROCEDURES

Materials

Materials purchased from vendors were as follows. Ham’s F-12/Dulbecco’s modified Eagle’s medium were from Life Technologies, Inc.Monoclonal antibodies to rat Bax (1D1) were kindly provided byDr. Richard J. Youle (National Institutes of Health, Bethesda, MD) andpolyclonal antibody to rat adenine nucleotide translocator was providedby Dr. H. H. Schmid (Hormel Institute, Austin, MN). Anti-cytochromec monoclonal antibody (clone 7H8.2C12) was from PharMingen (SanDiego, CA); anti-Bcl-2 polyclonal antibody (DC 21) and anti-Bax poly-clonal antibody (P-19) were from Santa Cruz Biotechnology Inc. (SantaCruz, CA); anti-porin 31 HL (a-VDAC) monoclonal antibodies Ab-1,Ab-2, Ab-3, and Ab-4 were from Calbiochem (San Diego, CA). Horse-radish peroxidase-conjugated and preadsorbed secondary antibodies tomouse and rabbit were obtained from Jackson Immunoresearch Labo-ratories (Westgrove, PA). Rat kidney proximal tubule cells (RPTC;SKPT-0193 clone 2), and human Bcl-2-overexpressing RPTC (Bcl-21)were described before (1). Membrane grade Triton X-100 and digitoninwere obtained from Roche Molecular Biochemicals. Other detergents andchemical cross-linkers disuccinimidyl tartarate, disuccinimidyl glutarate,DSP, DTSSP, DTBP, EGS, DPDPB, and SANPAH were purchased fromPierce. All other reagents were of the highest grade available.

ATP Depletion by CCCP

Cells were cultured in serum-supplemented Ham’s F-12/Dulbecco’smodified Eagle’s medium with 17.5 mM glucose as described (30) andplated at 105 cells/cm2 in 60- or 100-mm collagen-coated dishes. Afterovernight growth, cells were washed with phosphate-buffered salineand subjected to ATP depletion by incubation in glucose-free Krebs-Ringer bicarbonate buffer (in mM: 115 NaCl, 1 KH2PO4, 4 KCl, 1MgSO4, 1.25 CaCl2, and 25 NaHCO3; pre-gassed with 95% N2, air, and5% CO2) containing 15 mM CCCP at 37 °C under normoxic conditions.Glycine was included at 5 mM in the buffer to simulate glycine contentsof tissues in vivo (31, 32), thus preventing early necrotic injury duringincubation (33).

Preparation of Subcellular Fractions

Protocol I—Cytosolic and membrane fractions were prepared by se-lective plasma membrane permeabilization with digitonin (34), followedby membrane solubilization. Briefly, control and experimental cells indishes were treated with 0.05% digitonin in isotonic buffer A (10 mM

HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, pH 7.4; ; 107cells/ml) containing protease inhibitors (1 mM 4-(2-aminoethyl)benzenesul-fonyl fluoride hydrochloride, 0.8 mM aprotinin, 50 mM bestatin, 15 mM

E-64, 20 mM leupeptin, 10 mM pepstatin A), for 1–2 min at room tem-perature. Cell permeabilization by digitonin was standardized by meas-uring 100% release of lactate dehydrogenase and was also monitoredvisually under an inverted microscope. The permeabilized cells wereshifted to 4 °C, scraped with a rubber policeman, and collected intocentrifuge tubes. The supernatants (Dig/Cytosol) were routinely col-lected after centrifugation at 15,000 3 g for 10 min. Following centrif-ugation, the pellet was further extracted with ice-cold detergent (1%Nonidet P-40 or Triton X-100 or CHAPS) in buffer A containing prote-ase inhibitors for 60 min at 4 °C to release membrane- and organelle-bound proteins including mitochondrial cytochrome c. Both detergent-soluble (membrane) and insoluble fractions were collected by low speed(15,000 3 g) or high speed (500,000 3 g) centrifugation. The proteinpatterns of soluble membrane fractions, after low or high speed centrif-ugation, both by SDS-PAGE and Western blotting were indistinguish-able. Therefore, solubilized membrane fractions were routinely col-lected by centrifugation at 15,000 3 g for 10 min. The relative proteinlevels of Dig/Cytosol, membrane- and detergent-insoluble fractions are58 6 4%, 15 6 0.5%, 27 6 4% for control, and 48 6 6%, 14 6 0.5%, 38 66% for ATP-depleted cells (4 h CCCP), respectively (data from fourindependent experiments).

Protocol II—Subcellular fractionation of cells was also achieved byDounce homogenization in isotonic Buffer B (250 mM sucrose, 10 mM

HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, pH 7.4) and differen-tial centrifugation yielding nuclear (500 3 g pellet), mitochondrial(15,000 3 g pellet), microsomal (500,000 3 g pellet), and cytosol(500,000 3 g supernatant) fractions. Unbroken cells constituted ,1%,as monitored by light microscopy with trypan blue. Unlike control cells,ATP-depleted cells contained large numbers of altered mitochondriathat sedimented with nuclei. In order to make valid comparisons, nu-clear and mitochondrial fractions were collected together as 15,000 3 gpellet. Protein concentration was estimated with bicinchoninic acid(BCA) reagent (Pierce) following supplier’s protocol using bovine serumalbumin as standard.

Protein Cross-linking

All cross-linkers were dissolved in Me2SO just before using. Cross-linkers were added at 1 mM (0.1 mM for SANPAH) concentration tointact cells (;2 3 106 cells equivalent to 1 mg of total protein), cellspermeabilized with digitonin or detergent extracts of membrane. Opti-mal cross-linking conditions for cytosol and membrane extracts weredetermined after testing different cross-linker to protein ratios. 1 mM

concentration of all cross-linkers except for SANPAH (0.1 mM) wasfound to be optimal for a range of protein concentrations (100–500 mg)in the extracts. After the addition of cross-linkers, cells or extracts wereincubated on a head-to-head rocker for 30 min at room temperature.Amine targeting cross-linkers (NHS esters and imido esters) werequenched by adding 0.1 volume of 2 M Tris-HCl (pH 7.4) and incubatedwith rocking for another 30 min at room temperature. Sulfhydryl tar-geting cross-linker DPDPB was removed from cells by washing thepellets or from extracts by protein precipitation with 3% trichloroaceticacid or 80% acetone. In case of SANPAH, all the incubations werecarried at 4 °C with 20 min of incubation to react NHS esters and 10min of exposure to UV light (360 nm) to generate the nonspecificallyreactive nitrenes. After cross-linking, cytosol and membrane fractionsfrom intact cells were collected as described above. Cleavage of -S–S-bridge-containing cross-linkers was achieved by incubating extractswith 50 mM DTT for 30 min at 37 °C.

Immunoanalysis

Proteins were resolved by non-reducing or reducing (50 mM DTT)SDS-PAGE in Xcell II mini cell on 10% or 4–12% (gradient) NuPAGEgels (Invitrogen, CA) using MES or MOPS running buffer as recom-mended by the manufacturer. After electrophoresis, proteins from thegel were electroblotted onto 0.2-mm PVDF membranes following man-ufacturer’s directions. Western blotting using appropriate primary an-tibodies and peroxidase-conjugated suitable secondary antibodies wasperformed to analyze proteins. Chemiluminescent substrates (Pierce)were used to detect antigen-antibody complexes on the PVDF mem-

SANPAH, N-succinimidyl 6-[49-azido-29-nitrophenylamino]hexanoate;CCCP, carbonyl cyanide-m-chlorophenylhydrazone; DTT, dithiothre-itol; IEF, isoelectric focusing; MOPS, 4-morpholinepropanesulfonicacid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesul-fonic acid; MES, 4-morpholineethanesulfonic acid; PVDF, polyvinyli-dene difluoride; PAGE, polyacrylamide gel electrophoresis; NHS,N-hydroxysuccinimide.

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brane. Immunoprecipitation was carried out as follows. Solubilizedextracts in lysis buffer (1% Nonidet P-40 in buffer A with proteaseinhibitors) were pre-cleared by mixing with 50 ml of protein G-Sepha-rose beads (1:1 diluted suspension) for 1 h at 4 °C, and beads wereremoved by centrifugation. The resultant supernatants were incubatedwith primary antibodies (2 mg of antibody) at 4 °C for 2 h or overnight.Immunoprecipitates were collected by incubating with proteinG-Sepharose for 1 h, followed by centrifugation for 2 min at 4 °C. Thepellets were washed with lysis buffer three times, followed by threewashes with same buffer containing 500 mM NaCl to increase strin-gency and reduce nonspecific binding of proteins to immunoprecipi-tates. In some cases, high salt washes were omitted deliberately topreserve proteins in immunoprecipitates that might have associatednonspecifically or with weak affinity. After final wash, the proteinG-Sepharose beads with immunoprecipitates were suspended in SDS/sample buffer and analyzed by SDS-PAGE and Western blotting asdescribed above.

Protein Analysis by Isoelectric Focusing

For IEF, a 6% acrylamide (30% acrylamide, 1.8% N,N-methylenebisacrylamide) slab gel was prepared with 7.5% Ampholine (1:1 (v/v)mix of ampholytes pH 3.5–9.5 and pH 5.0–8.0; Amersham PharmaciaBiotech) with 1% CHAPS in a gel cassette. Cytosol and membraneextracts with or without cross-linking were applied to IEF gels asfollows. Protein samples (10–30 mg) were diluted with an equal volumeof 23 sample buffer (40 mM arginine, 40 mM lysine, 30% glycerol) andwere applied on top of the gel. The gel was focused for 1 h at 100 V, 3 hat 200 V, and 0.5 h at 500 V using anode (7 mM phosphoric acid) and

cathode (20 mM arginine, 20 mM lysine) buffers at room temperature.Proteins in IEF gels were electroblotted onto a 0.2-mm PVDF membranefor 1 h in 0.7%(v/v) acetic acid, pH 3.0, and were detected by appropriateantibodies. The pH gradient on the IEF gel was determined either bysurface electrode or by pH measurement of deionized water eluate offocused gel slices (0.5-cm width).

RESULTS

Relative Distribution of Bax in Normal and ATP-depletedCells—We have shown previously that cultured rat kidneyproximal tubule cells express high concentrations of Bax, whichis localized in the cytosol (1). ATP depletion by either hypoxiaor treatment with CCCP in the absence of glucose causes Baxtranslocation to mitochondria and cytochrome c release intocytosol (1). Provision of growth medium after hypoxia or chem-ically induced ATP depletion allows resynthesis of ATP byglycolysis and causes apoptotic death in cells with cytosoliccytochrome c (1). As shown in Fig. 1A, Bax is predominantlycytosolic in control cells (.99% soluble) and migrates to mem-branes (mitochondria) in ATP-depleted cells (.90% membrane-bound; Fig. 1A, lanes 2 and 3). The relative percentages ofsoluble and membrane-bound Bax in control and ATP-depletedRPTC were determined by densitometric analysis of chemilu-minescence signals on films after Western blotting. From sev-eral experiments, we found that, after 4 h of ATP depletion, the

FIG. 1. Bax redistribution during ATP depletion. RPTC were exposed to 15 mM CCCP in glucose-free medium for 4 h. Cell fractions wereanalyzed for Bax, cytochrome oxidase (COX), and cytochrome c after Western blotting with chemiluminescence detection. If more than one antigenwas probed, blots were exposed sequentially to antibodies of indicated antigens. A, relative distribution of Bax from normal and CCCP-treated cellsafter differential detergent fractionation. Cytosol from digitonin-permeabilized cells was obtained by collecting supernatant (Dig/Cyto) aftercentrifugation at 15,000 3 g for 10 min (lane 2) or 500,000 3 g for 15 min (lane 5). The cytosol-depleted cell pellet was then extracted with eitherNonidet P-40 or CHAPS and collected as detergent-soluble (NP/Mem or CH/Mem) and insoluble matrix fractions (NP/Insol or CH/Insol) aftercentrifugation as described under “Experimental Procedures.” Insoluble matrix fraction was dissolved in SDS/sample buffer, and all the fractionswere analyzed by loading proportional amounts of each fraction on reducing SDS-PAGE gels in relation to 100 mg of total cellular protein. Theaverage protein distribution in cytosol, membrane, and insoluble fractions is 58:15:27 for normal and 48:14:38 for 4 h ATP-depleted cells. B, relativedistribution of Bax and cytochrome oxidase in different cell fractions of control and CCCP-treated RPTC. Dig/Cyto (lane 2) and NP/Mem (lane 3)fractions obtained by protocol I are compared with cytosol (lane 4), combined nuclei 1 mitochondria (lane 5) and microsomal (lane 6) fractionsacquired by protocol II in relation to total cell lysate (lane 1). C, Bax and cytochrome c distribution in cell fractions of control (lanes 1–3) andCCCP-treated (lanes 4–6) RPTC.

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average amount of Bax translocated to membranes varies from80% to 100% of total. The zwitterionic detergent CHAPS ex-tracted membrane-inserted Bax almost as efficiently (.95%) asnonionic detergent Nonidet P-40 (Fig. 1A, see lanes 3 and 4 andlanes 6 and 7). Digitonin-released cytosol collected after cen-trifugation at 15,000 3 g for 10 min (Fig. 1A, lane 2) or500,000 3 g for 15 min (Fig. 1A, lane 5) contained the sameamount of Bax, although total protein was marginally reducedby 12 6 3% (four independent determinations) after 500,000 3g centrifugation. These results further confirmed the solublenature of Bax protein in normal cells and validated the use ofdigitonin to obtain cytosol from whole cells.

Double immunostaining, using an anti-Bax antibody and anantibody to cytochrome oxidase, a mitochondrial marker, re-vealed that the localization of Bax coincided exactly with thatof cytochrome oxidase during ATP depletion, showing that Baxtranslocates exclusively to the mitochondria (1). In accordancewith our earlier results, Bax and cytochrome oxidase wereshown in the current study to be present in different fractionsin normal cells but co-localized to the same fractions in ATP-depleted cells (Fig. 1B, lanes 2 and 3). In order to assess Baxtranslocation by conventional cell fractionation, Dounce ho-mogenization followed by differential centrifugation was car-ried out. Control cells provided clean fractions of nuclei, mito-chondria, microsomes, and cytosol. Integrity and specificity ofmitochondrial fractions were confirmed by the presence cyto-chrome c (data not shown). Interestingly, although mitochon-dria appeared filamentous in normal cells, they were round andaggregated around the nucleus in ATP-depleted cells (1), sug-gesting that ATP depletion has changed the shape and densi-ties of mitochondria. The modified shape and densities of mi-tochondria posed a challenge in obtaining clean mitochondrialfractions in ATP-depleted cells. Therefore, mitochondria andnuclei were collected together to assess Bax translocation. To-gether with our previous immunocytochemical observationsshowing that nuclei of ATP-depleted cells are devoid of Bax andthat the protein is visualized exclusively in mitochondria (1),these fractionation studies provided good evidence that Baxtranslocates to mitochondria, but not nuclei or the microsomalmembranes during ATP depletion (Fig. 1B, lanes 4–6). Basedon these results, we routinely used the crude membrane frac-tions that remained after the removal of digitonin-releasedcytosol to assess Bax localization. Separate studies showed thatmembrane bound Bax was efficiently extracted with deter-gents, and remained in the supernatants to the same extentregardless of whether the extracts were centrifuged at15,000 3 g or 500,000 3 g. Thus assay of detergent extracts ofthe crude membrane fraction represented a valid method toassess Bax in mitochondrial membranes. Double immuno-staining using anti-Bax and anti-cytochrome c antibodies re-vealed that, in 100% of the cells with Bax in mitochondria, adiffuse cytosolic cytochrome c staining was observed, whereascells with Bax in cytosol displayed a filamentous mitochondrialstaining (1). Data presented in Fig. 1C confirm these publishedobservations and show that, after Bax translocation, cyto-chrome c is released from mitochondria into the cytosol.

Mitochondrially Localized Bax Forms Oligomers in the Mem-brane—Although the Bax molecule has mitochondrial target-ing signals in its sequence, it remains unclear what factorskeep it in cytosol in normal cells. The molecular modificationsthat Bax might undergo before or during translocation to mi-tochondria are also unknown. In vitro studies have shown thattreatment of liposomes with Bax can permeabilize lipid mem-branes to allow transit of cytochrome c and dextrans (35).Calculation of the sizes of Bax-induced membrane pores hassuggested that homo-oligomers of at least four Bax molecules

are required to account for the results (35). These observationsand other considerations suggested to us that translocated Baxin mitochondrial membranes might exist in the form of oli-gomers. To preserve the possible oligomeric state of Bax in thecytosol or mitochondrial membranes, we employed a battery ofeight different bifunctional protein cross-linkers. Cross-linkingwas usually performed prior to solubilization of the mem-branes, since detergent treatment, by itself, can artificiallyinduce dimerization of Bax with other Bax molecules and withBcl-2 (26). These studies showed that Nonidet P-40 and TritonX-100, but not CHAPS, induced spurious homodimers and het-erodimers (26). As a control to confirm these prior observations,and to test the efficacy of cross-linkers to stabilize putativeoligomers, we also solubilized membranes in Nonidet P-40,Triton X-100, and CHAPS prior to cross-linking in someexperiments.

In our initial studies, control and CCCP-treated cells weresubjected to chemical cross-linking with a membrane-perme-able linker (DSP) at different time points. Cytosol was collectedand analyzed for Bax by SDS-PAGE and immunoblotting un-der non-reducing conditions. As shown in Fig. 2A, after pro-longed incubation with CCCP, Bax disappeared from the cy-tosol. However, we did not detect any slow-moving Baxcontaining adducts in the cytosol at any of these time points. Aphotoactivatable cross-linker SANPAH, one of whose reactivegroups can interact with any atom in the vicinity during cross-linking, also failed to demonstrate Bax adducts in the cytosol(data not shown), supporting the monomeric nature of cytosolicBax. Analysis of DSP cross-linked membrane fractions of ATP-depleted cells showed that progressively greater amounts ofBax are present in membranes with increasing durations ofATP depletion (Fig. 2B). In contrast to cytosolic Bax (Fig. 2A),the membrane-inserted Bax formed slow-moving adducts (Fig.2B, lanes 3–6). When the cross-linker was cleaved under re-ducing conditions with 50 mM DTT, only the 21-kDa species ofBax was present in these samples (Fig. 2C), suggesting that theslow-moving adducts indeed represent Bax-containing com-plexes. Interestingly, increasing the length of the cross-linkersfrom 6.4 to 16.1 Å permitted the demonstration of higher orderoligomers possibly containing six or more molecules of Bax inmembranes (Fig. 2D). The molecular sizes of these adductsobtained from various experiments were estimated to be mul-tiples of ;21 kDa (Fig. 2E), suggesting the formation of Baxhomo-oligomers. In order to rule out the possibility that pre-treatment of cells with digitonin might induce Bax oligomerformation, membranes were prepared with or without digitonintreatment. As shown in Fig. 2F, digitonin is not responsible forBax adduct formation in mitochondrial membranes of ATP-depleted cells.

Isoelectric Focusing Supports Homo-oligomerization of Baxin the Membrane—Although data presented in Fig. 2E suggestthat Bax monomers make up membrane-bound oligomers, itdoes not rule out the possibility that Bax may form oligomerswith other proteins of similar molecular size. To investigatethis possibility, SDS-PAGE analysis was complemented withisoelectric focusing to distinguish homo-oligomers from hetero-oligomers. This approach assumes that Bax-interacting pro-teins must have isoelectric points different from Bax. The pro-apoptotic Bax is an acidic protein with a theoreticallyestimated pI of 4.69, and this value correlated well with themeasured value of ;4.75 for untreated Bax by isoelectric fo-cusing (Fig. 3B). We employed two types of cross-linkers toanalyze oligomeric Bax. The sulfhydryl reactive agent DPDPB,which does not alter the net charge on reacting proteins, andthe amine-reactive agent EGS, which lowers the pI of cross-linked proteins by reacting with basic (amine) groups, were

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used. CHAPS extracted membrane oligomeric Bax treatedwithout or with DPDPB (SDS-PAGE; Fig. 3A, lanes 1 and 2)focused at a pI of ;4.75 (Fig. 3B, lanes 1 and 2), a value similarto that of monomeric Bax from cytosol (Fig. 3B, lane 7). Sincenonionic detergents but not CHAPS were shown to inducehomo- and hetero-oligomerization of Bax (26), cytosolic Baxwas exposed to either Triton X-100 or CHAPS prior to cross-linking with DPDPB. In either case, the cross-linked proteinfocused at the same pI as untreated monomeric cytosolic Bax(Fig. 3B, lanes 5–7). Results using the amine-reactive cross-linker EGS were similar, except that Bax migrated as a moreacidic species at a pI of ;4.4 due to loss of free basic aminegroups(s) in the protein. All other amine-reactive cross-linkersalso reduced the pI of Bax (data not shown). Membrane-boundoligomeric Bax, stabilized by EGS and extracted with CHAPS

(Fig. 3A, lane 3), and cytosolic Bax, artificially dimerized in thepresence of Triton X-100 and stabilized by EGS (see Fig. 5A,lane 1), co-migrated during isoelectric focusing (Fig. 3B, lanes 3and 4).

Bcl-2 Expression Does Not Prevent Translocation of Bax toMitochondria during ATP Depletion—We have shown earlierby immunocytochemistry and Western blotting that Bcl-2 doesnot prevent Bax translocation to mitochondria following hy-poxia or chemically induced ATP depletion in cultured proxi-mal tubule cells, but is able to block the release of cytochromec (1). Bax translocation is also a critical event in neuronalapoptosis and is not prevented by overexpression of Bcl-2 dur-ing nerve growth factor deprivation (36). However, Bcl-2 inhib-ited the release of cytochrome c, caspase activation, and celldeath in these neurons (36). These results contradict other

FIG. 2. Membrane translocation and oligomerization of Bax in membrane fractions. Following incubation of RPTC with CCCP for 0, 1,2, 2.5, 3, or 4 h, cells were treated with cleavable membrane-permeable cross-linker DSP (1 mM). Cytosol and membrane fractions were obtainedas described under “Experimental Procedures,” and proportional amounts corresponding to total protein were analyzed for Bax by Western blottingunder non-reducing conditions unless indicated. A, prolonged exposure to CCCP resulted in the disappearance of Bax from cytosol and did not showany slow moving adducts at all time points (lanes 1–6). B, progressively larger amounts of Bax accumulated in the membrane fraction (NonidetP-40 extract) as slow moving adducts (lanes 1–6). C, slow moving Bax adducts in DSP cross-linked cells are derived from Bax monomers. Slowmoving Bax adducts under non-reducing conditions (lanes 2–5) are converted to Bax monomers if disulfide bonds in the cross-linked preparationsare cleaved by incubation with 50 mM DTT (lanes 7–10). D, cross-linkers with longer spacer arms demonstrated the presence of increased amountsof higher order Bax oligomers. Membrane extracts of ATP-depleted cells (4 h CCCP) after incubating with cross-linkers of different spacer lengthsas indicated. E, correlation of molecular weights of Bax adducts with expected number of Bax molecules in the corresponding adduct. The molecularweights of adducts containing Bax were calculated by plotting their migrations against migrations of molecular weight standards in semi-logarithmic plots from eight different experiments. Inset shows two representative lanes of Bax ladders obtained by reducing SDS-PAGE andWestern blotting of EGS cross-linked membranes from ATP-depleted cells. F, digitonin pre-treatment does not induce Bax adduct formation in themembranes. Membranes from digitonin-permeabilized cells (Dig1) or heavy membrane fraction containing mitochondria and nuclei (Dig2) weretreated with (CL1) or without (CL2) cross-linker (SANPAH) before extraction with Nonidet P-40. Samples (20 mg) were analyzed for Bax underreducing conditions. Both preparations showed presence of similar Bax adducts (lanes 2 and 4). Number of Bax monomers in all slow movingadducts is indicated in appropriate panels.

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published results where Bax migration into mitochondria wasblocked by Bcl-2 overexpression (25, 37). The relative levels oftotal Bax in RPTC and Bcl-21 cells are not significantly differ-ent. With extended durations of ATP depletion, Bax transloca-tion in Bcl-21 cells reaches similar levels as in RPTC (Fig. 4A,compare lanes 1–5 and 6–10), as we have reported previously(1). The studies presented here had greater than 70% of totalBax translocated to membranes.

Bcl-2 Prevents Oligomerization of Translocated Bax in theMembrane without Forming Hetero-oligomers with Bax—Wehave reported before that translocated Bax molecules in mem-brane fractions of ATP-depleted Bcl-21 cells have little or noassociation with Bcl-2 as shown by cross-immunoprecipitationstudies (1). When Bax antibodies were used, the immunopre-cipitates did not contain Bcl-2; conversely, when Bcl-2 antibod-ies were used, only trace amounts of Bax were occasionallyfound in the precipitates. Even these trace amounts of Bax thatwere sometimes present in Bcl-2 immunoprecipitates are likelyto represent low affinity nonspecific adsorption, or Bax/Bcl-2heterodimers induced artificially by nonionic detergents (26,38). We studied the issue further by using eight protein cross-linkers of different spacer lengths and chemical reactivities todetect associations of Bax and Bcl-2. Membrane-translocatedBax failed to form oligomers with either Bcl-2 or other Baxmolecules in cells overexpressing Bcl-2 (Fig. 4, B and C). Lackof Bax oligomerization was evident with four different chemicalcross-linkers, as shown in Fig. 4B, the photo cross-linker SAN-PAH (Fig. 4C), and three other cross-linkers (data not shown).On the other hand, in RPTC, which do not overexpress Bcl-2,translocated Bax readily forms large molecular weight adductseven when ,15% of total Bax is present in the membrane (Fig.2B, lane 3). Moreover, when these membranes of ATP-depletedRPTC are cross-linked with the photo-activable cross-linkerSANPAH, oligomeric Bax adducts are demonstrated readily incontrast to the behavior of the same protein in ATP-depleted

Bcl-21 cell membranes (Fig. 4C, inset). The failure of SANPAHto cross-link Bax to other Bax molecules or to Bcl-2 is not likelyto be related to the availability of specific reactive groupsnearby in the putative partner. One of the reactive groups inSANPAH is amine-specific, but the other forms a highly reac-tive nitrene that forms adducts nonspecifically with any atomin the vicinity. Thus, SANPAH has the ability to cross-linkamines in one partner to a wide spectrum of structures in theother partner within the reach of the spacer arm. On the otherhand, steric factors related to cross-linker spacer lengths anddistances between reactive groups in the partners also need tobe considered. A partial answer to this question is provided bythe results using SANPAH, as discussed above. Additionally,we deliberately solubilized ATP-depleted Bcl-21 cell mem-branes in Nonidet P-40, Triton X-100, or CHAPS prior to cross-linking. Under these conditions as reported previously byYoule’s group (26), Bax/Bax, Bax/Bcl-2, and Bcl-2/Bcl-2 dimersare induced artificially by Nonidet P-40 and Triton X-100 butnot by CHAPS. As the results in Fig. 5 show, Bax/Bax, Bax/Bcl-2, and Bcl-2/Bcl-2 adducts were readily demonstrable whencross-linking was done after Nonidet P-40 or Triton X-100solubilization, but not CHAPS treatment. Finally, the spacerlengths of the eight cross-linkers that we used vary between 6.4and 19.9 Å, distances that should cover a wide range of sepa-ration of reactive groups in the partners. Together with theimmunoprecipitation results, these observations provide im-portant data that argue strongly for the validity of not only thepositive observations with respect oligomeric Bax adducts, butalso their prevention by Bcl-2, and the lack of demonstrableBax/Bcl-2 associations when cross-linking was performed be-fore detergent solubilization.

Detergent-induced Bax Homodimerization Is Inhibited byBcl-2—It has been reported previously that detergents induceBax homodimerization and Bax/Bcl-2 heterodimerization (26,38). We have extended our studies to test whether Bax oli-

FIG. 3. Analysis of Bax by SDS-PAGE (A) and isoelectric focusing (B) to identify the nature of Bax oligomers. ATP-depleted cells werefirst treated with or without indicated cross-linkers, and membrane proteins were then extracted with CHAPS. Cytosol from control cells was firstexposed to Triton X-100 or CHAPS followed by cross-linking and immunoblotting for Bax after SDS-PAGE (A) or isoelectric focusing (B) undernon-reducing conditions. CH, CHAPS; TX, Triton X-100; CL1, DPDPB; CL2, EGS; 2, no cross-linker. The sulfhydryl-reacting cross-linker, DPDPB,does not change the pI of Bax, whereas EGS altered the pI of modified Bax.

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gomerizes in the presence of detergents. Whole cells with cyto-solic Bax (normal RPTC) were extracted with the detergentsTriton X-100, Nonidet P-40, and CHAPS and then subjected tochemical cross-linking with DSP. Bax analysis by immunoblot-ting showed that cytosolic Bax formed homodimers in the pres-ence of detergents Triton X-100 and Nonidet P-40 (Fig. 5A).However, the zwitterionic detergent CHAPS failed to induceBax homodimerization (Fig. 5A, lanes 2 and 5). These resultsagree completely with previous reports that showed differentialeffects of detergents on Bax dimerization (26, 38).

Since Bcl-2 prevented Bax oligomerization in the mitochon-drial membrane, we tested whether Bcl-2 also interferes withdetergent-induced Bax homodimerization. Normal Bcl-21 cellswere solubilized with different detergents to allow membraneBcl-2 and cytosolic Bax to interact with each other, cross-linked, and then analyzed by SDS-PAGE and Western blotting(Fig. 5B, lanes 1–6 (anti-Bax) and lane 7 (anti-Bcl-2)). In cellextracts from Bcl-2-overexpressing cells (Fig. 5B), Bax ho-modimerization was partially inhibited (Fig. 5, compare lanes 1and 3 in panels A and B). Although Bcl-2 was able to formheterodimers with Bax in these detergents, the total amount ofBax/Bcl-2 heterodimers could not account for the entire de-crease in Bax/Bax homodimer formation. Speculatively, itseems reasonable to consider that Bcl-2 may reduce Bax ho-modimerization by competing with Bax for space in detergentmicelles. Although the results show that Bcl-2 has the ability toform heterodimers with Bax, they also show that the amountsof heterodimers that form are relatively sparse, considering theconcentrations of the partners in the detergent, suggesting thatthey cannot form tight complexes. Further studies are clearlynecessary to address the questions regarding Bax and Bcl-2interactions among themselves and each other in detergents aswell as in membranes. The Bax homodimers seen in TritonX-100 extracts without cross-linker (Fig. 5, lane 4 in panels Aand B) are probably due to presence of oxidizing contaminantseven in the membrane grade detergent. These dimers disap-peared with pretreatment of extracts with 50 mM DTT for 30min at 37 °C (but not boiling for 10 min). Similarly, the trace

amounts of dimers seen after non-reducing SDS-PAGE of oli-gomerized membrane Bax not subjected to cross-linking (Figs.2D and 3A, lane 1), which are probably due to incompletedissociation and/or partial oxidation, also disappeared underreducing conditions (Fig. 2F, lanes 1 and 3).

In order to investigate the physical state of Bcl-2 in mito-chondrial membranes, Bcl-21 cells were treated with DSP fol-lowed by membrane extraction with Triton X-100, NonidetP-40, or CHAPS. Bcl-2 migrated as a monomeric protein onSDS-PAGE with (data not shown) or without cross-linking (Fig.5C, lane 1). However, when the membrane extracts in abovedetergents were subjected to cross-linking, small amounts ofBcl-2/Bcl-2 homodimers were detected in Triton X-100 or Non-idet P-40 extracts (Fig. 5C, lanes 2 and 4) but not in CHAPSextract (Fig. 5C, lane 3). Together, these results show thatdetergents such as Nonidet P-40 and Triton X-100 can inducethe formation of homo- and heterodimers of Bax and Bcl-2.

Dimer-forming Detergents Reduce Higher Order Bax Oli-gomers to Dimers—The data presented above indicate that thestructural conformation of Bcl-2 and Bax in natural mem-branes is different from that in detergents. An important dif-ference between detergents and membranes is that detergentspredominantly form micellar structures, whereas membranesare organized as lipid bilayers with asymmetric distribution ofproteins and lipids. We therefore tested whether detergentsolubilization of membrane-inserted Bax would change its oli-gomeric properties. Stabilization of complexes by chemicalcross-linking was carried out before or after detergent solubi-lization of membranes of ATP-depleted RPTC and Bcl-2-over-expressing cells. The data presented in Fig. 6A show thattranslocated Bax in natural membranes exists in oligomericform. The higher orders of oligomers were reduced mainly todimers when cross-linking was performed after solubilizationwith either Nonidet P-40 or Triton X-100 (Fig. 6A, comparelanes 1 and 2 or lanes 3 and 4). Lane 5 containing TritonX-100-solubilized membranes without cross-linker shows Baxdimers; as discussed earlier, dimer formation in this case isalso attributable to incomplete dissociation and oxidants pres-

FIG. 4. Effect of Bcl-2 overexpres-sion on Bax translocation and oli-gomerization. A, time course of Baxtranslocation in Bcl-21 cells and RPTC an-alyzed by Western blotting after reducingSDS-PAGE. B, cross-linkers of diversespacer lengths and reactivities failed toreveal slow moving Bax adducts in mem-branes from Bcl-21 cells analyzed on non-reducing gels. Results with four cross-link-ers are shown after 4 h of CCCP treatment.C, nonspecifically reactive photoactivat-able cross-linker SANPAH also failed toreveal Bax oligomers in membranes ofBcl-2-overexpressing cells. At varioustime points of CCCP incubation, Bcl-21cells were permeabilized with digitoninand treated with photo-cross-linker SAN-PAH before membrane extraction withNonidet P-40. Unlike Bcl-21 cells, CCCP-treated (4 h) RPTC readily revealed slowmoving Bax adducts after cross-linkingwith SANPAH (see inset).

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ent in Triton X-100 (Fig. 5). As shown before, Bax oligomeriza-tion was dramatically reduced in membranes from ATP-de-pleted Bcl-2-overexpressing cells (Fig. 6B, lanes 1 and 3). It isworth noting again that the Bcl-2 protein did not form eitherhomo-oligomers or hetero-oligomers with Bax in such cells (Fig.6B, lanes 1 and 3). However, if membranes had been solubilizedwith Nonidet P-40 and Triton X-100 before cross-linking, Bax/Bcl-2 heterodimers formed readily (Fig. 6B, lanes 2 and 4). Theidentity of Bax/Bcl-2 heterodimers was confirmed both by mo-lecular weight calculation and Western blotting with anti-Bcl-2antibodies (data not shown). In contrast, membranes solubi-lized in CHAPS before cross-linking still contained higher or-der Bax oligomers in RPTC (Fig. 6C, lane 1) but in ATP-depleted Bcl-21 cells, no Bcl-2 containing adducts were detected(Fig. 6C, lane 2). Controls with cross-linking followed byCHAPS extraction also showed similar results (Fig. 3A, lane 3).Overall, our results suggest that Bax oligomerization in themitochondrial outer membrane is prevented under natural con-ditions (i.e. without prior exposure to detergents) by Bcl-2without requirement for stable associations with Bax.

VDAC or ANT Form Homodimers but Not Heterodimers withBax—Recently it has been reported that Bax may interact withan outer membrane protein, the voltage-dependent anion chan-nel protein (also known as porin), to induce cytochrome c re-lease (27). Another study reported that Bax might interact withan inner membrane protein, the adenine nucleotide transloca-tor (28). This interaction has also been suggested to be anantecedent factor responsible for cytochrome c release. There-fore we searched for Bax and Bcl-2 interactions with VDAC orANT in our ATP depletion model. Although our cross-linkingand isoelectrofocusing studies have indicated no association ofBax with proteins other than itself, further studies were car-

ried out to identify Bax-associated proteins. Membrane ex-tracts were immunoprecipitated with antibodies to Bax orBcl-2, and the resulting precipitates were analyzed for thepresence of VDAC or ANT by immunoblotting. As shown in Fig.7A, antibodies to Bax and Bcl-2 immunoprecipitated only Baxand Bcl-2, respectively, and did not bring down even traces ofVDAC protein (Fig. 7A, lanes 2 and 3 and lanes 4 and 5). Ourattempts to immunoprecipitate VDAC from nonionic detergentmembrane extracts with four commercially available antibod-ies failed, though these antibodies could recognize VDAC inimmunoblots (Fig. 7A, lane 1). However, when the membraneproteins were solubilized with SDS and renatured by dilutingwith Nonidet P-40-containing buffer, these same antibodiessuccessfully immunoprecipitated VDAC (data not shown), sug-gesting that partial renaturation of VDAC exposes otherwiseburied antibody-binding epitopes. Available antibodies againstVDAC were therefore not usable for co-immunoprecipitationstudies to identify association partners, at least in our modelsystem. Conceivably, steric hindrance related to the complexityof the outer membrane protein microenvironment may alsohave been responsible for failed immunoprecipitation of VDACwith these antibodies. Immunoblotting of Bax and Bcl-2 immu-noprecipitates for ANT also gave negative results (data notshown), suggesting that neither ANT nor VDAC is associatedwith translocated Bax in mitochondria. Immunoprecipitationunder less stringent (150 mM NaCl washes only) as well asstringent (0.5 M NaCl washes included) conditions failed toreveal the presence of VDAC or ANT in the immunoprecipi-tates (data not shown). Therefore, chemical cross-linking ap-proach was used to stabilize any weak interactions betweenproteins and identify Bax-associated proteins. Our results, pre-sented in Fig. 7B, show that Bax and VDAC each can form

FIG. 5. Detergents induce homo-and heterodimers of Bax and Bcl-2that can be cross-linked. Control RPTC(A) or control Bcl-21 cells (B) were firstsolubilized with indicated detergents Tri-ton X-100 (TX), CHAPS (CH), or NonidetP-40 (NP) without (2CL) or with (1CL)chemical cross-linking with DSP. Sam-ples were analyzed for Bax and Bcl-2 afterseparation on non-reducing SDS-PAGE.A, the nonionic detergents Triton X-100(lane 1) and Nonidet P-40 (lane 3) induceddimerization of Bax, whereas CHAPS didnot (lane 2). B, analysis of Bax (lanes 1–6)and Bcl-2 (lane 7) shows that in Bcl-2-overexpressing cells, Triton X-100 (lane 1)and Nonidet P-40 (lanes 3 and 7) inducedBax homodimers and Bax/Bcl-2 het-erodimers as demonstrated by cross-link-ing. Bax dimerization is reduced by Bcl-2.CHAPS failed to induce homodimers orheterodimers. C, nonionic detergents, Tri-ton X-100 or Nonidet P-40 (lanes 2 and 4),induced weak homodimerization of Bcl-2in membrane fractions after extraction,but CHAPS does not (lane 3). Note thathomodimers of Bax or Bcl-2 were fre-quently observed following extractionwith Triton X-100, but not with NonidetP-40 or CHAPS even without the use ofchemical cross-linkers. These dimerswere readily disrupted by treatment with50 mM DTT for 30 min at 37 °C.

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homo-oligomers; in the same fractions, Bax/VDAC associationswere not observed as there were no adducts of intermediatesize (;54 kDa). Such associations between Bax and VDAC orANT should have produced heterodimers in the size range of52–56 kDa. We did not detect complexes in that size range inBax containing mitochondrial membranes from ATP-depletedcells. Detection of VDAC homo-oligomers probably supportstheir role as channel-forming proteins. Additionally, Bcl-2/VDAC associations could not be demonstrated in Bcl-2-overex-

pressing control cells (Fig. 7B, lanes 8 and 9). Similarly, ANTprotein also did not form associations with Bax or Bcl-2 (Fig.7C). In control cells, DTBP did not cross-link ANT molecules,whereas DPDPB, an agent with longer linker length, did (Fig.7C, lanes 1 and 2 and lanes 5 and 6). On the other hand, inATP-depleted cells, both DTBP and DPDPB reacted withANT and revealed dimers (Fig. 7C, lanes 3, 4, 7, and 8). Thisresult suggests that ANT molecules, during ATP depletion,either come closer or undergo conformational change or both

FIG. 6. Pre-exposure of Bax oligomers to nonionic detergents disintegrates higher order oligomers to dimers. Membranes fromCCCP-treated (4 h) RPTC and Bcl-21 cells were either first subjected to cross-linking followed by extraction with nonionic detergents (CL1NP orCL1TX) or were solubilized first with different detergents and then subjected to cross-linking (NP1CL, TX1CL, or CH1CL). All samples(cross-linked with DSP) were analyzed under non-reducing conditions. A, higher order oligomers diminished markedly when membranes wereexposed to nonionic detergents before being subjected to cross-linking (lanes 2 and 4) compared with cross-linking followed by extraction (lanes 1and 3). Lane 5 represents Triton X-100 extract (membrane fraction) of CCCP-treated cells, without cross-linking, containing translocated Bax.Dimer represents artificial disulfide linkage not disrupted by SDS-PAGE under non-reducing conditions. B, in Bcl-2-overexpressing cell mem-branes, membrane-translocated Bax did not form higher order homo-oligomers or heterodimers with Bcl-2, as demonstrated by cross-linkingfollowed by solubilization (lanes 1 and 3). Moreover, only trace amounts of Bax dimers could be seen, in contrast to the abundance of dimers andhigher order oligomers that form in membranes in wild type (RPTC) cells (A). However, when membranes were exposed to the nonionic detergentsNonidet P-40 (lane 2) or Triton X-100 (lane 4) before cross-linking, Bax/Bcl-2 heterodimers appeared. The identity of the faint bands seen spanningbelow the 31-kDa region is not known. C, CHAPS solubilization prior to cross-linking still preserved Bax oligomers, which formed aftertranslocation to the membrane (lane 1). Results with cross-linking followed by CHAPS solubilization are identical (see Fig. 3A, lane 3). In Bcl-21cell membranes containing translocated Bax, Bcl-2 did not form any slow moving adducts (lane 2). CH, CHAPS; NP, Nonidet P-40; TX, TritonX-100.

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to allow cross-linking by DTBP. Our failure to identify inter-mediate forms of ANT/Bax or ANT/Bcl-2 complexes with fourother cross-linkers of different spacer arm lengths and reac-tive specificities, including the nonspecific highly reactivephoto cross-linker SANPAH (data not shown), together withunequivocal negative immunoprecipitation results, arguesagainst the necessity for such interactions for the function ofBax or Bcl-2 proteins.

Isoelectric Focusing Analysis to Identify Homo-oligomeriza-tion Versus Hetero-oligomerization of Bax—The calculated val-ues of isoelectric points (pI) of rat Bax (4.69) and human Bcl-2

(7.32) are close to experimental values of 4.76 (rat Bax) and7.39 (human Bcl-2, see Fig. 8A). The calculated pI values of theproteins that have been reported to interact with Bax, e.g.VDAC (9.04), ANT (10.54), Bcl-2 (7.32) and proteins that areknown to be released by Bax from the mitochondria, e.g. cyto-chrome c (10.39), adenylate kinase (9.86), and apoptosis-induc-ing factor (9.63) suggest that all of these proteins are basic innature. In contrast, Bax is an acidic protein (pI 4.75) both inRPTC (see Fig. 3) and Bcl-2-overexpressing cells (Fig. 8A, lanes1–3) before or after translocation to membrane. Bcl-2, in nor-mal cells, has two fast moving, minor isoforms with pI of 6.61

FIG. 7. Mitochondrial VDAC and ANT proteins failed to co-immunoprecipitate with Bax and did not form cross-linked adductswith Bax but formed homo-oligomeric adducts. A, immunoprecipitation of detergent extracts from ATP-depleted RPTC with anti-Bax (mousemonoclonal, 1D1) and ATP-depleted Bcl-21 cells with anti-Bcl-2 (rabbit polyclonal DC 21) antibodies were carried out as described under“Experimental Procedures.” The immunoprecipitates were analyzed under non-reducing or reducing (data not shown) SDS-PAGE, followed byimmunoblotting with anti-porin 31HL antibody, and subsequently probed for either Bax or Bcl-2. Membrane fraction from RPTC was included asa control for VDAC signal (lane 1). Both a-Bax (lanes 2 and 3) and a-Bcl-2 (lanes 4 and 5) antibodies failed to co-precipitate VDAC (shown byabsence of signal). On the other hand, probing these blots with Bax or Bcl-2 antibodies showed signal for the presence of Bax (lanes 2 and 3) andBcl-2 (lanes 4 and 5), respectively, in these immunoprecipitates. Intact IgG present in the precipitates is not shown. Analyzing samples underreducing conditions (data not shown) revealed extra bands of IgG light (25 kDa) and heavy (50 kDa) chains with no bands in the 33-kDa region,indicating lack of VDAC in Bax immunoprecipitates. Similar results were obtained with ANT (data not shown). B, chemical cross-linking with DSPor DTSSP was carried out after releasing cytosol with digitonin. Digitonin permeabilization allowed water-soluble DTSSP to react withintracellular membranes. Membrane fractions from RPTC after ATP depletion and control Bcl-21 cells were analyzed for Bax and VDAC. VDACwas visualized as homo-oligomeric adducts in both ATP-depleted RPTC (lanes 2 and 3) as well as in control Bcl-21 cells (lanes 8 and 9). Lack ofassociation of VDAC with Bax in ATP-depleted RPTC or control Bcl-21 cells was suggested by the absence of adducts with molecular mass of ;54kDa (Bax/VDAC dimer) or ;59 kDa (Bcl-2/VDAC dimer). Bax oligomers in ATP-depleted RPTC were present (a-Bax), with no evidence ofBax-VDAC heterodimer formation (absence of ;54 kDa; lanes 5 and 6). C, the inner mitochondrial membrane protein ANT was visualizedpredominantly as monomers and homodimers, with smaller amounts of higher order complexes when treated with DPDPB in both control andATP-depleted membranes from Bcl-21 cells (lanes 1 and 3) or RPTC (lanes 5 and 7). In contrast, DTBP, a cross-linker with a shorter spacer, failedto stabilize ANT dimers and its complexes in control cells (lanes 2 and 6), but stabilized them in membranes from ATP-depleted cells (lanes 4 and8). Complexes in the size range expected for Bax/ANT (;55 kDa) or Bcl-2/ANT (;60 kDa) heterodimers were not visualized. When the membraneswere sequentially probed for Bax, homo-oligomers of Bax were visualized in ATP-depleted cells; again, complexes in the size range of Bax/ANToligomers (55 kDa) were not found (data not shown). Similarly, sequential probing for Bcl-2 also failed to reveal Bcl-2/ANT complexes (data notshown).

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and 6.85 (Fig. 8). These two minor forms may represent phos-phorylation variants of Bcl-2 since phosphorylation of Bcl-2 atboth tyrosine and serine/threonine residues has been reported(39, 40). Disappearance of these two isoforms with DPDPBcross-linking (Fig. 8A, lane 5) probably suggests that theseisoforms may have been cross-linked to non-soluble cell struc-tures (41). Amine-reactive cross-linkers like EGS have changedBcl-2 into acidic species (Fig. 8A, lane 6; pI 6.16–6.85). Basedon the formation of small amounts of Bax/Bcl-2 dimers in thepresence of Nonidet P-40 detergent (Fig. 5B), we consideredthat such a complex would have an intermediate pI that is anaverage of Bax and Bcl-2 pI values. As shown in Fig. 8B, wewere able to detect such a complex with a pI value of ;5.8 on anIEF gel in DPDPB-cross-linked detergent-solubilized mem-branes. However, in ATP-depleted Bcl-21 cell membranes, wefailed to find Bax/Bcl-2 complexes not only in SDS-PAGE gels(Fig. 4, B and C) but also in IEF gels (Fig. 8A), suggesting thatsuch complexes could only form under artificial conditions. IfVDAC or ANT were to form such complexes with Bax, weshould have been able to detect Bax protein at a pI ;6.8 or ;7.5, respectively. However, our results (Fig. 8A, lanes 2 and 3)clearly indicate that no such complexes are formed in cross-linked membranes, suggesting that Bax does not interact witheither VDAC or ANT in the mitochondria.

Based on our results, we propose a model (Fig. 9) to explainhow Bax may permeabilize the mitochondrial outer membraneto release cytochrome c and other intermembrane space pro-teins. The model assumes a channel formed by Bax that canpermeate intermembrane proteins across the mitochondrialouter membrane.

DISCUSSION

Regulation of apoptosis by the Bcl-2 family of proteins occursprimarily at the mitochondrial outer membrane and involvesmitochondrial permeabilization or its prevention. The studiespresented in this paper are attempts to understand the mech-anisms by which Bax induces cytochrome c efflux from mito-

chondria of ATP-depleted cells. Although the signaling mech-anisms responsible for Bax translocation during ATP depletionremain unclear, the experiments reported here have revealedimportant insights into the physical states and associations ofBax molecules after they have been inserted into mitochondrialmembranes. Using chemical cross-linkers, we were able to de-tect higher order homo-oligomers of Bax in membrane fractionsof ATP-depleted cells. The presence of Bax oligomers fromtetramers to decamers (Figs. 2, 3, 4, and 6) in the membranefraction suggests that Bax may be able to form large structureswith potential to allow the passage of proteins at least of thesize of cytochrome c (;12 kDa). Recently, it has been demon-strated that at least 4 molecules of Bax can form a pore of size22 Å that is capable of transporting cytochrome c, a moleculewith a Stokes diameter of 17 Å (35). Similar oligomers wereidentified with recombinant Bax protein in the presence of octylglucoside (42). Isoelectric focusing analysis of these cross-linked oligomers suggests that Bax multimers contain homo-geneous populations of Bax molecules (Figs. 3 and 8). Overall,with respect to the current studies, our results using cross-linkers clearly favor homo-oligomerization of Bax in the mito-chondrial outer membrane.

In contrast, cytosolic Bax failed to form oligomers (Fig. 2A).These findings suggest that Bax containing adducts in cytosolmove instantaneously into mitochondria soon after they haveformed, or that the unique conformation of cytosolic Bax doesnot allow reactivity with cross-linkers. The latter possibilitywas ruled out by the fact that amine-reactive cross-linkerswere able to alter the pI of cytosolic Bax.2 Cross-linking atdifferent time points after ATP depletion failed to reveal Baxadducts in the cytosol, and this may rule out the first possibil-ity. On the other hand, nonionic detergents such as NonidetP-40 and Triton X-100 were able to induce dimerization of

2 V. Mikhailov, M. Mikhailova, D. J. Pulkrabek, Z. Dong, M. A.Venkatachalam, and P. Saikumar, unpublished data.

FIG. 8. Isoelectric focusing analysis of membrane fractions to identify hetero-oligomers of Bax. Isoelectric focusing methodology wasdescribed under “Experimental Procedures.” A, CHAPS extract of membranes from ATP-depleted and control Bcl-21 cells were subjected to IEFwith or without cross-linking, as described under “Experimental Procedures” (2CL, no cross-linker; CL, 1, DPDPB; CL 2, EGS). The sulfhydryl-reacting cross-linker, DPDPB, does not change the pI of Bax or Bcl-2 (lanes 2 and 5), whereas the amine-reactive cross-linker, EGS, changes thepI of these proteins (lanes 3 and 6). Bcl-2 has two minor isoforms with pI lower than the predominant form indicated by arrowheads (lane 4). AfterDPDPB cross-linking, these two isoforms were not seen, suggesting possible cross-linking of these forms to non-soluble cell structures withfree-sulfhydryl groups. In either case, complexes with pI greater or lesser than the pI of Bax or Bcl-2 were not detected with or withoutcross-linking, suggesting lack of interaction with other proteins including VDAC and ANT. B, control Bcl-21 cells were solubilized in Nonidet P-40followed by cross-linking with DPDPB. Detergent-induced Bax/Bcl-2 heterodimers (see inset for sample analyzed by SDS-PAGE and immunoblot-ting) were detected by immunoblotting after IEF. Both a-Bax and a-Bcl-2 antibodies were utilized sequentially to identify Bax and Bcl-2 in theimmunoblot. The pI of the Bax/Bcl-2 complex is the average of Bax and Bcl-2 pI values. CL 1, DPDPB; CL 2, EGS; 2CL, no cross-linker.

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cytosolic Bax with itself or Bcl-2, as demonstrated by cross-linking, suggesting that detergents were altering the confor-mation of cytosolic Bax (Fig. 5). However, CHAPS, a zwitteri-onic detergent, did not induce Bax or Bcl-2 oligomerization,which suggests that this detergent has actions different fromthose of nonionic detergents with respect to its ability to modifyBax or Bcl-2 molecules. These results confirm earlier work fromYoule’s group (26, 38) and suggest that Bax monomers move tomitochondrial membranes in response to unknown stimuli andform homo-oligomers in situ. Our results also suggest that,although there may be similarities between lipid bilayers anddetergent micelles in their effects on Bax with respect to theirshared ability to induce and maintain oligomers, there are alsokey differences. When cross-linking of membrane proteins wascarried out after membrane solubilization in Nonidet P-40 orTriton X-100, multimers of Bax were either absent or presentin trace amounts, the vast majority of Bax being present asdimers and monomers (Fig. 6A). This suggests that only planarmembrane bilayers allow Bax multimerization to higher orderstructures, and that nonionic detergents might have dissoci-ated these pre-existing Bax multimers. In contrast, CHAPS,which does not induce oligomerization of cytosolic Bax, also

failed to dissociate higher order Bax oligomers in mitochondrialmembranes (Fig. 6C).

In contrast to results obtained by others, we have shown thatBcl-2 overexpression does not abolish Bax translocation to mi-tochondria (Fig. 4A). The results presented in this paper dem-onstrate that Bcl-2 cannot block Bax association with mito-chondria in the ATP depletion model. However, Bcl-2 cancompletely prevent Bax oligomerization in the mitochondrialouter membrane (Figs. 4 (B and C) and 6B). Thus, at least inour experimental model, Bcl-2 may inhibit cytochrome c releaseand protect cells by preventing oligomerization of Bax ratherthan by blocking Bax insertion into mitochondrial membrane.Moreover, we also found that Bax translocation to mitochon-dria in rat proximal tubule cells is independent of apoptoticstimuli. UV (80 J/m2) exposure also induces Bax translocationin both RPTC and Bcl-21 cells but oligomerization is seen onlyin RPTC but not in Bcl-21 cells.2 This suggests that preventionof Bax translocation is not a necessary prerequisite for theprotective actions of Bcl-2 in mitochondrial membranes.

Our results suggest that prevention of Bax homo-oligomer-ization by Bcl-2 does not involve direct interactions of Bcl-2with Bax, at least to an extent of close proximity and affinity

FIG. 9. Hypothetical model for Bax-induced mitochondrial outer membrane permeabilization and Bcl-2 protection. TranslocatedBax undergoes conformational changes in mitochondrial outer membranes to form oligomers. Based on our results and published work, we proposethat higher order oligomers ($4 Bax molecules) form channels that permit the transit of apoptosis-activating factors such as cytochrome c.Although not conclusively demonstrated, our results also suggest that Bax is not associated with other mitochondrial channel-forming proteins(VDAC and ANT) and that such interactions may not be required for the permeabilizing actions of Bax. Alternatively, it is possible but unlikelythat oligomeric Bax do not form pores, but somehow induce pore formation by rearranging other mitochondrial outer membrane proteins (data notshown). In Bcl-2-overexpressing cells, oligomerization of Bax is prevented by Bcl-2 even after being inserted into the membrane. We suggest thatsteric hindrance imposed by abundant and possibly saturating concentrations of Bcl-2 in membrane microdomains may prevent inserted Bax fromundergoing conformational changes required for oligomerization. This model can also explain, in other examples of apoptosis where ATP levels arenot completely compromised, the apparent prevention of Bax insertion by saturating concentrations of Bcl-2 in membrane (25, 37) by assuming thatpartially inserted Bax may return to cytosol due to failed conformational changes required for Bax anchoring in the membrane. OM, outermembrane; IM, inner membrane; IMS, intermembrane space.

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that could have been revealed by co-immunoprecipitation, ordemonstrable by eight different cross-linkers with varyingspacer arms (6.4–19.9 Å) and reactivities (imido and NHSesters for amines, pyridyldithio for sulfhydryls, and photo-activable nitrophenylazide for non-selective linking to atoms inthe vicinity). All of these cross-linkers were able to form Bcl-2adducts, visualized as Bcl-2 homodimers and Bcl-2/Bax het-erodimers, only when pretreated with nonionic detergentsprior to cross-linking; but failed to detect such adducts whenmembranes were cross-linked prior to detergent extraction.Together with immunoprecipitation studies, our results sug-gest strongly that mechanisms other than direct Bcl-2/Baxinteractions must be involved in Bcl-2 protection against Baxcytotoxicity. How then, could Bcl-2 prevent Bax-induced leak-age of cytochrome c? The inability of membrane-bound Bax toform oligomers in mitochondria containing Bcl-2 could conceiv-ably be explained by the abundance of Bcl-2 and possible sat-uration of membranes with the protein in Bcl-2-overexpressingcells (Fig. 9). Our failure to observe Bax oligomers in mem-branes with translocated Bax from Bcl-2-overexpressing cellssuggests that partially inserted Bax may not have undergoneconformational changes required to form oligomers/channels,perhaps by steric constraints imposed by abundant Bcl-2 mol-ecules occupying the same microenvironment(s). This explana-tion demands that Bax and Bcl-2 share some critical lipiddomains, and possibly cooperating proteins in mitochondrialmembranes. Conceivably, these membrane domains and co-operating proteins are required for full Bax insertion and func-tion, made possible by exposure of previously “masked” se-quences. If the “shared” domains are saturated with Bcl-2, Baxmolecules are either denied access totally, or provided accessfor limited insertion by steric restraints that prevent assump-tion of configurations necessary for oligomer/channel forma-tion. This hypothesis is consistent not only with our presentfindings, which show Bax insertion without oligomerization,but also with the findings of other laboratories, which havereported reduction and even prevention of Bax translocation byBcl-2 (37, 43).

Previous reports have suggested that Bax heterodimeriza-tion with mitochondrial outer membrane porin (VDAC) or in-ner membrane protein ANT may be involved in the release ofcytochrome c or other inter-membrane space proteins. How-ever, we failed to detect associations of Bax with either ANT orVDAC by immunoprecipitation of extracts from cells with mi-tochondrially translocated Bax. High salt washes during im-munoprecipitation were deliberately avoided to encourage lowaffinity or nonspecific protein interactions in some experi-ments; nevertheless, we failed to co-immunoprecipitate ANT orVDAC with Bax. Moreover, eight different cross-linkers of di-verse spacer lengths and chemical reactivities also failed toreveal adducts between Bax and VDAC or ANT. On the otherhand, these agents readily demonstrated homo-oligomers ofANT and VDAC. Additionally, the pI of monomeric Bax in thecytosol and oligomeric translocated Bax in membranes, with orwithout stabilization by cross-linkers, were identical. This sup-ports the idea that Bax oligomers were homogeneous in com-position and contained Bax exclusively. It is particularly in-structive to compare overall the behavior of oligomeric Bax,membrane-bound Bax extracted with CHAPS (which does notinduce oligomerization by itself), monomeric Bax in the cytosolexposed to CHAPS with or without cross-linker, cross-linkedcytosolic Bax after dimerization by Triton X-100, and untreatednatural Bax. The results show that neither the detergent northe cross-linker DPDPB had altered the net charge of theprotein species. Under these conditions, the identity of pIamong all the species argues forcefully for the formation of Bax

homo-oligomers in membrane fractions of ATP-depleted cellswith translocated protein.

In summary, our results are consistent with a model (Fig. 9)of Bax-dependent mitochondrial permeabilization in which for-mation of Bax oligomers in the outer mitochondrial membranetriggers the release of cytochrome c. The anti-apoptotic protein,Bcl-2, when overexpressed, may either interfere with Bax in-sertion into membranes (37, 43), or prevent Bax oligomeriza-tion in the membrane following ATP depletion or UV irradia-tion by mechanisms related to steric exclusion from criticalmicrodomains in mitochondrial membranes. This model alsoconsiders the possibility of oligomeric Bax somehow inducingpore formation by other outer membrane proteins to permeabi-lize cytochrome c. In the light of recent data, the latter possi-bility is considered unlikely, suggesting that Bax oligomers bythemselves in liposomes are capable of transporting cyto-chrome c across the lipid bilayer (35). According to our model,Bcl-2 prevents such oligomerization by physically interferingwith complete insertion of Bax into the membrane, a require-ment for oligomerization, without forming stable heteromericcomplexes. Therefore, partially inserted but loosely bound Bax,in Bcl-2-loaded mitochondria, may quickly equilibrate withcytosolic Bax in other models of apoptosis where Bax associa-tion with mitochondria in Bcl-2-overexpressing cells is seem-ingly reduced. Future studies on purified, stably cross-linkedmultimers of Bax and systems that permit regulated expres-sion of Bcl-2 should help to clarify these issues.

Acknowledgments—We greatly acknowledge the generous gift of Baxmonoclonal antibody 1D1 from Dr. Richard J. Youle and ANT polyclonalantibody from Dr. H. H. Schmid.

REFERENCES

1. Saikumar, P., Dong, Z., Patel, Y., Hall, K., Hopfer, U., Weinberg, J. M., andVenkatachalam, M. A. (1998) Oncogene 17, 3401–3415

2. Susin, S. A., Zamzami, N., Castedo, M., Hirsch, T., Marchetti, P., Macho, A.,Daugas, E., Geuskens, M., and Kroemer, G. (1996) J. Exp. Med. 184,1331–1341

3. Reed, J. C., Jurgensmeier, J. M., and Matsuyama, S. (1998) Biochim. Biophys.Acta 1366, 127–137

4. Scarlett, J. L., and Murphy, M. P. (1997) FEBS Lett. 418, 282–2865. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T. I.,

Jones, D. P., and Wang, X. (1997) Science 275, 1129–11326. Eskes, R., Antonsson, B., Osen-Sand, A., Montessuit, S., Richter, C., Sadoul,

R., Mazzei, G., Nichols, A., and Martinou, J. C. (1998) J. Cell Biol. 143,217–224

7. Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491–5018. Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998) Cell 94,

481–4909. Hsu, Y. T., Wolter, K. G., and Youle, R. J. (1997) Proc. Natl. Acad. Sci. U. S. A.

94, 3668–367210. Finucane, D. M., Bossy-Wetzel, E., Waterhouse, N. J., Cotter, T. G., and Green,

D. R. (1999) J. Biol. Chem. 274, 2225–223311. Jurgensmeier, J. M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D., and Reed,

J. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4997–500212. Cregan, S. P., MacLaurin, J. G., Craig, C. G., Robertson, G. S., Nicholson,

D. W., Park, D. S., and Slack, R. S. (1999) J. Neurosci. 19, 7860–786913. Marchetti, P., Castedo, M., Susin, S. A., Zamzami, N., Hirsch, T., Macho, A.,

Haeffner, A., Hirsch, F., Geuskens, M., and Kroemer, G. (1996) J. Exp. Med.184, 1155–1160

14. Saikumar, P., Dong, Z., Weinberg, J. M., and Venkatachalam, M. A. (1998)Oncogene 17, 3341–3349

15. Hengartner, M. O. (1999) Recent Prog. Horm. Res. 54, 213–22216. von Ahsen, O., Renken, C., Perkins, G., Kluck, R. M., Bossy-Wetzel, E., and

Newmeyer, D. D. (2000) J. Cell Biol. 150, 1027–103617. Petit, P. X., Goubern, M., Diolez, P., Susin, S. A., Zamzami, N., and Kroemer,

G. (1998) FEBS Lett. 426, 111–11618. Vander Heiden, M. G., Chandel, N. S., Williamson, E. K., Schumacker, P. T.,

and Thompson, C. B. (1997) Cell 91, 627–63719. Kluck, R. M., Esposti, M. D., Perkins, G., Renken, C., Kuwana, T.,

Bossy-Wetzel, E., Goldberg, M., Allen, T., Barber, M. J., Green, D. R., andNewmeyer, D. D. (1999) J. Cell Biol. 147, 809–822

20. Zha, H., and Reed, J. C. (1997) J. Biol. Chem. 272, 31482–3418821. Cheng, E. H., Levine, B., Boise, L. H., Thompson, C. B., and Hardwick, J. M.

(1996) Nature 379, 554–55622. Schendel, S. L., Montal, M., and Reed, J. C. (1998) Cell Death Differ. 5,

372–38023. Antonsson, B., Conti, F., Ciavatta, A., Montessuit, S., Lewis, S., Martinou, I.,

Bernasconi, L., Bernard, A., Mermod, J. J., Mazzei, G., Maundrell, K.,Gambale, F., Sadoul, R., and Martinou, J. C. (1997) Science 277, 370–372

24. Schlesinger, P. H., Gross, A., Yin, X. M., Yamamoto, K., Saito, M., Waksman,G., and Korsmeyer, S. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,

Bax Oligomerization 18373

by guest on August 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from

11357–1136225. Gross, A., Jockel, J., Wei, M. C., and Korsmeyer, S. J. (1998) EMBO J. 17,

3878–388526. Hsu, Y. T., and Youle, R. J. (1998) J. Biol. Chem. 273, 10777–1078327. Shimizu, S., Narita, M., and Tsujimoto, Y. (1999) Nature 399, 483–48728. Marzo, I., Brenner, C., Zamzami, N., Jurgensmeier, J. M., Susin, S. A., Vieira,

H. L., Prevost, M. C., Xie, Z., Matsuyama, S., Reed, J. C., and Kroemer, G.(1998) Science 281, 2027–2031

29. Basanez, G., Nechushtan, A., Drozhinin, O., Chanturiya, A., Choe, E., Tutt, S.,Wood, K. A., Hsu, Y., Zimmerberg, J., and Youle, R. J. (1999) Proc. Natl.Acad. Sci. U. S. A. 96, 5492–5497

30. Woost, P. G., Orosz, D. E., Jin, W., Frisa, P. S., Jacobberger, J. W., Douglas,J. G., and Hopfer, U. (1996) Kidney Int. 50, 125–134

31. Nissim, I., and Weinberg, J. M. (1996) Kidney Int. 49, 684–9532. Duran, M. A., Spencer, D., Weise, M., Kronfol, N. O., Spencer, R. F., and Oken,

D. E. (1990) Toxicol. Appl. Pharmacol. 105, 183–19433. Venkatachalam, M. A., and Weinberg, J. M. (1993) in Surviving Hypoxia,

(Hochachka, P. W., Lutz, P. L., Sick, T., Rosenthal, M., and van den

Thillart, G., eds) pp. 473–494, CRC Press, Boca Raton, FL34. Fiskum, G., Craig, S. W., Decker, G. L., and Lehninger, A. L. (1980) Proc. Natl.

Acad. Sci. U. S. A. 77, 3430–343435. Saito, M., Korsmeyer, S. J., and Schlesinger, P. H. (2000) Nat. Cell Biol. 2,

553–55536. Putcha, G. V., Deshmukh, M., and Johnson, E. M., Jr. (1999) J. Neurosci. 19,

7476–748537. Murphy, K. M., Ranganathan, V., Farnsworth, M. L., Kavallaris, M., and Lock,

R. B. (2000) Cell Death Differ. 7, 102–11138. Hsu, Y. T., and Youle, R. J. (1997) J. Biol. Chem. 272, 13829–1383439. Haldar, S., Chintapalli, J., and Croce, C. M. (1996) Cancer Res. 56, 1253–125540. Poommipanit, P. B., Chen, B., and Oltvai, Z. N. (1999) J. Biol. Chem. 274,

1033–103941. Puthalakath, H., Huang, D. C., O’Reilly, L. A., King, S. M., and Strasser, A.

(1999) Mol. Cell 3, 287–29642. Antonsson, B., Montessuit, S., Lauper, S., Eskes, R., and Martinou, J. C. (2000)

Biochem. J. 345, 271–27843. Murphy, K. M., Streips, U. N., and Lock, R. B. (1999) Oncogene 18, 5991–5999

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by guest on August 30, 2020

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nloaded from

Venkatachalam and Pothana SaikumarValery Mikhailov, Margarita Mikhailova, Donna J. Pulkrabek, Zheng Dong, Manjeri A.

Bcl-2 Prevents Bax Oligomerization in the Mitochondrial Outer Membrane

doi: 10.1074/jbc.M100655200 originally published online February 20, 20012001, 276:18361-18374.J. Biol. Chem. 

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