4 The abbreviations used are: tbid, truncated 16-kDa C-terminal fragment;

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1 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 38, pp , September 21, by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. The Mitochondrial TOM Complex Is Required for tbid/bax-induced Cytochrome c Release * Received for publication, April 13, 2007, and in revised form, June 21, 2007 Published, JBC Papers in Press, July 16, 2007, DOI /jbc.M Martin Ott 1, Erik Norberg, Katharina M. Walter 2, Patrick Schreiner, Christian Kemper, Doron Rapaport, Boris Zhivotovsky, and Sten Orrenius 3 From the Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, SE Stockholm, Sweden, Institute for Physiological Chemistry, Ludwig Maximilians University, D Munich, Germany, and the Interfacultary Institute for Biochemistry, Eberhard Karls University, D Tübingen, Germany Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tbid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tbid/bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release. The release of cytochrome c and other pro-apoptotic proteins from the mitochondrial intermembrane space into the cytosol is a key event in the activation of apoptosis. Once in the cytosol, cytochrome c interacts with APAF-1, in the presence of datp, to form the apoptosome and trigger the activation of pro-caspase-9 (1). Caspase-9 then cleaves and activates procaspase-3, the main executioner caspase. Although cytochrome c-mediated activation of the caspases is regarded as the main outcome of mitochondrial membrane permeabilization, other intermembrane space proteins play additional, independent roles in cell death signaling (2). During the early phase of apoptosis, only the mitochondrial outer membrane is permeabilized, whereas other intracellular membranes as well as the plasma membrane remain intact. * This work was supported by grants from the Swedish Research Council (Grants 3IX and ), the Swedish Cancer Society (Grant 3829-B0409XAC) and Stockholm Cancer Society (Grant ), and the EP-6 (Grant GSHC-CT ). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 A post-doctoral fellow of the Wenner-Gren Foundation, Stockholm, Sweden. 2 The recipient of a stipend for academic exchange from the Deutscher Akademischer Austauschdienst, Germany. 3 To whom correspondence should be addressed: Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE Stockholm, Sweden. Sten.Orrenius@ki.se. The mechanism(s) by which the pro-apoptotic Bcl-2 family proteins permeabilize the mitochondrial outer membrane have been studied extensively during recent years. A major pathway involves the soluble cytoplasmic proteins Bid and Bax. Upon cleavage of Bid by proteases, e.g. caspase-8, a truncated 16-kDa C-terminal fragment (tbid) 4 is formed, which subsequently can promote the oligomerization/insertion of Bax into the outer mitochondrial membrane (3, 4). The oligomeric form of Bax is believed to form a pore that allows the extrusion of intermembrane space proteins into the cytosol (5). Oligomerization results in a change in Bax structure with exposure of an N-terminal domain, which is normally not exposed to the cytosol, and the insertion of the C-terminal domain and of the central pore-forming helices 5 and 6 into the mitochondrial outer membrane (6, 7). The C terminus is of critical importance for the pro-apoptotic function of Bax, and it shows some similarity to the signals that normally direct tail-anchored proteins to mitochondria (8). Several hypotheses have been elaborated to explain the specificity by which Bax targets the mitochondria. One of them involves an interaction of Bax with the mitochondrial porin, also called voltage-dependent anion channel, a barrel protein in the outer mitochondrial membrane (9). Another claims that cardiolipin, a phospholipid present in the mitochondrial inner membrane and in the contact sites between the two membranes, is important for tbid/bax-induced cytochrome c release (10). In addition, it has recently been reported that Bax interacts via its N terminus with Tom22, a receptor of the translocase of the outer mitochondrial membrane (TOM) complex (11). In this study, we have used the yeast Saccharomyces cerevisiae to investigate which proteins in the mitochondrial outer membrane might be responsible for the specific interaction with tbid/bax. Yeast was instrumental in the discovery of several biologically important mechanisms, which turned out to be highly conserved between yeast and mammalian systems. The straightforward genetics allow the usage of mutants that are not available at a comparable quality in any eukaryotic system. By employing an in vitro system consisting of yeast mitochondria and recombinant proteins, we found that neither cardiolipin nor porin is required for tbid/bax-induced cytochrome c release. In contrast, we report that a functional translocase of 4 The abbreviations used are: tbid, truncated 16-kDa C-terminal fragment; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TOM, translocase of the outer mitochondrial membrane. SEPTEMBER 21, 2007 VOLUME 282 NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 27633

2 the outer membrane, the TOM complex, is involved in tbid/ Bax-induced cytochrome c release from yeast as well as mammalian mitochondria. EXPERIMENTAL PROCEDURES Yeast Strains and Growth Conditions, Mitochondrial Preparation The wild type strain used in this study was W303-A, and the CRD1-mutant and the porin double deletion mutant were described earlier (12, 13). Yeast cultures were grown in a lactate medium at 30 C, except for the strains tom40-4 and tom40-2, and the parental wild type, which were grown at 25 C. Mitochondria from yeast and rat liver were isolated as described previously (14, 15). Recombinant Proteins Full-length human Bax and human tbid were expressed in Escherichia coli strain (BL21 DE3) as N-terminal hexahistidine-tagged proteins from the vectors pet23bax and pet23tbid, respectively. The proteins were purified in the absence of any detergent, using standard chromatographic techniques. The proteins were dialyzed against 30% glycerol, 100 mm NaCl, 0.2 mm EDTA, 25 mm HEPES/ KOH, ph 7.4, and stored at 80 C. To obtain oligomeric Bax, the recombinant, monomeric Bax was incubated prior to the experiments with 1% octylglycoside for 1hat4 C(7). Cytochrome c Release and Bax Insertion Isolated mitochondria from yeast or rat liver were incubated in SHKCl-buffer (600 mm sorbitol, 100 mm KCl, 25 mm HEPES/KOH, ph 7.4) at a protein concentration of 1 mg/ml. To suppress permeability transition of rat liver mitochondria, 1 M cyclosporin A was present. Recombinant proteins were added at a concentration of 10 nm (tbid) or 150 nm (monomeric Bax), and the suspension was incubated for 15 min at 30 C; in one series of experiments, incubation was for 1hat37 C. Thereafter, the samples were fractionated into supernatant and pellet by centrifugation (15 min, 16,000 g, 4 C) and applied to SDS-PAGE and Western blotting using the indicated antibodies. To analyze for the insertion of Bax, the mitochondrial pellet was resuspended in 100 mm sodium carbonate and incubated with continuous shaking at 4 C for 30 min. Then, the membranes were recovered by centrifugation (30 min, 100,000 g, 4 C) and dissolved in loading buffer. Import of Precursor Proteins and Proteinase K Treatment Precursor proteins were synthesized in reticulocyte lysate (TNT, Promega) in the presence of [ 35 S]methionine and imported into isolated mitochondria, essentially as described previously (16). To remove proteins and protein domains exposed to the outside of mitochondria, the organelles were incubated with 50 g/ml proteinase K for 30 min at 0 C. Then, 2 mm phenylmethylsulfonyl fluoride was added, and the sample was incubated for an additional 5 min. Mitochondria were reisolated by centrifugation (10 min, g, 4 C) and either suspended in loading buffer or used for subsequent experiments. Co-immunoprecipitation of Bax with the Isolated TOM Complex The TOM core complex of Neurospora crassa strain GR-107 was purified as described (17) and stored at 80 C. For co-immunoprecipitation, the purified TOM core complex, as well as Bax and tbid were incubated at the same protein concentration of 15 g/ml in immunoprecipitation buffer (140 mm KCl, 1 mm EDTA, 0.1% CHAPS, 10 mm HEPES/KOH, ph 7.4) for 15 min at 30 C. The reaction was cleared by centrifugation (15 min, 16,000 g, 4 C) and split, and the indicated antibodies were added together with protein G-Sepharose beads. Samples were gently rotated for 1.5 h at 4 C. The beads were washed three times with the immunoprecipitation buffer before bound proteins were eluted with loading buffer without reducing agent. Samples were analyzed by SDS-PAGE and immunodecoration. Co-immunoprecipitation of Bax with the TOM Complex during Protein Insertion Isolated wild type yeast mitochondria were incubated with Bax and tbid in 140 mm KCl, 1 mm EDTA, 500 mm sorbitol, 10 mm HEPES/KOH, ph 7.4 for the indicated time at 4 C. Mitochondria were lysed directly by the addition of 0.5% CHAPS and cleared by centrifugation (15 min, 16,000 g, 4 C) and split, and the indicated antibodies were added together with protein G-Sepharose beads. Samples were gently rotated for 1.5 h at 4 C. The beads were washed three times with the immunoprecipitation buffer before bound proteins were eluted with loading buffer without reducing agent. Samples were analyzed by SDS-PAGE and immunodecoration. Antibody Competition with Mitochondria and Permeabilized Cells Bax/Bak double knock-out murine fibroblasts were grown in Dulbecco s modified Eagle s medium supplemented with 10% fetal calf serum, 2 mm glutamine, and antibiotics cells were resuspended in 100 l of 500 mm sorbitol, 80 mm KCl, 10 mm MgCl 2,1mM KPO 4, 2.5 mm EDTA, 2.5 mm MnCl 2, 2 mm ATP, 10 mm succinate, 50 mm HEPES/KOH, ph 7.4. Cells were permeabilized by the addition of 0.01% digitonin. The permeabilized cells were preincubated with polyclonal antibodies against rat Tom20 (a gift from Henry Weiner, Indiana) or porin (Calbiochem) for 30 min on ice. The sample was divided, and one half was treated with the recombinant proteins. The other half was incubated with radiolabeled psu9-dhfr. The samples were fractionated by centrifugation (15 min, 16,000 g, 4 C). Alternatively, yeast mitochondria were incubated in the same way with polyclonal antibodies against yeast Tom20 and Tom22 and tested for cytochrome c release induced by the recombinant proteins. RESULTS AND DISCUSSION Cytochrome c Release from Isolated Yeast Mitochondria Induced by Recombinant tbid and Bax To investigate mitochondrial components that might be required for tbid/baxinduced cytochrome c release, we decided to use yeast mitochondria as a model system. First, we tested whether yeast mitochondria release cytochrome c in a fashion similar to rat liver mitochondria, when incubated with recombinant, purified proteins. Rat liver mitochondria released no cytochrome c if exposed to monomeric Bax for 15 min at 30 C. In contrast, treatment with a combination of tbid and monomeric Bax caused substantial cytochrome c release (Fig. 1A), which is in accordance with the notion that monomeric Bax has to be activated by tbid to mediate cytochrome c release. Monomeric Bax induced a minor release of cytochrome c with mitochondria from wild type yeast under similar conditions. However, a combination of tbid and Bax triggered the release of a substantial amount of cytochrome c from these organelles (Fig. 1B). These JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 NUMBER 38 SEPTEMBER 21, 2007

3 FIGURE 1. Characterization of tbid/bax-mediated cytochrome c release from yeast mitochondria. A, rat liver mitochondria were incubated with recombinant proteins as described under Experimental Procedures and fractionated into pellet and supernatant. The fractions were separated by SDS-PAGE and analyzed by Western blotting. B, wild type yeast mitochondria were incubated and processed as in panel A. C, wild type yeast mitochondria were treated with proteinase K (PK) at50 g/ml for 30 min on ice, as described under Experimental Procedures. After inhibition of the protease, mitochondria were incubated with tbid and Bax. D, tbid/bax-induced cytochrome c release from mitochondria isolated from yeast mutants lacking cardiolipin ( crd1) or the mitochondrial porins ( por1 por2) was analyzed as in panel A. mbax, monomeric Bax. findings encouraged us to use wild type yeast, and the various mutants available, to study the molecular requirements for tbid/bax-induced cytochrome c release. Exposed Domains of Mitochondrial Outer Membrane Proteins Are Not Required for tbid/bax-induced Cytochrome c Release To address the question whether domains of mitochondrial outer membrane proteins, which are exposed to the cytosol, are involved in tbid/bax targeting, we removed such domains by controlled proteinase K treatment of the isolated mitochondria. Thereby, protease-accessible domains of, for example, the receptor proteins Tom20, Tom22, and Tom70, could be removed, whereas integral membrane proteins, such as the barrel proteins Tom40 and the porins, were not affected (Fig. 1C). When mitochondria were incubated with recombinant tbid and Bax after pretreatment with proteinase K, the amount of cytochrome c released was similar to that in mock-treated mitochondria. Therefore, we concluded that exposed domains of mitochondrial outer membrane proteins are not required for tbid/bax-induced cytochrome c release. Notably, it was recently reported that Tom22 may interact with Bax during apoptosis (11). Tom22 recognizes cryptic targeting signals of inner membrane or matrix proteins and is, at least in yeast, not required for the biogenesis of outer membrane proteins. In our system, removal of the cytosolic domain of Tom22 by proteinase K treatment did not influence cytochrome c release. Neither Cardiolipin nor Porin Is Required for tbid/bax-induced Cytochrome c Release in Yeast As mentioned above, the uniquely mitochondrial phospholipid, cardiolipin, has been TOM Complex Mediates Bax Insertion suggested to act as a possible mitochondrial target for tbid in mammalian systems (18). It has also been shown that cardiolipin is required for tbid/bax-induced membrane permeabilization in a reconstituted system containing liposomes and the recombinant proteins (10). Using a yeast mutant lacking the gene for cardiolipin synthase, CRD1, we demonstrated earlier that cardiolipin is not required for cytochrome c release induced by treatment with chemically oligomerized Bax (12). To test whether cardiolipin is required for tbid/bax-induced cytochrome c release, mitochondria isolated from this mutant were now incubated with a combination of tbid and Bax. The induced release of cytochrome c was similar to that seen in wild type mitochondria (Fig. 1D, upper panel). Thus, it appears that cardiolipin is not required for cytochrome c release in yeast, which is in line with a recent study in mammalian cells demonstrating that down-regulation of the mammalian cardiolipin synthase by small interfering RNA did not abolish apoptosis signaling via Fas ligation, caspase-8 activation, and Bid cleavage (19). Next,wetestedwhetherthemitochondrialporins,thevoltagedependent anion channels, might be required in the release process. It has previously been proposed that these proteins are targets for Bcl-2 family proteins (9, 20). However, when isolated mitochondria from a yeast strain lacking both porins were incubated with tbid and Bax, the extent of cytochrome c release was similar to that observed with wild type mitochondria (Fig. 1D, lower panel). This finding suggests that the porins are not involved in tbid/bax-induced cytochrome c release, at least not in yeast. The Mitochondrial TOM Complex Is Required for tbid/baxinduced Cytochrome c Release Next, we tested whether other mitochondrial barrel proteins could possibly be required for the action of tbid/bax on mitochondria. To investigate the role of Tom40, the pore-forming subunit of the TOM complex, we used yeast mutants expressing a temperature-sensitive version of Tom40, which is inactivated by incubation at a non-permissive temperature 37 C (21, 22); since Tom40 is essential for cell viability, there is no yeast mutant completely devoid of this protein. Incubation at non-permissive temperature in the absence of tbid/bax did not lead to cytochrome c release from either mutant or wild type mitochondria (Fig. 2A). Further, when mitochondria from both strains were incubated with tbid and Bax at permissive temperature, the extent of cytochrome c release was similar. However, when the mitochondria were preincubated at non-permissive temperature, which destroys the function of the mutant Tom40 protein, tbid/bax-induced cyto- SEPTEMBER 21, 2007 VOLUME 282 NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 27635

4 FIGURE 2. The mitochondrial TOM complex is required for tbid/bax-induced cytochrome c release. A, mitochondria from the tom40-4 strain, or from the corresponding wild type, were incubated at either 25 or 37 C before the addition of recombinant tbid and Bax. After incubation at 30 C for 15 min, the samples were fractionated and analyzed as in Fig. 1A. B, the amount of tbid/bax-induced cytochrome c release after preincubation of mitochondria at 37 C from five independent experiments was quantified by densitometry and compared with the amount of cytochrome c released from mock-treated counterparts. C, membrane insertion of Bax into mitochondria isolated from either the wild type strain or the tom40-4 mutant was monitored with alkaline extraction. Immunodecoration of the membrane fraction is presented. D, import of psu9-dhfr into tom40-4 mitochondria. psu9-dhfr and Su9-DHFR, precursor and mature forms, respectively. E, cytochrome c release from tom40-4 mitochondria induced by chemically oligomerized Bax. Mitochondria were incubated as in panel A. F, at harsher incubation conditions, monomeric Bax (mbax) is sufficient to release cytochrome c from both wild type and tom 40-4 mitochondria. Mitochondria were incubated at 37 C for 1hinthepresence of recombinant proteins. Samples were fractionated into supernatant (S) and pellet (P) and analyzed by Western blotting. chrome c release from wild type mitochondria was not affected, whereas mitochondria from the tom40-4 strain, as well as those from the tom40-2 strain (not shown), reproducibly released less cytochrome c when exposed to tbid/bax (Fig. 2, A and B). Thus, it appears that functional Tom40 is required for the release process in yeast. Bax Insertion Is Affected in tom40-4 Mitochondria It has previously been shown that Tom40 is involved not only in the translocation of precursor proteins across the mitochondrial outer membrane but also in the insertion of proteins into this membrane (for review, see Ref. 23). Interestingly, the insertion of outer membrane signal-anchored proteins, such as Tom20 and Tom70, does not require receptors of the TOM complex but is mediated by the Tom40 protein (16). To investigate whether Bax insertion was defective in the tom40 mutants, we employed fractionation experiments with alkaline treatment. Bax was recovered in similar quantities in the membrane fraction of wild type mitochondria, independently of whether they had been preincubated at 37 C or not. In contrast, Bax insertion was strongly reduced in the heat-treated tom40-4 mitochondria (Fig. 2C). This finding suggests that a functional Tom40 is required for Bax insertion into the outer mitochondrial membrane. To verify the defective protein import in the tom40-4 strain, we investigated the import of [ 35 S]methionine-labeled psu9- DHFR into these mitochondria. Import of this model precursor protein can be monitored by the formation of the mature polypeptide that is resistant to externally added trypsin. Import proceeded normally in mitochondria from the tom40-4 strain incubated at permissive temperature. However, if the mitochondria were preincubated at non-permissive temperature, the import of this protein was markedly decreased (Fig. 2D). As expected, wild type mitochondria that had been preincubated at 37 C showed no import defect (not shown). The TOM Complex Is Not Required for Cytochrome c Release Induced by Chemically Oligomerized Bax To exclude the possibility that heat-treated mitochondria from the tom40-4 strain are unable to release proteins from their intermembrane space, we incubated mitochondria from this strain with chemically oligomerized Bax. This form of Bax is known to have undergone conformational changes that resemble those found under apoptotic conditions (7). When these mitochondria were incubated with oligomeric Bax, the resulting cytochrome c release was identical, whether or not the mitochondria contained functional Tom40 (Fig. 2E). Thus, it seems that mitochondria from the tom40-4 strain are in fact able to release cytochrome c from their intermembrane space and that permeabilization of the outer membrane by chemically oligomerized Bax and by tbid-activated monomeric Bax occurs by distinct mechanisms. At Harsher Incubation Conditions, the TOM Complex, but not tbid, Is Required for Bax to Release Cytochrome c It was recently reported that during prolonged (1 h) incubation of yeast mitochondria at 37 C, tbid was not required for Bax to release cytochrome c and that the release process was unaffected by tom40 mutations (24). Thus, we performed a series of experiments under similar conditions. Indeed, when the yeast mitochondria were incubated at 37 C for 1 h, monomeric Baxinduced cytochrome c release was observed and occurred independently of tbid (Fig. 2F). However, in our experiments (Fig. 1B), the addition of tbid still enhanced Bax-induced cyto JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 NUMBER 38 SEPTEMBER 21, 2007

5 FIGURE 3. Bax interacts directly with the TOM complex. A, isolated TOM core complex was incubated with tbid and Bax for 15 min at 30 C. The sample was then split and subjected to immunoprecipitation (IP) using antibodies against Bax, Tom40, or a control antibody (subunit F1 of the ATPase). Immunocomplexes were recovered with protein G-Sepharose, washed carefully, and analyzed by Western blotting. Total represents 40% of material per immunoprecipitation. B, mitochondria were incubated with tbid and Bax at 4 C. After 2 and 30 min, one half of the incubation was solubilized, the lysate was cleared by centrifugation and subjected to co-immunoprecipitation using antibodies against Tom22 and porin. Samples were analyzed using Western blotting with antibodies against Tom22, Bax, or Tom40. The signal of IgG is shown as input control. * indicates cross-reacting IgG in the Tom40 immunoblot. chrome c release, a finding that more accurately reflects the situation prevailing in the mammalian and liposome systems. A possible explanation for this discrepancy between yeast and mammalian mitochondria is that yeast does not contain any anti-apoptotic Bcl-2-like proteins, which might inhibit accidental insertion of Bax and/or its assembly into functional oligomeres (24). The low effectiveness of this process in the absence of tbid seen under our conditions (Fig. 1B) could increase in a time- and temperature-dependent manner and thus lead to a more efficient Bax-induced cytochrome c release under those harsher conditions. However, in contrast to the findings of Sanjuan Szklarz et al. (24), the amount of cytochrome c released from tom40-4 mitochondria (and from tom40-2 mitochondria, not shown) under those harsher conditions was lower as compared with that found with wild type mitochondria, which showed almost complete release (Fig. 2F). From this, we concluded that also during prolonged incubation at higher temperature, the functionality of the TOM complex affects the amount of cytochrome c that can be released by Bax. Bax Interacts Directly with the TOM Complex To analyze whether Bax can interact directly with the TOM complex in vitro, we incubated isolated TOM core complex (consisting of Tom40, Tom22, Tom5, Tom6, and Tom7 from N. crassa) with tbid and Bax. After incubation for 15 min at 30 C, the sample was split and subjected to immunoprecipitation with antibodies against Tom40, Bax, or a control antibody. Antibodies against Tom40 recovered Tom40 and, to a lesser extent, Bax. Similarly, antibodies against Bax could precipitate Bax together with a lower, but significant, amount of Tom40. The control antibody, however, did not precipitate any detectable Bax or Tom40 (Fig. 3A). From this, we concluded that isolated TOM core complex and Bax can interact with each other in vitro. Next, we asked whether Bax might interact directly with the TOM complex during import into isolated yeast mitochondria. As the interactions of protein insertases with their substrates are normally transient and occur with low affinities, we performed the experiment at low temperature to slow down the speed of the reaction. To do so, mitochondria were incubated at 4 C with the recombinant proteins and permeabilized after 2 and 30 min. Next, the lysates were subjected to coimmunoprecipitation using antibodies against Tom22 and porin (Fig. 3B). Antibodies against Tom22 allowed isolating Tom22 together with Tom40. Bax was only co-immunoprecipitated with the TOM complex from lysates of mitochondria that were exposed to Bax for 2 min but not from lysates of mitochondria that were incubated with Bax for 30 min. Antibodies against porin did not co-immunoprecipitate Bax or the TOM complex. From this, we concluded that Bax interacts transiently with the TOM complex during its insertion into the outer mitochondrial membrane. Antibodies against the TOM Complex Inhibit Cytochrome c Release from Mammalian Mitochondria To investigate whether the TOM complex is required for tbid/bax-induced cytochrome c release by another, independent approach, we performed antibody competition experiments. Thus, we preincubated isolated yeast mitochondria with antibodies against Tom20 and Tom22 and probed for tbid/bax-induced cytochrome c release. Both Tom20 and Tom22 are subunits of the TOM complex that expose a large domain into the cytosol. Preincubation with antibodies against these proteins markedly decreased cytochrome c release (Fig. 4A), suggesting that antibodies against the receptors of the TOM complex can inhibit cytochrome c release from yeast mitochondria. Next, we investigated whether the TOM complex might also be involved in tbid/bax-induced cytochrome c release from mammalian mitochondria. For this purpose, we used embryonic fibroblasts from Bax/Bak double knock-out mice (25). When the plasma membrane was permeabilized with low concentrations of digitonin, which left the mitochondrial outer membrane intact, there was no cytochrome c released from the mitochondria (Fig. 4B). As expected, cytochrome c release could be induced by the combined addition of tbid and Bax but not with tbid or Bax alone (not shown). When these permeabilized cells were preincubated with antibodies directed against Tom20, cytochrome c release could be blocked efficiently. In contrast, antibodies against mitochondrial porin did not protect from cytochrome c release. Similarly, preincubation with Tom20 antibodies decreased the amount of Bax associated with SEPTEMBER 21, 2007 VOLUME 282 NUMBER 38 JOURNAL OF BIOLOGICAL CHEMISTRY 27637

6 FIGURE 4. Antibodies against the TOM complex inhibit protein import and tbid/bax-induced cytochrome c release from mammalian mitochondria. A, wild type mitochondria from yeast were incubated with or without antibodies against Tom20 or Tom22 for 30 min at 4 C. Then, the mitochondria were incubated with the recombinant proteins for 15 min at 30 C and analyzed for cytochrome c release. B, Bax/Bak double knockout murine embryonic fibroblasts (DKO-MEF) were permeabilized and preincubated with the different antibodies. tbid and Bax were then added, and the samples were fractionated and probed for cytochrome c and Bax. C, psu9-dhfr was incubated with permeabilized double knock-out cells in the presence or absence of the indicated antibodies. The cells were recovered, and the proteins were analyzed by autoradiography. mbax, monomeric Bax. FIGURE 5. Working model. Exposure of the C-terminal membrane anchor of Bax allows the interaction with the TOM complex, which mediates its insertion into the outer mitochondrial membrane (OMM). The membrane-inserted Bax then oligomerizes into pores, which allow the release of mitochondrial intermembrane space proteins such as cytochrome c (Cyt c). Caspase-8-generated tbid facilitates this process in a yet unidentified manner. the mitochondria, whereas incubation with a porin antibody did not show any effect. These findings are similar to those reported for the biogenesis of the single membrane-spanning protein Tom22, whose insertion into the outer membrane of rat liver mitochondria could be blocked by antibodies against Tom20 (26). Finally, we investigated the effect of the antibodies on import of psu9- DHFR. Although antibodies against Tom20 completely inhibited import and maturation of this model protein, antibodies against porin had no effect (Fig. 4C). From this, we can conclude that inhibition of the mammalian TOM complex with antibodies leads to a decreased protein import into mitochondria and also reduces tbid/bax-induced cytochrome c release. Concluding Remarks In this study, we show that a functional TOM complex is required for optimal tbid/bax-induced cytochrome c release from yeast and mammalian mitochondria and that the combination of tbid and monomeric Bax is sufficient to trigger this release process, as no other Bcl-2 family proteins are present in yeast. Cardiolipin, mitochondrial porins, and cytosolic domains of outer membrane proteins appear not to play an important role in the targeting of Bax to yeast mitochondria. The finding that Bax requires the TOM complex for insertion is reminiscent of a similar requirement for insertion of single membrane-spanning proteins, such as Tom20, Tom22, and Tom70, into the mitochondrial outer membrane (16). Thus, interaction with the TOM complex could be a crucial step in the targeting of Bax to the mitochondrial outer membrane, which is an important event during apoptosis signaling (Fig. 5). In addition, it appears likely that the interaction of tbid with Bax may facilitate the membrane insertion of Bax via the TOM complex (27). Such interaction might result in conformational changes, which allow targeting signals in Bax to be exposed and recognized by the TOM complex. The TOM complex might then mediate the insertion of monomeric Bax into the mitochondrial outer membrane, where, possibly with the help of tbid or solely by the close contact with the membrane (7), Bax can oligomerize to form pores. Notably, tbid seems not to mediate, but rather facilitate, the process of Bax insertion and oligomerization in the yeast outer membrane, as monomeric Bax alone can provoke cytochrome c release, although with a lower effectiveness as compared with a combination of monomeric Bax and tbid (Figs. 1B and 3F). The observation that cytochrome c release induced by chemically oligomerized Bax did not display a requirement for the TOM complex suggests that this form of Bax has already undergone the conformational changes needed for membrane integration and assembly. The nature of the targeting signal of Bax is yet unclear. Bax possesses a C-terminal helix that shares some homology with the signals known to direct tail-anchored proteins to mitochondria. Deletion of this domain abolishes the proapoptotic activity of the molecule (8). However, the N termi JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 NUMBER 38 SEPTEMBER 21, 2007

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