The Mitochondrial TOM Complex Is Required for tBid/Bax-induced Cytochrome c Release

Abstract

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. 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 (1Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar). 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 (2Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4060) Google Scholar). 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. 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) 4The abbreviations used are: tBidtruncated 16-kDa C-terminal fragmentCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidTOMtranslocase of the outer mitochondrial membrane is formed, which subsequently can promote the oligomerization/insertion of Bax into the outer mitochondrial membrane (3Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar, 4Desagher S. Osen-Sand A. Nichols A. Eskes R. Montessuit S. Lauper S. Maundrell K. Antonsson B. Martinou J.C. J. Cell Biol. 1999; 144: 891-901Crossref PubMed Scopus (1093) Google Scholar). The oligomeric form of Bax is believed to form a pore that allows the extrusion of intermembrane space proteins into the cytosol (5Antonsson 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. Martinou J.C. Science. 1997; 277: 370-372Crossref PubMed Scopus (932) Google Scholar). 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 α helices 5 and 6 into the mitochondrial outer membrane (6Annis M.G. Soucie E.L. Dlugosz P.J. Cruz-Aguado J.A. Penn L.Z. Leber B. Andrews D.W. EMBO J. 2005; 24: 2096-2103Crossref PubMed Scopus (326) Google Scholar, 7Yethon J.A. Epand R.F. Leber B. Epand R.M. Andrews D.W. J. Biol. Chem. 2003; 278: 48935-48941Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). 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 (8Nechushtan A. Smith C.L. Hsu Y.T. Youle R.J. EMBO J. 1999; 18: 2330-2341Crossref PubMed Scopus (629) Google Scholar). truncated 16-kDa C-terminal fragment 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid translocase of the outer mitochondrial membrane 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 β barrel protein in the outer mitochondrial membrane (9Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1928) Google Scholar). 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 (10Kuwana T. Mackey M.R. Perkins G. Ellisman M.H. Latterich M. Schneiter R. Green D.R. Newmeyer D.D. Cell. 2002; 111: 331-342Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar). 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 (11Bellot G. Cartron P.F. Er E. Oliver L. Juin P. Armstrong L.C. Bornstein P. Mihara K. Manon S. Vallette F.M. Cell Death Differ. 2006; 14: 785-794Crossref PubMed Scopus (132) Google Scholar). 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 the outer membrane, the TOM complex, is involved in tBid/Bax-induced cytochrome c release from yeast as well as mammalian mitochondria. 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 (12Iverson S.L. Enoksson M. Gogvadze V. Ott M. Orrenius S. J. Biol. Chem. 2004; 279: 1100-1107Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 13Blachly-Dyson E. Song J. Wolfgang W.J. Colombini M. Forte M. Mol. Cell Biol. 1997; 17: 5727-5738Crossref PubMed Scopus (140) Google Scholar). 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 (14Daum G. Bohni P.C. Schatz G. J. Biol. Chem. 1982; 257: 13028-13033Abstract Full Text PDF PubMed Google Scholar, 15Ott M. Robertson J.D. Gogvadze V. Zhivotovsky B. Orrenius S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1259-1263Crossref PubMed Scopus (796) Google Scholar). 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 1 h at 4°C (7Yethon J.A. Epand R.F. Leber B. Epand R.M. Andrews D.W. J. Biol. Chem. 2003; 278: 48935-48941Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). 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 μm 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 1 h at 37°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 [35S]methionine and imported into isolated mitochondria, essentially as described previously (16Ahting U. Waizenegger T. Neupert W. Rapaport D. J. Biol. Chem. 2005; 280: 48-53Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To remove proteins and protein domains exposed to the outside of mitochondria, the organelles were incubated with 50 μg/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, 10 000 × 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 (17Ahting U. Thun C. Hegerl R. Typke D. Nargang F.E. Neupert W. Nussberger S. J. Cell Biol. 1999; 147: 959-968Crossref PubMed Scopus (171) Google Scholar) 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 μg/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 were with the immunoprecipitation buffer proteins were with loading buffer Samples were by SDS-PAGE and Co-immunoprecipitation of Bax with the TOM during Insertion-Isolated wild type yeast mitochondria were incubated with Bax and tBid in mm KCl, 1 mm EDTA, mm sorbitol, 10 mm HEPES/KOH, pH for the indicated at 4 °C. Mitochondria were by the of 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 were with the immunoprecipitation buffer proteins were with loading buffer Samples were by SDS-PAGE and with Mitochondria and double were grown in medium with 2 mm and 1 × were resuspended in 100 of mm sorbitol, mm KCl, 10 mm 1 mm mm EDTA, mm 2 mm 10 mm 50 mm HEPES/KOH, pH were by the of The were with antibodies against rat from or porin for 30 min The sample was and one was with the recombinant proteins. The other was incubated with The samples were fractionated by centrifugation (15 min, 16,000 × g, 4 yeast mitochondria were incubated in the same with antibodies against yeast and and for cytochrome c release by the recombinant proteins. Cytochrome c Release from Isolated Yeast Mitochondria by Recombinant tBid and investigate mitochondrial components that might be required for tBid/Bax-induced cytochrome c we to yeast mitochondria as a system. we yeast mitochondria release cytochrome c in a to rat liver mitochondria, incubated with recombinant, purified proteins. liver mitochondria cytochrome c exposed to monomeric Bax for 15 min at 30 °C. In contrast, with a of tBid and monomeric Bax cytochrome c release which is in with the that monomeric Bax has to be by tBid to cytochrome c release. Bax a release of cytochrome c with mitochondria from wild type yeast a of tBid and Bax the release of a of cytochrome c from organelles to wild type and the mutants to study the for tBid/Bax-induced cytochrome c release. of Mitochondrial Proteins for tBid/Bax-induced Cytochrome c the domains of mitochondrial outer membrane proteins, which are exposed to the cytosol, are involved in we domains by proteinase K of the isolated mitochondria. domains for the receptor proteins Tom22, and be whereas membrane proteins, as the β barrel proteins and the were not mitochondria were incubated with recombinant tBid and Bax with proteinase the of cytochrome c was to that in mitochondria. we that exposed domains of mitochondrial outer membrane proteins are not required for tBid/Bax-induced cytochrome c release. it was recently reported that with Bax during apoptosis (11Bellot G. Cartron P.F. Er E. Oliver L. Juin P. Armstrong L.C. Bornstein P. Mihara K. Manon S. Vallette F.M. Cell Death Differ. 2006; 14: 785-794Crossref PubMed Scopus (132) Google Scholar). targeting signals of inner membrane or proteins and at in not required for the of outer membrane proteins. In of the domain of by proteinase K not cytochrome c release. nor for tBid/Bax-induced Cytochrome c Release in the mitochondrial cardiolipin, has been to as a mitochondrial for tBid in mammalian M. M. X. M. X. Wang X. Cell Biol. PubMed Scopus Google Scholar). has also been that cardiolipin is required for tBid/Bax-induced membrane in a system and the recombinant proteins (10Kuwana T. Mackey M.R. Perkins G. Ellisman M.H. Latterich M. Schneiter R. Green D.R. Newmeyer D.D. Cell. 2002; 111: 331-342Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar). Using a yeast mutant the for cardiolipin we earlier that cardiolipin is not required for cytochrome c release by with Bax (12Iverson S.L. Enoksson M. Gogvadze V. Ott M. Orrenius S. J. Biol. Chem. 2004; 279: 1100-1107Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). To cardiolipin is required for tBid/Bax-induced cytochrome c mitochondria isolated from this mutant were now incubated with a of tBid and Bax. The release of cytochrome c was to that in wild type mitochondria it that cardiolipin is not required for cytochrome c release in which is in with a recent study in mammalian that of the mammalian cardiolipin by not apoptosis signaling via and Bid cleavage F. C. D. D. Cell Death Differ. 2006; 14: PubMed Scopus Google Scholar). we the mitochondrial the voltage-dependent anion might be required in the release has previously been that proteins are targets for Bcl-2 family proteins (9Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1928) Google Scholar, W.J. Korsmeyer S.J. Science. 2003; PubMed Scopus Google Scholar). isolated mitochondria from a yeast strain were incubated with tBid and Bax, the of cytochrome c release was to that with wild type mitochondria that the are not involved in tBid/Bax-induced cytochrome c at not in The Mitochondrial TOM for tBid/Bax-induced Cytochrome c we other mitochondrial β barrel proteins be required for the of mitochondria. To investigate the of the pore-forming of the TOM complex, we used yeast mutants a of which is by incubation at a °C T. Rapaport D. C. E. Forte M. M.G. Neupert W. Nargang F.E. J. Cell Biol. PubMed Scopus Google Scholar, W. M.G. EMBO J. PubMed Scopus Google is for cell is yeast mutant of this at in the absence of not to cytochrome c release from either mutant or wild type mitochondria mitochondria from strains were incubated with tBid and Bax at the of cytochrome c release was the mitochondria were at which the function of the mutant tBid/Bax-induced cytochrome c release from wild type mitochondria was not whereas mitochondria from the tom40-4 as well as from the strain cytochrome c exposed to A and it that functional is required for the release in Bax in tom40-4 has previously been that is involved not only in the of proteins the mitochondrial outer membrane also in the insertion of proteins into this membrane D. J. Cell Biol. 2005; PubMed Scopus Google Scholar). the insertion of outer membrane proteins, as and not of the TOM complex is by the protein (16Ahting U. Waizenegger T. Neupert W. Rapaport D. J. Biol. Chem. 2005; 280: 48-53Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To investigate Bax insertion was in the we experiments with Bax was recovered in in the membrane of wild type mitochondria, of been at °C or In contrast, Bax insertion was in the tom40-4 mitochondria that a functional is required for Bax insertion into the outer mitochondrial To the protein in the tom40-4 we investigated the of into mitochondria. Import of this protein can be by the formation of the that is to added Import normally in mitochondria from the tom40-4 strain incubated at the mitochondria were at the of this protein was wild type mitochondria that been at °C The TOM for Cytochrome c Release by the that mitochondria from the tom40-4 strain are to release proteins from intermembrane we incubated mitochondria from this strain with Bax. form of Bax is to have that found (7Yethon J.A. Epand R.F. Leber B. Epand R.M. Andrews D.W. J. Biol. Chem. 2003; 278: 48935-48941Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). mitochondria were incubated with oligomeric Bax, the cytochrome c release was or not the mitochondria functional it that mitochondria from the tom40-4 strain are in to release cytochrome c from intermembrane space and that of the outer membrane by Bax and by monomeric Bax by Conditions, the TOM not for Bax to Release Cytochrome was recently reported that during incubation of yeast mitochondria at °C, tBid was not required for Bax to release cytochrome c and that the release was by V. A. D. S. M. B. B. Martinou J.C. C. C. J. Mol. Biol. PubMed Scopus Google Scholar). we a series of experiments the yeast mitochondria were incubated at °C for 1 monomeric cytochrome c release was and of tBid in experiments the of tBid cytochrome c a that the in the mammalian and systems. A for this between yeast and mammalian mitochondria is that yeast not any proteins, which might insertion of Bax its into functional V. A. D. S. M. B. B. Martinou J.C. C. C. J. Mol. Biol. PubMed Scopus Google Scholar). The of this in the absence of tBid in a and and to a cytochrome c release in to the of V. A. D. S. M. B. B. Martinou J.C. C. C. J. Mol. Biol. PubMed Scopus Google the of cytochrome c from tom40-4 mitochondria from mitochondria, not was as with that found with wild type mitochondria, which release we that also during incubation at the of the TOM complex the of cytochrome c that can be by Bax. Bax with the TOM analyze Bax can with the TOM complex in we incubated isolated TOM core complex of Tom22, and from with tBid and Bax. incubation for 15 min at 30 °C, the sample was and to immunoprecipitation with antibodies against Bax, or a against recovered to a Bax. antibodies against Bax Bax together with a of The not any Bax or we that isolated TOM core complex and Bax can with other in we Bax might with the TOM complex during into isolated yeast mitochondria. the of protein with are normally and with we the at to the of the To mitochondria were incubated at 4 °C with the recombinant proteins and 2 and 30 min. the were to using antibodies against and porin against together with Bax was only with the TOM complex from of mitochondria that were exposed to Bax for 2 min not from of mitochondria that were incubated with Bax for 30 min. against porin not Bax or the TOM we that Bax interacts with the TOM complex during its insertion into the outer mitochondrial against the TOM Cytochrome c Release from investigate the TOM complex is required for tBid/Bax-induced cytochrome c release by independent we experiments. we isolated yeast mitochondria with antibodies against and and for tBid/Bax-induced cytochrome c release. and are of the TOM complex that a domain into the with antibodies against proteins cytochrome c release that antibodies against the of the TOM complex can cytochrome c release from yeast mitochondria. we investigated the TOM complex might also be involved in tBid/Bax-induced cytochrome c release from mammalian mitochondria. For this we used from double T. V. Korsmeyer S.J. Science. PubMed Scopus Google Scholar). the plasma membrane was with of which the mitochondrial outer membrane was cytochrome c from the mitochondria cytochrome c release be by the of tBid and Bax not with tBid or Bax were with antibodies against cytochrome c release be In contrast, antibodies against mitochondrial porin not from cytochrome c release. with antibodies the of Bax with the mitochondria, whereas incubation with a porin not show any are to reported for the of the protein Tom22, insertion into the outer membrane of rat liver mitochondria be by antibodies against Y. H. M. Mihara K. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). we investigated the of the antibodies of Although antibodies against and of this antibodies against porin we can that of the mammalian TOM complex with antibodies leads to a protein into mitochondria and also tBid/Bax-induced cytochrome c release. this study, we show that a functional TOM complex is required for tBid/Bax-induced cytochrome c release from yeast and mammalian mitochondria and that the of tBid and monomeric Bax is to trigger this release as other Bcl-2 family proteins are present in mitochondrial and domains of outer membrane proteins not to play an important in the targeting of Bax to yeast mitochondria. The that Bax the TOM complex for insertion is of a for insertion of proteins, as Tom22, and into the mitochondrial outer membrane (16Ahting U. Waizenegger T. Neupert W. Rapaport D. J. Biol. Chem. 2005; 280: 48-53Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). interaction with the TOM complex be a in the targeting of Bax to the mitochondrial outer membrane, which is an important event during apoptosis signaling In addition, it that the interaction of tBid with Bax the membrane insertion of Bax via the TOM complex M. A. Korsmeyer S.J. H. J. Cell Death Differ. PubMed Scopus Google Scholar). interaction might in which allow targeting signals in Bax to be exposed and by the TOM The TOM complex might then the insertion of monomeric Bax into the mitochondrial outer membrane, with the of tBid or by the contact with the membrane (7Yethon J.A. Epand R.F. Leber B. Epand R.M. Andrews D.W. J. Biol. Chem. 2003; 278: 48935-48941Abstract Full Text Full Text PDF PubMed Scopus (174) Google Bax can to form tBid not to the of Bax insertion and in the yeast outer membrane, as monomeric Bax can cytochrome c with a as with a of monomeric Bax and tBid and The that cytochrome c release by Bax not a for the TOM complex that this form of Bax has the for membrane and The of the targeting of Bax is yet Bax a C-terminal α that some with the signals to direct tail-anchored proteins to mitochondria. of this domain the of the (8Nechushtan A. Smith C.L. Hsu Y.T. Youle R.J. EMBO J. 1999; 18: 2330-2341Crossref PubMed Scopus (629) Google Scholar). the N terminus was also to targeting P.F. M. Oliver L. K. Manon S. Vallette F.M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google this domain not show a found in proteins. is also required to the mechanism(s) by which the TOM complex, with the of tBid and other the insertion of Bax into the mitochondrial outer Antonsson for the of and and for protein Gogvadze for the of rat liver mitochondria, for the double and for the