Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Dec 15;368(Pt 3):915–921. doi: 10.1042/BJ20020972

Bax oligomerization in mitochondrial membranes requires tBid (caspase-8-cleaved Bid) and a mitochondrial protein.

Xavier Roucou 1, Sylvie Montessuit 1, Bruno Antonsson 1, Jean-Claude Martinou 1
PMCID: PMC1223025  PMID: 12193163

Abstract

In response to various apoptotic stimuli, Bax, a pro-apoptotic member of the Bcl-2 family, is oligomerized and permeabilizes the mitochondrial outer membrane to apoptogenic factors, including cytochrome c. Bax oligomerization can also be induced by incubating isolated mitochondria containing endogenous Bax with recombinant tBid (caspase-8-cleaved Bid) in vitro. The mechanism by which Bax oligomerizes under these conditions is still unknown. To address this question, recombinant human full-length Bax was purified as a monomeric protein. Bax failed to oligomerize spontaneously in isolated mitochondria or in liposomes composed of either cardiolipin or lipids extracted from mitochondria. However, in the presence of tBid, the protein formed large complexes in mitochondrial membranes and induced the release of cytochrome c. tBid also induced Bax oligomerization in isolated mitochondrial outer membranes, but not in other membranes, such as plasma membranes or microsomes. Moreover, tBid-induced Bax oligomerization was inhibited when mitochondria were pretreated with protease K. The presence of the voltage-dependent anion channel was not required either for Bax oligomerization or for Bax-induced cytochrome c release. Finally, Bax oligomerization was reconstituted in proteoliposomes made from mitochondrial membrane proteins. These findings imply that tBid is necessary but not sufficient for Bax oligomerization; a mitochondrial protein is also required.

Full Text

The Full Text of this article is available as a PDF (235.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams J. M., Cory S. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci. 2001 Jan;26(1):61–66. doi: 10.1016/s0968-0004(00)01740-0. [DOI] [PubMed] [Google Scholar]
  2. Antonsson B., Montessuit S., Lauper S., Eskes R., Martinou J. C. Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochem J. 2000 Jan 15;345(Pt 2):271–278. [PMC free article] [PubMed] [Google Scholar]
  3. Antonsson B., Montessuit S., Sanchez B., Martinou J. C. Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem. 2001 Jan 2;276(15):11615–11623. doi: 10.1074/jbc.M010810200. [DOI] [PubMed] [Google Scholar]
  4. Blachly-Dyson E., Peng S., Colombini M., Forte M. Selectivity changes in site-directed mutants of the VDAC ion channel: structural implications. Science. 1990 Mar 9;247(4947):1233–1236. doi: 10.1126/science.1690454. [DOI] [PubMed] [Google Scholar]
  5. Cheng E. H., Wei M. C., Weiler S., Flavell R. A., Mak T. W., Lindsten T., Korsmeyer S. J. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell. 2001 Sep;8(3):705–711. doi: 10.1016/s1097-2765(01)00320-3. [DOI] [PubMed] [Google Scholar]
  6. De Giorgi FrancesaA, Lartigue Lydia, Bauer Manuel K. A., Schubert Alexis, Grimm Stefan, Hanson George T., Remington S. James, Youle Richard J., Ichas François. The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB J. 2002 Apr;16(6):607–609. doi: 10.1096/fj.01-0269fje. [DOI] [PubMed] [Google Scholar]
  7. Desagher S., Martinou J. C. Mitochondria as the central control point of apoptosis. Trends Cell Biol. 2000 Sep;10(9):369–377. doi: 10.1016/s0962-8924(00)01803-1. [DOI] [PubMed] [Google Scholar]
  8. Du C., Fang M., Li Y., Li L., Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell. 2000 Jul 7;102(1):33–42. doi: 10.1016/s0092-8674(00)00008-8. [DOI] [PubMed] [Google Scholar]
  9. Ekert P. G., Silke J., Hawkins C. J., Verhagen A. M., Vaux D. L. DIABLO promotes apoptosis by removing MIHA/XIAP from processed caspase 9. J Cell Biol. 2001 Feb 5;152(3):483–490. doi: 10.1083/jcb.152.3.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eskes R., Antonsson B., Osen-Sand A., Montessuit S., Richter C., Sadoul R., Mazzei G., Nichols A., Martinou J. C. Bax-induced cytochrome C release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions. J Cell Biol. 1998 Oct 5;143(1):217–224. doi: 10.1083/jcb.143.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eskes R., Desagher S., Antonsson B., Martinou J. C. Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol. 2000 Feb;20(3):929–935. doi: 10.1128/mcb.20.3.929-935.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Esposti M. D., Erler J. T., Hickman J. A., Dive C. Bid, a widely expressed proapoptotic protein of the Bcl-2 family, displays lipid transfer activity. Mol Cell Biol. 2001 Nov;21(21):7268–7276. doi: 10.1128/MCB.21.21.7268-7276.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  14. Fivaz M., Abrami L., Tsitrin Y., van der Goot F. G. Not as simple as just punching a hole. Toxicon. 2001 Nov;39(11):1637–1645. doi: 10.1016/s0041-0101(01)00151-9. [DOI] [PubMed] [Google Scholar]
  15. Golstein P. Controlling cell death. Science. 1997 Feb 21;275(5303):1081–1082. doi: 10.1126/science.275.5303.1081. [DOI] [PubMed] [Google Scholar]
  16. Graham J. M. Fractionation of a microsomal fraction from rat liver or cultured cells. Methods Mol Biol. 1993;19:51–57. doi: 10.1385/0-89603-236-1:51. [DOI] [PubMed] [Google Scholar]
  17. Griffiths G. J., Dubrez L., Morgan C. P., Jones N. A., Whitehouse J., Corfe B. M., Dive C., Hickman J. A. Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J Cell Biol. 1999 Mar 8;144(5):903–914. doi: 10.1083/jcb.144.5.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grinberg Michal, Sarig Rachel, Zaltsman Yehudit, Frumkin Dan, Grammatikakis Nicholas, Reuveny Eitan, Gross Atan. tBID Homooligomerizes in the mitochondrial membrane to induce apoptosis. J Biol Chem. 2002 Jan 22;277(14):12237–12245. doi: 10.1074/jbc.M104893200. [DOI] [PubMed] [Google Scholar]
  19. Gross A., Jockel J., Wei M. C., Korsmeyer S. J. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 1998 Jul 15;17(14):3878–3885. doi: 10.1093/emboj/17.14.3878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gross A., Pilcher K., Blachly-Dyson E., Basso E., Jockel J., Bassik M. C., Korsmeyer S. J., Forte M. Biochemical and genetic analysis of the mitochondrial response of yeast to BAX and BCL-X(L). Mol Cell Biol. 2000 May;20(9):3125–3136. doi: 10.1128/mcb.20.9.3125-3136.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Harris M. H., Vander Heiden M. G., Kron S. J., Thompson C. B. Role of oxidative phosphorylation in Bax toxicity. Mol Cell Biol. 2000 May;20(10):3590–3596. doi: 10.1128/mcb.20.10.3590-3596.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hegde Ramesh, Srinivasula Srinivasa M., Zhang ZhiJia, Wassell Richard, Mukattash Rula, Cilenti Lucia, DuBois Garrett, Lazebnik Yuri, Zervos Antonis S., Fernandes-Alnemri Teresa. Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein-caspase interaction. J Biol Chem. 2001 Oct 17;277(1):432–438. doi: 10.1074/jbc.M109721200. [DOI] [PubMed] [Google Scholar]
  23. Hsu Y. T., Youle R. J. Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J Biol Chem. 1998 Apr 24;273(17):10777–10783. doi: 10.1074/jbc.273.17.10777. [DOI] [PubMed] [Google Scholar]
  24. Hsu Y. T., Youle R. J. Nonionic detergents induce dimerization among members of the Bcl-2 family. J Biol Chem. 1997 May 23;272(21):13829–13834. doi: 10.1074/jbc.272.21.13829. [DOI] [PubMed] [Google Scholar]
  25. Hu Y., Benedict M. A., Ding L., Núez G. Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis. EMBO J. 1999 Jul 1;18(13):3586–3595. doi: 10.1093/emboj/18.13.3586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Joza N., Susin S. A., Daugas E., Stanford W. L., Cho S. K., Li C. Y., Sasaki T., Elia A. J., Cheng H. Y., Ravagnan L. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature. 2001 Mar 29;410(6828):549–554. doi: 10.1038/35069004. [DOI] [PubMed] [Google Scholar]
  27. Korsmeyer S. J., Wei M. C., Saito M., Weiler S., Oh K. J., Schlesinger P. H. Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ. 2000 Dec;7(12):1166–1173. doi: 10.1038/sj.cdd.4400783. [DOI] [PubMed] [Google Scholar]
  28. Kudla G., Montessuit S., Eskes R., Berrier C., Martinou J. C., Ghazi A., Antonsson B. The destabilization of lipid membranes induced by the C-terminal fragment of caspase 8-cleaved bid is inhibited by the N-terminal fragment. J Biol Chem. 2000 Jul 28;275(30):22713–22718. doi: 10.1074/jbc.M003807200. [DOI] [PubMed] [Google Scholar]
  29. Lee A. C., Xu X., Blachly-Dyson E., Forte M., Colombini M. The role of yeast VDAC genes on the permeability of the mitochondrial outer membrane. J Membr Biol. 1998 Jan 15;161(2):173–181. doi: 10.1007/s002329900324. [DOI] [PubMed] [Google Scholar]
  30. Li H., Zhu H., Xu C. J., Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. 1998 Aug 21;94(4):491–501. doi: 10.1016/s0092-8674(00)81590-1. [DOI] [PubMed] [Google Scholar]
  31. Li L. Y., Luo X., Wang X. Endonuclease G is an apoptotic DNase when released from mitochondria. Nature. 2001 Jul 5;412(6842):95–99. doi: 10.1038/35083620. [DOI] [PubMed] [Google Scholar]
  32. Luo X., Budihardjo I., Zou H., Slaughter C., Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell. 1998 Aug 21;94(4):481–490. doi: 10.1016/s0092-8674(00)81589-5. [DOI] [PubMed] [Google Scholar]
  33. Lutter M., Fang M., Luo X., Nishijima M., Xie X., Wang X. Cardiolipin provides specificity for targeting of tBid to mitochondria. Nat Cell Biol. 2000 Oct;2(10):754–761. doi: 10.1038/35036395. [DOI] [PubMed] [Google Scholar]
  34. Martinou J. C., Green D. R. Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol. 2001 Jan;2(1):63–67. doi: 10.1038/35048069. [DOI] [PubMed] [Google Scholar]
  35. Mikhailov V., Mikhailova M., Pulkrabek D. J., Dong Z., Venkatachalam M. A., Saikumar P. Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane. J Biol Chem. 2001 Feb 20;276(21):18361–18374. doi: 10.1074/jbc.M100655200. [DOI] [PubMed] [Google Scholar]
  36. Montessuit S., Mazzei G., Magnenat E., Antonsson B. Expression and purification of full-length human Bax alpha. Protein Expr Purif. 1999 Mar;15(2):202–206. doi: 10.1006/prep.1998.1010. [DOI] [PubMed] [Google Scholar]
  37. Nechushtan A., Smith C. L., Lamensdorf I., Yoon S. H., Youle R. J. Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol. 2001 Jun 11;153(6):1265–1276. doi: 10.1083/jcb.153.6.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Priault M., Chaudhuri B., Clow A., Camougrand N., Manon S. Investigation of bax-induced release of cytochrome c from yeast mitochondria permeability of mitochondrial membranes, role of VDAC and ATP requirement. Eur J Biochem. 1999 Mar;260(3):684–691. doi: 10.1046/j.1432-1327.1999.00198.x. [DOI] [PubMed] [Google Scholar]
  39. Reed J. C., Jurgensmeier J. M., Matsuyama S. Bcl-2 family proteins and mitochondria. Biochim Biophys Acta. 1998 Aug 10;1366(1-2):127–137. doi: 10.1016/s0005-2728(98)00108-x. [DOI] [PubMed] [Google Scholar]
  40. Roucou Xavier, Rostovtseva Tatiana, Montessuit Sylvie, Martinou Jean-Claude, Antonsson Bruno. Bid induces cytochrome c-impermeable Bax channels in liposomes. Biochem J. 2002 May 1;363(Pt 3):547–552. doi: 10.1042/0264-6021:3630547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Saito M., Korsmeyer S. J., Schlesinger P. H. BAX-dependent transport of cytochrome c reconstituted in pure liposomes. Nat Cell Biol. 2000 Aug;2(8):553–555. doi: 10.1038/35019596. [DOI] [PubMed] [Google Scholar]
  42. Saleh A., Srinivasula S. M., Acharya S., Fishel R., Alnemri E. S. Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation. J Biol Chem. 1999 Jun 18;274(25):17941–17945. doi: 10.1074/jbc.274.25.17941. [DOI] [PubMed] [Google Scholar]
  43. Shimizu S., Narita M., Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature. 1999 Jun 3;399(6735):483–487. doi: 10.1038/20959. [DOI] [PubMed] [Google Scholar]
  44. Srinivasula S. M., Ahmad M., Fernandes-Alnemri T., Alnemri E. S. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell. 1998 Jun;1(7):949–957. doi: 10.1016/s1097-2765(00)80095-7. [DOI] [PubMed] [Google Scholar]
  45. Sundararajan R., Cuconati A., Nelson D., White E. Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K. J Biol Chem. 2001 Sep 24;276(48):45120–45127. doi: 10.1074/jbc.M106386200. [DOI] [PubMed] [Google Scholar]
  46. Susin S. A., Lorenzo H. K., Zamzami N., Marzo I., Snow B. E., Brothers G. M., Mangion J., Jacotot E., Costantini P., Loeffler M. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature. 1999 Feb 4;397(6718):441–446. doi: 10.1038/17135. [DOI] [PubMed] [Google Scholar]
  47. Suzuki Y., Imai Y., Nakayama H., Takahashi K., Takio K., Takahashi R. A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell. 2001 Sep;8(3):613–621. doi: 10.1016/s1097-2765(01)00341-0. [DOI] [PubMed] [Google Scholar]
  48. Verhagen A. M., Ekert P. G., Pakusch M., Silke J., Connolly L. M., Reid G. E., Moritz R. L., Simpson R. J., Vaux D. L. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell. 2000 Jul 7;102(1):43–53. doi: 10.1016/s0092-8674(00)00009-x. [DOI] [PubMed] [Google Scholar]
  49. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev. 2001 Nov 15;15(22):2922–2933. [PubMed] [Google Scholar]
  50. Wei M. C., Lindsten T., Mootha V. K., Weiler S., Gross A., Ashiya M., Thompson C. B., Korsmeyer S. J. tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev. 2000 Aug 15;14(16):2060–2071. [PMC free article] [PubMed] [Google Scholar]
  51. Wei M. C., Zong W. X., Cheng E. H., Lindsten T., Panoutsakopoulou V., Ross A. J., Roth K. A., MacGregor G. R., Thompson C. B., Korsmeyer S. J. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science. 2001 Apr 27;292(5517):727–730. doi: 10.1126/science.1059108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wolter K. G., Hsu Y. T., Smith C. L., Nechushtan A., Xi X. G., Youle R. J. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol. 1997 Dec 1;139(5):1281–1292. doi: 10.1083/jcb.139.5.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yang J., Liu X., Bhalla K., Kim C. N., Ibrado A. M., Cai J., Peng T. I., Jones D. P., Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997 Feb 21;275(5303):1129–1132. doi: 10.1126/science.275.5303.1129. [DOI] [PubMed] [Google Scholar]
  54. Zou H., Li Y., Liu X., Wang X. An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem. 1999 Apr 23;274(17):11549–11556. doi: 10.1074/jbc.274.17.11549. [DOI] [PubMed] [Google Scholar]
  55. van Loo G., Schotte P., van Gurp M., Demol H., Hoorelbeke B., Gevaert K., Rodriguez I., Ruiz-Carrillo A., Vandekerckhove J., Declercq W. Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation. Cell Death Differ. 2001 Dec;8(12):1136–1142. doi: 10.1038/sj.cdd.4400944. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES