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. 2002 May 1;363(Pt 3):547–552. doi: 10.1042/0264-6021:3630547

Bid induces cytochrome c-impermeable Bax channels in liposomes.

Xavier Roucou 1, Tatiana Rostovtseva 1, Sylvie Montessuit 1, Jean-Claude Martinou 1, Bruno Antonsson 1
PMCID: PMC1222507  PMID: 11964155

Abstract

Bax is a proapoptotic member of the Bcl-2 family of proteins. The Bax protein is dormant in the cytosol of normal cells and is activated upon induction of apoptosis. In apoptotic cells, Bax gets translocated to mitochondria, inserts into the outer membrane, oligomerizes and triggers the release of cytochrome c, possibly by channel formation. The BH3 domain-only protein Bid induces a conformational change in Bax before its insertion into the outer membrane. The mechanism by which Bid promotes Bax activation is not understood, and whether Bid is the only protein required for Bax activation is unclear. Here we report that recombinant full-length Bax (Bax(FL)) does not form channels in lipid bilayers when purified as a monomer. In contrast, in the presence of Bid cut with caspase 8 (cut Bid), Bax forms ionic channels in liposomes and planar bilayers. This channel-forming activity requires an interaction between cut Bid and Bax, and is inhibited by Bcl-x(L). Moreover, in the absence of the putative transmembrane C-terminal domain, Bax does not form ionic channels in the presence of cut Bid. Cut Bid does not induce Bax oligomerization in liposomes and the Bax channels formed in the presence of cut Bid are not large enough to permeabilize vesicles to cytochrome c. In conclusion, our results suggest that monomeric Bax(FL) can form channels only in the presence of cut Bid. Cut Bid by itself is unable to induce Bax oligomerization in lipid membranes. It is suggested that another factor that might be present in mitochondria is required for Bax oligomerization.

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Selected References

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  1. Antonsson B., Conti F., Ciavatta A., Montessuit S., Lewis S., Martinou I., Bernasconi L., Bernard A., Mermod J. J., Mazzei G. Inhibition of Bax channel-forming activity by Bcl-2. Science. 1997 Jul 18;277(5324):370–372. doi: 10.1126/science.277.5324.370. [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. Basañez G., Nechushtan A., Drozhinin O., Chanturiya A., Choe E., Tutt S., Wood K. A., Hsu Y., Zimmerberg J., Youle R. J. Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5492–5497. doi: 10.1073/pnas.96.10.5492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bratton S. B., Walker G., Srinivasula S. M., Sun X. M., Butterworth M., Alnemri E. S., Cohen G. M. Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes. EMBO J. 2001 Mar 1;20(5):998–1009. doi: 10.1093/emboj/20.5.998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cain K., Brown D. G., Langlais C., Cohen G. M. Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspase-activating complex. J Biol Chem. 1999 Aug 6;274(32):22686–22692. doi: 10.1074/jbc.274.32.22686. [DOI] [PubMed] [Google Scholar]
  7. Chou J. J., Li H., Salvesen G. S., Yuan J., Wagner G. Solution structure of BID, an intracellular amplifier of apoptotic signaling. Cell. 1999 Mar 5;96(5):615–624. doi: 10.1016/s0092-8674(00)80572-3. [DOI] [PubMed] [Google Scholar]
  8. Deckwerth T. L., Elliott J. L., Knudson C. M., Johnson E. M., Jr, Snider W. D., Korsmeyer S. J. BAX is required for neuronal death after trophic factor deprivation and during development. Neuron. 1996 Sep;17(3):401–411. doi: 10.1016/s0896-6273(00)80173-7. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Desagher S., Osen-Sand A., Nichols A., Eskes R., Montessuit S., Lauper S., Maundrell K., Antonsson B., Martinou J. C. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol. 1999 Mar 8;144(5):891–901. doi: 10.1083/jcb.144.5.891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. Marzo I., Brenner C., Zamzami N., Jürgensmeier J. M., Susin S. A., Vieira H. L., Prévost M. C., Xie Z., Matsuyama S., Reed J. C. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science. 1998 Sep 25;281(5385):2027–2031. doi: 10.1126/science.281.5385.2027. [DOI] [PubMed] [Google Scholar]
  17. McDonnell J. M., Fushman D., Milliman C. L., Korsmeyer S. J., Cowburn D. Solution structure of the proapoptotic molecule BID: a structural basis for apoptotic agonists and antagonists. Cell. 1999 Mar 5;96(5):625–634. doi: 10.1016/s0092-8674(00)80573-5. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Minn A. J., Vélez P., Schendel S. L., Liang H., Muchmore S. W., Fesik S. W., Fill M., Thompson C. B. Bcl-x(L) forms an ion channel in synthetic lipid membranes. Nature. 1997 Jan 23;385(6614):353–357. doi: 10.1038/385353a0. [DOI] [PubMed] [Google Scholar]
  20. Montal M., Mueller P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3561–3566. doi: 10.1073/pnas.69.12.3561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Muchmore S. W., Sattler M., Liang H., Meadows R. P., Harlan J. E., Yoon H. S., Nettesheim D., Chang B. S., Thompson C. B., Wong S. L. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature. 1996 May 23;381(6580):335–341. doi: 10.1038/381335a0. [DOI] [PubMed] [Google Scholar]
  23. Narita M., Shimizu S., Ito T., Chittenden T., Lutz R. J., Matsuda H., Tsujimoto Y. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14681–14686. doi: 10.1073/pnas.95.25.14681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nechushtan A., Smith C. L., Hsu Y. T., Youle R. J. Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J. 1999 May 4;18(9):2330–2341. doi: 10.1093/emboj/18.9.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pastorino J. G., Tafani M., Rothman R. J., Marcinkeviciute A., Hoek J. B., Farber J. L., Marcineviciute A. Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore. J Biol Chem. 1999 Oct 29;274(44):31734–31739. doi: 10.1074/jbc.274.44.31734. [DOI] [PubMed] [Google Scholar]
  26. Petros A. M., Medek A., Nettesheim D. G., Kim D. H., Yoon H. S., Swift K., Matayoshi E. D., Oltersdorf T., Fesik S. W. Solution structure of the antiapoptotic protein bcl-2. Proc Natl Acad Sci U S A. 2001 Feb 27;98(6):3012–3017. doi: 10.1073/pnas.041619798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rostovtseva T. K., Bezrukov S. M. ATP transport through a single mitochondrial channel, VDAC, studied by current fluctuation analysis. Biophys J. 1998 May;74(5):2365–2373. doi: 10.1016/S0006-3495(98)77945-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Schendel S. L., Azimov R., Pawlowski K., Godzik A., Kagan B. L., Reed J. C. Ion channel activity of the BH3 only Bcl-2 family member, BID. J Biol Chem. 1999 Jul 30;274(31):21932–21936. doi: 10.1074/jbc.274.31.21932. [DOI] [PubMed] [Google Scholar]
  31. Schendel S. L., Xie Z., Montal M. O., Matsuyama S., Montal M., Reed J. C. Channel formation by antiapoptotic protein Bcl-2. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5113–5118. doi: 10.1073/pnas.94.10.5113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schlesinger P. H., Gross A., Yin X. M., Yamamoto K., Saito M., Waksman G., Korsmeyer S. J. Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11357–11362. doi: 10.1073/pnas.94.21.11357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Suzuki M., Youle R. J., Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell. 2000 Nov 10;103(4):645–654. doi: 10.1016/s0092-8674(00)00167-7. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Vander Heiden M. G., Li X. X., Gottleib E., Hill R. B., Thompson C. B., Colombini M. Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane. J Biol Chem. 2001 Mar 20;276(22):19414–19419. doi: 10.1074/jbc.M101590200. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Wang K., Yin X. M., Chao D. T., Milliman C. L., Korsmeyer S. J. BID: a novel BH3 domain-only death agonist. Genes Dev. 1996 Nov 15;10(22):2859–2869. doi: 10.1101/gad.10.22.2859. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]

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