Abstract
Because of structural homology with the transmembrane domain of Bcl-2, the proapoptotic protein Bax has been proposed to be anchored to the outer membrane of mitochondria through its carboxy-terminal end (CT). We took advantage of the absence of Bcl-2 family members in yeast to further investigate the role of Bax CT in its mitochondrial association and function. The complete deletion or the addition of a C-terminal c-myc tag as well as the replacement of CT by a random coiled sequence enhanced membrane insertion of Bax. It has previously been suggested that conformational change in the N-terminal end of Bax would allow the C-terminal end to play its anchoring function. We found that a mutant truncated in both N- and C-termini still exhibited a strong binding activity to mitochondria. In mammals, Bax interaction with the caspase-8-generated truncated form of Bid (tc-Bid) is believed to promote a conformational change necessary for the insertion of Bax into mitochondria. We coexpressed Bax and tc-Bid in yeast and found that native Bax functions are not stimulated by tc-Bid, whereas functions of an active variant with a modified CT are. We propose that Bax CT has to undergo a conformational change to allow the insertion of Bax in mitochondria but, contrary to current views, is not a bona fide membrane anchor.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- CT:
-
C-terminal α-helix
- DPMS:
-
dolichol-phosphate mannose synthase
- MAC:
-
mitochondrial apoptosis-induced channel
- PGK:
-
phosphoglycerate kinase
- PVDF:
-
polyvinyl difluoride
- SDS-PAGE:
-
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
- TCA:
-
trichloroacetic acid
References
Gross A, McDonnell JM and Korsmeyer SJ (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 13: 1899–1911
Martinou JC and Green DR (2001) Breaking the mitochondrial barrier. Nat. Rev. Mol. Cell. Biol. 2: 63–67
Antonsson B and Martinou JC (2000) The Bcl-2 protein family. Exp. Cell Res. 256: 50–57
Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG and Youle RJ (1997) Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol. 139: 1281–1292
Desagher S and Martinou JC (2001) Mitochondria as the central control point of apoptosis. Trends Cell. Biol. 10: 369–377
Roucou X and Martinou JC (2001) Conformational change of Bax: a question of life or death. Cell Death Diff. 8: 875–877
Suzuki M, Youle RJ and Tjandra N (2000) Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 103: 645–654
Greenhalf W, Stephan C and Chaudhuri B (1996) Role of mitochondria and C-terminal membrane anchor of Bcl-2 in Bax induced growth arrest and mortality in Saccharomyces cerevisiae. FEBS Lett. 380: 169–175
Clow A, Greenhalf W and Chaudhuri B (1998) Under respiratory growth conditions, Bcl-x(L) and Bcl-2 are unable to overcome yeast cell death triggered by a mutant Bax protein lacking the membrane anchor. Eur. J. Biochem. 258: 19–28
Priault M, Camougrand N, Chaudhuri B and Manon S (1999) Role of the C-terminal domain of Bax and Bcl-XL in their localization and function in yeast cells. FEBS Lett. 443: 225–228
Nguyen M, Millar DG, Yong VW, Korsmeyer SJ and Shore GC (1993) Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence. J. Biol. Chem. 268: 25265–25268
Janiak F, Leber B and Andrews DW (1994) Assembly of Bcl-2 into microsomal and outer mitochondrial membranes. J. Biol. Chem. 269: 9842–9849
Tremblais K, Oliver L, Juin P, Le Cabellec MT, Meflah K and Vallette FM (1999) The C-terminus of bax is not a membrane addressing/anchoring signal. Biochem. Biophys. Res. Commun. 260: 582–591
Nechushtan A, Smith CL, Hsu YT and Youle RJ (1999) Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J. 18: 2330–2341
Martinez-Senac M, Corbalan-Garcia S and Gomez-Fernandez JC (2001) Conformation of the C-terminal domain of the pro-apoptotic protein Bax and mutants and its interaction with membranes. Biochemistry 40: 9983–9992
Goping IS, Gross A, Lavoie JN, Nguyen M, Jemmerson R, Roth K, Korsmeyer SJ and Shore GC (1998) Regulated targeting of BAX to mitochondria. J. Cell Biol. 143: 207–215
Wood DE and Newcomb EW (2000) Cleavage of Bax enhances its cell death function. Exp. Cell Res. 256: 375–382
Cartron PF, Moreau C, Oliver L, Mayat E, Meflah K and Vallette FM (2002) Involvement of the N-terminus of Bax in its intracellular localization and function. FEBS Lett. 512: 95–100
Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B and Martinou JC (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144: 891–901
Eskes R, Desagher S, Antonsson B and Martinou JC (2000) Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol. Cell. Biol. 20: 929–935
Roucou X, Montessuit S, Antonsson B and Martinou JC (2002) Bax oligomerization in mitochondrial membranes requires tBid and a mitochondrial protein. Biochem. J. 368: 915–921
Manon S, Chaudhuri B and Guérin M (1997) Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-xL. FEBS Lett. 415: 29–32
Priault M, Chaudhuri B, Clow A, Camougrand N and Manon S (1999) Investigation of bax-induced release of cytochrome c from yeast mitochondria permeability of mitochondrial membranes, role of VDAC and ATP requirement. Eur. J. Biochem. 260: 684–691
Oliver L, Priault M, Tremblais K, Le Cabellec MT, Meflah K, Manon S and Vallette FM (2000) The substitution of the C-terminus of bax by that of bcl-xL does not affect its subcellular localization but abrogates its pro-apoptotic properties. FEBS Lett. 487: 161–165
Li H, Zhu H, Xu CJ and Yuan J (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94: 491–501
Priault M, Camougrand N, Chaudhuri B, Schaeffer J and Manon S (1999) Comparison of the effects of bax-expression in yeast under fermentative and respiratory conditions: investigation of the role of adenine nucleotides carrier and cytochrome c. FEBS Lett. 456: 232–238
Roucou X, Prescott M, Devenish RJ and Nagley P (2000) A cytochrome c-GFP fusion is not released from mitochondria into the cytoplasm upon expression of Bax in yeast cells. FEBS Lett. 471: 235–239
Pavlov EV, Priault M, Pietkiewicz D, Cheng EHY, Antonsson B, Manon S, Korsmeyer SJ, Mannella CA and Kinnally KW (2001) A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast. J. Cell Biol. 155: 725–731
Cartron PF, Priault M, Oliver L, Meflah K, Manon S and Vallette FM (2003) The N-terminal end of bax contains a mitochondrial-targeting signal. J. Biol. Chem. 278: 11633–11641
Roucou X, Rostovtseva T, Montessuit S, Martinou JC and Antonsson B (2002) Bid induces cytochrome c-impermeable Bax channels in liposomes. Biochem. J. 363: 547–552
Appaix F, Guerrero K, Rampal D, Izikki M, Kaambre T, Sikk P, Brdiczka D, Riva-Lavieille C, Olivares J, Longuet M, Antonsson B and Saks VA (2002) Bax and heart mitochondria: uncoupling and inhibition of respiration without permeability transition. Biochim. Biophys. Acta 1556: 155–167
Acknowledgements
This work was supported by grants from the Centre National de la Recherche Scientifique, the Association pour la Recherche contre le Cancer, the Conseil Régional d'Aquitaine and the Université de Bordeaux 2. MP was the recipient of a fellowship from the Ministère de la Recherche et de la Technologie.
Author information
Authors and Affiliations
Corresponding author
Additional information
Edited by S Roy
Rights and permissions
About this article
Cite this article
Priault, M., Cartron, PF., Camougrand, N. et al. Investigation of the role of the C-terminus of Bax and of tc-Bid on Bax interaction with yeast mitochondria. Cell Death Differ 10, 1068–1077 (2003). https://doi.org/10.1038/sj.cdd.4401270
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/sj.cdd.4401270
Keywords
This article is cited by
-
Robust autoactivation for apoptosis by BAK but not BAX highlights BAK as an important therapeutic target
Cell Death & Disease (2020)
-
Utilizing mitochondrial events as biomarkers for imaging apoptosis
Cell Death & Disease (2011)
-
Bid: a Bax-like BH3 protein
Oncogene (2008)
-
The mitochondrial pathway in yeast apoptosis
Apoptosis (2007)
-
Bax and Bid, two proapoptotic Bcl-2 family members, inhibit homologous recombination, independently of apoptosis regulation
Oncogene (2006)
