Molecular characterization of mitochondrial apoptosis-inducing factor (original) (raw)

Nature volume 397, pages 441–446 (1999)Cite this article

Abstract

Mitochondria play a key part in the regulation of apoptosis (cell death)1,2. Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in the degradation phase of apoptosis3,4,5,6,7,8,9. Here we report the identification and cloning of an apoptosis-inducing factor, AIF5, which is sufficient to induce apoptosis of isolated nuclei. AIF is a flavoprotein of relative molecular mass 57,000 which shares homology with the bacterial oxidoreductases; it is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced. Recombinant AIF causes chromatin condensation in isolated nuclei and large-scale fragmentation of DNA. It induces purified mitochondria to release the apoptogenic proteins cytochrome c and caspase-9. Microinjection of AIF into the cytoplasm of intact cells induces condensation of chromatin, dissipation of the mitochondrial transmembrane potential, and exposure of phosphatidylserine in the plasma membrane. None of these effects is prevented by the wide-ranging caspase inhibitor known as Z-VAD.fmk. Overexpression of Bcl-2, which controls the opening of mitochondrial permeability transition pores, prevents the release of AIF from the mitochondrion but does not affect its apoptogenic activity. These results indicate that AIF is a mitochondrial effector of apoptotic cell death.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 51 print issues and online access

$199.00 per year

only $3.90 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Kroemer, G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nature Med. 3, 614–620 (1997).
    Article CAS Google Scholar
  2. Green, D. R. & Reed, J. C. Mitochondria and apoptosis. Science 281, 1309–1312 (1998).
    Article CAS Google Scholar
  3. Zamzami, N. et al. Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183, 1533–1544 (1996).
    Article CAS Google Scholar
  4. Liu, X. S., Kim, C. N., Yang, J., Jemmerson, R. & Wang, X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147–157 (1996).
    Article CAS Google Scholar
  5. Susin, S. A. et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184, 1331–1342 (1996).
    Article CAS Google Scholar
  6. Kluck, R. M., Bossy-Wetzel, E., Green, D. R. & Newmeyer, D. D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132–1136 (1997).
    Article CAS Google Scholar
  7. Vander Heiden, M. G., Chandal, N. S., Williamson, E. K., Schumacker, P. T. & Thompson, C. B. Bcl-XL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91, 627–637 (1997).
    Article CAS Google Scholar
  8. Mancini, M. et al. The caspase-3 precursor has a cytosolic and mitochondrial distribution: Implications for apoptotic signaling. J. Cell Biol. 140, 1485–1495 (1998).
    Article CAS Google Scholar
  9. Susin, S. A. et al. Mitochondrial release of caspases-2 and -9 during the apoptotic process. J. Exp. Med. 189, 381–394 (1999).
    Article CAS Google Scholar
  10. Ducret, A., Van Ostveen, I., Eng, J. K., Yates, J. R. & Aebersold, R. High-throughput protein characterization by automated reverse-phase chromatography electrospray tandem mass spectrometry. Protein Sci. 7, 706–719 (1998).
    Article CAS Google Scholar
  11. Claros, M. G. & Vincens, P. Computation method to predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem. 241, 779–786 (1996).
    Article CAS Google Scholar
  12. Cedano, J., Aloy, P., Pérez-Pons, J. A. & Quero, E. Relation between amino acid composition and cellular location of proteins. J. Mol. Biol. 266, 594–600 ((1997)).
    Article CAS Google Scholar
  13. Boulikas, T. Nuclear localization signals (NLS). Crit. Rev. Euk. Gene Exp. 3, 193–227 (1993).
    CAS Google Scholar
  14. Li, P. et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apopotic protease cascade. Cell 91, 479–489 (1997).
    Article CAS Google Scholar
  15. Yasuhara, N. et al. Essential role of active nuclear transport in apoptosis. Genes to Cells 2, 55–64 (1997).
    Article CAS Google Scholar
  16. Enari, M. et al. Acaspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43–50 (1998).
    Article ADS CAS Google Scholar
  17. Scaffidi, C. et al. Two CD95 (APO-1/Fas) signalling pathways. EMBO J. 17, 1675–1687 (1998).
    Article CAS Google Scholar
  18. Kluck, R. M. et al. Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system. EMBO J. 16, 4639–4649 (1997).
    Article CAS Google Scholar
  19. Robinson, K. M. & Lemire, B. D. Arequirement for matrix processing peptidase but not for mitochondrial chaperonin in the covalent attachment of FAD to yeast succinate dehydrogenase flavoprotein. J. Biol. Chem. 271, 4061–4067 (1996).
    Article CAS Google Scholar
  20. Liu, X., Zou, H., Slaughter, C. & Wang, X. DFF, a heterodimeric protein that functions downstream of caspase 3 to trigger DNA fragmentation during apoptosis. Cell 89, 175–184 (1997).
    Article CAS Google Scholar
  21. Samejima, K. et al. Transition from caspase-dependent to caspase-independent mechanisms at the onset of apoptotic execution. J. Cell Biol. 143, 225–239 (1998).
    Article CAS Google Scholar
  22. Oberhammer, F. et al. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12, 3679–3684 (1993).
    Article CAS Google Scholar
  23. Lagarkova, M. A., Iarvaia, O. V. & Razin, S. V. Large-scale fragmentation of mammalian DNA in the course of apoptosis proceeds via excision of chromosomal DNA loops and their oligomers. J. Biol. Chem. 270, 20239–20241 (1995).
    Article CAS Google Scholar
  24. Trbovich, A. M. et al. High and low molecular weight DNA cleavage in ovarian granulosa cells: characterization and protease modulation in intact cells and in cell-free nuclear autodigestion assays. Cell Death Differ. 5, 38–49 (1998).
    Article CAS Google Scholar
  25. Susin, S. A. et al. The central executioner of apoptosis. Multiple links between protease activation and mitochondria in Fas/Apo-1/CD95- and ceramide-induced apoptosis. J. Exp. Med. 186, 25–37 (1997).
    Article CAS Google Scholar
  26. Bossy-Wetzel, E., Newmeyer, D. D. & Green, D. R. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17, 37–49 (1998).
    Article CAS Google Scholar
  27. Marzo, I. et al. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281, 2027–2031 (1998).
    Article ADS CAS Google Scholar
  28. Enari, M., Hase, A. & Nagata, S. Apoptosis by a cytosolic extract from Fas-activated cells. EMBO J. 14, 5201–5208 (1995).
    Article CAS Google Scholar
  29. Wada, J. & Kanwar, Y. S. Characterization of mammalian translocase of inner mitochondrial membrane (Tim44) isolated from diabetic newborn mouse kidney. Proc. Natl Acad. Sci. USA 95, 144–149 (1998).
    Article ADS CAS Google Scholar
  30. Zhu, W. et al. Bcl-2 mutants with restricted subcellular localization reveal spatially distinct pathways for apoptosis in different cell types. EMBO J. 15, 4130–4141 (1996).
    Article CAS Google Scholar

Download references

Acknowledgements

We thank P. M. Alzari for suggestions, M. Geuskens for electron microscopy, G.Salvesen for caspase-8, D. Andrews for Rat-1 cells, and S. Arya and S. Chung for expression constructs. This work was supported by grants from ANRS, ARC, CNRS, FF, FRM, INSERM, LNC, and PRFMMIP (to G.K.), the NIH, the NSF Science and Technology Center for Molecular Biotechnology (to R.A.), and Amgen (to D.P.S. and J.M.P.). S.A.S. and I.M. hold postdoctoral fellowships from the European Commission and from the Spanish Ministry of Science, respectively.

Author information

Authors and Affiliations

  1. Centre National de la Recherche Scientifique, UPR 420, 19 rue Guy Mquet, F-94801, Villejuif, France
    Santos A. Susin, Naoufal Zamzami, Isabel Marzo, Etienne Jacotot, Paola Costantini, Markus Loeffler, Nathanael Larochette & Guido Kroemer
  2. Unit de Biochimie Structurale, Institut Pasteur, 25 rue du Dr Roux, Paris, F-75724 Cedex 15, France
    Hans K. Lorenzo
  3. Department of Medical Biophysics and Immunology, The Amgen Institute and Ontario Cancer Institute, University of Toronto, 620 University Avenue, Suite 706, Toronto, M5G 2C1, Ontario, Canada
    Bryan E. Snow, Greg M. Brothers, Joan Mangion, David P. Siderovski & Josef M. Penninger
  4. Department of Molecular Biotechnology, University of Washington, Seattle, 98195, Washington, USA
    David R. Goodlett & Ruedi Aebersold

Authors

  1. Santos A. Susin
    You can also search for this author inPubMed Google Scholar
  2. Hans K. Lorenzo
    You can also search for this author inPubMed Google Scholar
  3. Naoufal Zamzami
    You can also search for this author inPubMed Google Scholar
  4. Isabel Marzo
    You can also search for this author inPubMed Google Scholar
  5. Bryan E. Snow
    You can also search for this author inPubMed Google Scholar
  6. Greg M. Brothers
    You can also search for this author inPubMed Google Scholar
  7. Joan Mangion
    You can also search for this author inPubMed Google Scholar
  8. Etienne Jacotot
    You can also search for this author inPubMed Google Scholar
  9. Paola Costantini
    You can also search for this author inPubMed Google Scholar
  10. Markus Loeffler
    You can also search for this author inPubMed Google Scholar
  11. Nathanael Larochette
    You can also search for this author inPubMed Google Scholar
  12. David R. Goodlett
    You can also search for this author inPubMed Google Scholar
  13. Ruedi Aebersold
    You can also search for this author inPubMed Google Scholar
  14. David P. Siderovski
    You can also search for this author inPubMed Google Scholar
  15. Josef M. Penninger
    You can also search for this author inPubMed Google Scholar
  16. Guido Kroemer
    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toGuido Kroemer.

Rights and permissions

About this article

Cite this article

Susin, S., Lorenzo, H., Zamzami, N. et al. Molecular characterization of mitochondrial apoptosis-inducing factor.Nature 397, 441–446 (1999). https://doi.org/10.1038/17135

Download citation

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.