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Molecular characterization of mitochondrial apoptosis-inducing factor

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.

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Figure 1: Alignment of mouse and human AIF amino-acid sequences withbenzene 1,2-dioxygenase system ferredoxin NADH reductase from Pseudomonas putida (BDSF; 30% amino-acid identity with mouse AIF).
Figure 2: Subcellular and submitochondrial distribution of AIF.
Figure 3: Effects of AIF on isolated nuclei and mitochondria.
Figure 4: Effects of AIF on intact cells.

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.

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Correspondence to Guido Kroemer.

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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

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