Abstract
We have characterised the apoptotic defects in cells null for cytochrome c (cyt c−/−). Such cells treated with staurosporine (STS) exhibited translocation to the mitochondria and activation of the proapoptotic signalling molecule Bax but failed to release Smac/DIABLO from these organelles, judged by both confocal microscopy and Western blotting. While reference cells expressing cytochrome c released both it and Smac/DIABLO under a variety of conditions of apoptotic induction, we have never observed release of Smac/DIABLO from cyt c−/− cells. We eliminate the possibility that proteasomal degradation of cytosolically localised Smac/DIABLO is responsible for our failure to visualise the protein outside the mitochondria. Our findings indicate an unanticipated nexus between release of cytochrome c and Smac/DIABLO from mitochondria, previously thought to be a more or less synchronised event early in apoptosis. We suggest that the failure of cyt c−/− cells to release Smac/DIABLO after recruitment of Bax to mitochondria represents an extreme manifestation of some inherent difference in the regulation of release of these two proteins from mitochondria.
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Abbreviations
- cyt c:
-
cytochrome c
- IMS:
-
intermembrane space
- OM:
-
outer membrane
- IAPs:
-
inhibitor of apoptosis proteins
- AIF:
-
apoptosis-inducing factor
- cyt c−/− cells:
-
cytochrome c knockout mouse embryonic fibroblast cells
- STS:
-
staurosporine
- MTR:
-
MitoTracker Red
- Tdt:
-
terminal deoxynucleotidyl transferase
- CHX:
-
cycloheximide
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Acknowledgements
The excellent technical assistance of Susan Ekkel is gratefully acknowledged. We thank Professor N Hoogenraad (La Trobe University) for his gift of antibodies specific for Hsp60, and Dr. R Baker (Australian National University) for helpful advice on proteasome inhibitors and ubiquitinylation. This work was supported by the Australian Research Council.
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Hansen, T., Smith, D. & Nagley, P. Smac/DIABLO is not released from mitochondria during apoptotic signalling in cells deficient in cytochrome c. Cell Death Differ 13, 1181–1190 (2006). https://doi.org/10.1038/sj.cdd.4401795
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DOI: https://doi.org/10.1038/sj.cdd.4401795
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