Smac/DIABLO is not released from mitochondria during apoptotic signalling in cells deficient in cytochrome c.
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.
Hansen
T M
TM
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Smith
D J
DJ
Nagley
P
P
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000172:deficiency / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000032:analysis / Q000378:metabolism*; D041681:NIH 3T3 Cells; D015398:Signal Transduction / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D020169:Caspases; D019311:Staurosporine