Pro-caspase-8 is predominantly localized in mitochondria and released intocytoplasm upon apoptotic stimulation

The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apopt osis, However, the source of pro-caspase-8 available for activation by apop totic triggers is unknown. In human fibroblasts and mouse clonal striatal c ells, confocal microscopy revealed that pro-caspase-8 immunofluorescence wa s colocalized with cytochrome c in mitochondria and was also distributed di ffusely in some nuclei. Biochemical analysis of subcellular fractions indic ated that pro-caspase-8 was enriched in mitochondria and in nuclei. Pro-cas pase-8 was found in the intermembrane space, inner membrane, and matrix of mitochondria after limited digestion of mitochondrial fractions, and this d istribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside, which opens the mi tochondria permeability transition pore. Release was blocked by the mitocho ndria permeability transition pore inhibitor cyclosporin A (CsA), After clo nal striatal cells were exposed for 6 h to an apoptotic inducer tumor necro sis factor-alpha (TNF-alpha), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, alo ng with pro-caspase-8 cleavage products, in the cytoplasm of the TNF-alpha -treated striatal cells. CsA blocked the TNF-alpha -induced release of pro- caspase 8 but not cytochrome c, Internucleosomal DNA fragmentation started at 6 h and peaked 12 h after TNF-alpha treatment. These results suggest tha t pro-caspase 8 is predominantly localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.