Mitochondrial release of caspase-2 and -9 during the apoptotic process

The barrier function of mitochondrial membranes is perturbed early during t he apoptotic process. Here we show that the mitochondria contain a caspase- like enzymatic activity cleaving the caspase substrate Z-VAD.afc, in additi on to three biological activities previously suggested to participate in th e apoptotic process: (a) cytochrome c; (b) an apoptosis-inducing factor (AI F) which causes isolated nuclei to undergo apoptosis in vitro; and (c) a DN Ase activity. All oi these factors, which are biochemically distinct, are r eleased upon opening of the permeability transition (PT) pore in a coordina te, Bcl-2-inhibitable fashion. Caspase inhibitors fully neutralize the Z-VA D.afc-cleaving activity, have a limited effect on the AIF activity, and hav e no effect at all on the DNase activities. Purification of proteins reacti ng with the biotinylated caspase substrate Z-VAD, immunodetection, and immu nodepletion experiments reveal the presence of procaspase-2 and -9 in mitoc hondria. Upon induction of PT pore opening, these procaspases are released from purified mitochondria and become activated. Similarly, upon induction of apoptosis, both procaspases redistribute from the mitochondrion to the c ytosol and are processed to generate enzymatically active caspases. This re distribution is inhibited by Bcl-2. Recombinant caspase-2 and -9 suffice to provoke full-blown apoptosis upon microinjection into cells. Altogether, t hese data suggest that caspase-2 and -9 zymogens are essentially localized in mitochondria and that the disruption of the outer mitochondrial membrane occurring early during apoptosis may be critical for their subcellular red istribution and activation.