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Unité de Biochimie Structurale, Institut Pasteur, F-75724 Paris Cedex 15, France; Centre National de la Recherche Scientifique, UPRES-A6022, Université de Technologie de Compiègne, F-60200 Compiègne, France; and || Unité d'Oncologie Virale, Institut Pasteur, F-75724 Paris Cedex 15, France
The barrier function of mitochondrial membranes is perturbed early during the apoptotic process. Here we show that the mitochondria contain a caspase-like enzymatic activity cleaving the caspase substrate Z-VAD.afc, in addition to three biological activities previously suggested to participate in the apoptotic process: (a) cytochrome c; (b) an apoptosis-inducing factor (AIF) which causes isolated nuclei to undergo apoptosis in vitro; and (c) a DNAse activity. All of these factors, which are biochemically distinct, are released upon opening of the permeability transition (PT) pore in a coordinate, Bcl-2–inhibitable fashion. Caspase inhibitors fully neutralize the Z-VAD.afc–cleaving activity, have a limited effect on the AIF activity, and have no effect at all on the DNase activities. Purification of proteins reacting with the biotinylated caspase substrate Z-VAD, immunodetection, and immunodepletion experiments reveal the presence of procaspase-2 and -9 in mitochondria. 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 cytosol and are processed to generate enzymatically active caspases. This redistribution is inhibited by Bcl-2. Recombinant caspase-2 and -9 suffice to provoke full-blown apoptosis upon microinjection into cells. Altogether, these 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 redistribution and activation.
Key Words: Bcl-2 • caspase • mitochondria • permeability transition • programmed cell death
S.A. Susin and H.K. Lorenzo contributed equally to this work.
Abbreviations used: afc, 7-amino-4-trifluoromethyl coumarin; AIF, apoptosis-inducing factor; ATA, aurinetricarboxylic acid; Atr, atractyloside; BA, bongkrekic acid; CAD, caspase-activated DNAse; CsA, cyclosporin A; DEX, dexamethasone; 
m, mitochondrial transmembrane potential; DFF, DNA fragmentation factor; fmk, fluoromethylketone; PT, permeability transition; PTPC, PT pore complex; RT, room temperature; Z-, N-benzyloxycarbonyl.
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