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In a cell-free apoptosis system, mitochondria spontaneously released cytochrome c, which activated DEVD-specific caspases, leading to fodrin cleavage and apoptotic nuclear morphology. Bcl-2 acted in situ on mitochondria to prevent the release of cytochrome c and thus caspase activation. During apoptosis in intact cells, cytochrome c translocation was similarly blocked by Bcl-2 but not by a caspase inhibitor, zVAD-fmk. In vitro, exogenous cytochrome c bypassed the inhibitory effect of Bcl-2. Cytochrome c release was unaccompanied by changes in mitochondrial membrane potential. Thus, Bcl-2 acts to inhibit cytochrome c translocation, thereby blocking caspase activation and the apoptotic process.
Bcl-2 and its relatives (for example, Bcl-x, E1B 19K, and CED-9) are potent inhibitors of apoptotic cell death (1-3). Bcl-2 is located predominantly in the outer mitochondrial membrane, the endoplasmic reticulum, and the nuclear membrane (4-8), and it appears to prevent apoptosis at a point in the process upstream of the activation of CED-3 family proteases such as caspase-3 (CPP32) (9-12). How Bcl-2 prevents protease activation is not known.
We used a cell-free system based on Xenopus egg extracts in which recombinant Bcl-2 prevents protease activation and subsequent apoptotic effects (13-16). This system is similar to other cell-free systems based on cytosol derived from apoptotic cultured cells (14, 17-20), but with one important exception: the spontaneous apoptosis in the Xenopus system depends on the presence of a heavy membrane fraction enriched in mitochondria (13). These organelles, when incubated in Xenopus egg cytosol, generate a soluble proteinaceous factor that can induce apoptosis rapidly in mitochondria-free extracts (16). Cytochrome c was recently identified as such a factor-it is released from mitochondria in apoptotic cells and can trigger activation of DEVD-specific caspases (DEVDases) and apoptotic effects in cell-free systems containing cytosol (16, Zl ).
We therefore examined the effect of exogenous Bcl-2 on cytochrome c release. Incubation of Xenopus egg mitochondria in the cell-free system resulted in the timeand temperature-dependent release of cytochrome c into the soluble fraction (Fig. 1, A and C). This release, which required the presence of cytosol (16), occurred after a lag period (-3 hours) corresponding to the time when DEVD-specific protease activity first appeared, which would be expected if cytochrome c is required for protease activation. About 1 hour later, apoptotic figures began to be...