CYTOCHROME-C ACTIVATION OF CPP32-LIKE PROTEOLYSIS PLAYS A CRITICAL ROLE IN A XENOPUS CELL-FREE APOPTOSIS SYSTEM

In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apop totic activity by preventing cytochrome c release from mitochondria. W e now describe in detail the crucial role of cytochrome c in this syst em, The mitochondrial fraction, when incubated with cytosol, releases cytochrome c, Cytochrome c in turn induces the activation of protease( s) resembling caspase-3 (CPP32), leading to downstream apoptotic event s, including the cleavage of fodrin and lamin B-1, CPP32-like protease activity plays an essential role in this system, as the caspase inhib itor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B-1 cleavage, a s well as nuclear morphology changes, Cytochrome c preparations from v arious vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis, Cytochrome c by itself was u nable to process the precursor form of CPP32; the presence of cytosol was required, The electron transport activity of cytochrome c is not r equired for its pro-apoptotic function, as Cu-and Zn-substituted cytoc hrome c had strong pro-apoptotic activity, despite being redox-inactiv e, However, certain structural features of the molecule were required for this activity, Thus, in the Xenopus cell-free system, cytosol-depe ndent mitochondrial release of cytochrome c induces apoptosis by activ ating CPP32-like caspases, via unknown cytosolic factors.