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 apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. 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 events, including the cleavage of fodrin and lamin B 1 . CPP32‐like protease activity plays an essential role in this system, as the caspase inhibitor, Ac‐DEVD‐CHO, strongly inhibited fodrin and lamin B 1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae , were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro‐apoptotic function, as Cu‐ and Zn‐substituted cytochrome c had strong pro‐apoptotic activity, despite being redox‐inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell‐free system, cytosol‐dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32‐like caspases, via unknown cytosolic factors.