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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 B1. CPP32‐like protease activity plays an essential role in this system, as the caspase inhibitor, Ac‐DEVD‐CHO, strongly inhibited fodrin and lamin B1 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.