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.
Kluck
R M
RM
Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, USA.
Martin
S J
SJ
Hoffman
B M
BM
Zhou
J S
JS
Green
D R
DR
Newmeyer
D D
DD
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases; D002474:Cell-Free System; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D066298:In Vitro Techniques; D034921:Lamin Type B; D034882:Lamins; D008840:Microfilament Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010063:Ovum / Q000378:metabolism; D010084:Oxidation-Reduction; D013049:Spectrin
D002352:Carrier Proteins; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D034921:Lamin Type B; D034882:Lamins; D008840:Microfilament Proteins; D009687:Nuclear Proteins; D013049:Spectrin