Caspase activation involves the formation of the aposome, a large (similarto 700 kDa) caspase-activating complex

In mammals, apoptotic protease-activating factor 1 (Apaf-1), cytochrome c, and dATP activate caspase-9, which initiates the postmitochondrial-mediated caspase cascade by proteolytic cleavage/activation of effector caspases to form active similar to 60-kDa heterotetramers. We now demonstrate that act ivation of caspases either in apoptotic cells or following dATP activation of cell lysates results in the formation of two large but different sized p rotein complexes, the "aposome" and the "microaposome". Surprisingly, most of the DEVDase activity in the lysate was present in the aposome and microa posome complexes with only small amounts of active caspase-3 present as its free similar to 60-kDa heterotetramer. The larger aposome complex (M-r = s imilar to 700,000) contained Apaf-1 and processed caspase-9, -3, and -7. Th e smaller microaposome complex (M-r = similar to 200,000-300,000) contained active caspase-3 and -7 but little if any Apaf-1 or active caspase-9. Lysa tes isolated from control THP.1 cells, prior to caspase activation, showed striking differences in the distribution of key apoptotic proteins. Apaf-1 and procaspase-7 may be functionally complexed as they eluted as an similar to 200-300-kDa complex, which did not have caspase cleavage (DEVDase) acti vity. Procaspase-3 and -9 were present as separate and smaller 60-90-kDa (d imer) complexes. During caspase activation, Apaf-1, caspase-9, and the effe ctor caspases redistributed and formed the aposome. This resulted in the pr ocessing of the effector caspases, which were then released, possibly bound to other proteins, to form the microaposome complex.