Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisitefor procaspase-9 activation

To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we prod uced recombinant full-length Apaf-1 and purified it to complete homogeneity , Here we show using gel filtration that full-length Apaf-1 exists as a mon omer that can be transformed to an oligomeric complex made of at least eigh t subunits after binding to cytochrome c and dATP, Apaf-1 binds to cytochro me c in the absence of dATP but does not form the oligomeric complex. Howev er, when dATP is added to the cytochrome c-bound Apaf-1 complex, complete o ligomerization occurs, suggesting that oligomerization is driven by hydroly sis of dATP, This was supported by the observation that ATP, but not the no nhydrolyzable adenosine 5'-O-(thiotriphosphate), can induce oligomerization of the Apaf-1-cytochrome c complex. Like the spontaneously oligomerizing A paf-530, which lacks its WD-40 domain, the oligomeric full-length Apaf-1-cy tochrome c complex can bind and process procaspase-9 in the absence of addi tional dATP or cytochrome c, However, unlike the truncated Apaf-530 compile r, the full-length Apaf-1 complex can release the mature caspase-9 after pr ocessing. Once released, mature caspase-9 can process procaspase-3, setting into motion the caspase cascade. These observations indicate that cytochro me c and dATP are required for oligomerization of Apaf-1 and suggest that t he WD-40 domain plays an important role in oligomerization of full-length A paf-1 and the release of mature caspase-9 from the Apaf-1 oligomeric comple x.