Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation.
To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we produced 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 monomer that can be transformed to an oligomeric complex made of at least eight subunits after binding to cytochrome c and dATP. Apaf-1 binds to cytochrome c in the absence of dATP but does not form the oligomeric complex. However, when dATP is added to the cytochrome c-bound Apaf-1 complex, complete oligomerization occurs, suggesting that oligomerization is driven by hydrolysis of dATP. This was supported by the observation that ATP, but not the nonhydrolyzable adenosine 5'-O-(thiotriphosphate), can induce oligomerization of the Apaf-1-cytochrome c complex. Like the spontaneously oligomerizing Apaf-530, which lacks its WD-40 domain, the oligomeric full-length Apaf-1-cytochrome c complex can bind and process procaspase-9 in the absence of additional dATP or cytochrome c. However, unlike the truncated Apaf-530 complex, the full-length Apaf-1 complex can release the mature caspase-9 after processing. Once released, mature caspase-9 can process procaspase-3, setting into motion the caspase cascade. These observations indicate that cytochrome c and dATP are required for oligomerization of Apaf-1 and suggest that the WD-40 domain plays an important role in oligomerization of full-length Apaf-1 and the release of mature caspase-9 from the Apaf-1 oligomeric complex.
Saleh
A
A
Center for Apoptosis Research and the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Srinivasula
S M
SM
Acharya
S
S
Fishel
R
R
Alnemri
E S
ES
D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000378:metabolism
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D003838:Deoxyadenine Nucleotides; D004792:Enzyme Precursors; D011506:Proteins; D011994:Recombinant Proteins; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; C026600:2'-deoxyadenosine triphosphate