An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9.
We report here the reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspase-9. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively. The hydrolysis of ATP/dATP and the binding of cytochrome c promote APAF-1 oligomerization, forming a large multimeric APAF-1.cytochrome c complex. Such a complex can be isolated using gel filtration chromatography and is by itself sufficient to recruit and activate procaspase-9. The stoichiometric ratio of procaspase-9 to APAF-1 is approximately 1 to 1 in the complex. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3.
Zou
H
H
Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
Li
Y
Y
Liu
X
X
Wang
X
X
D000255:Adenosine Triphosphate / Q000378:metabolism; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D001483:Base Sequence; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D003574:Cytochrome c Group / Q000502:physiology*; D017931:DNA Primers; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D015388:Organelles / Q000502:physiology*; D011485:Protein Binding; D011506:Proteins / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D017931:DNA Primers; D004792:Enzyme Precursors; D011506:Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases