Pro-apoptotic proteins released from the mitochondria regulate the protein composition and caspase-processing activity of the native Apaf-1/caspase-9 apoptosome complex

Davina Twiddy, David G Brown, Colin Adrain, Rebekah Jukes, Seamus J Martin, Gerald M Cohen, Marion MacFarlane, Kelvin Cain

Research output: Contribution to journalArticlepeer-review

93 Citations (Scopus)


The apoptosome is a large caspase-activating ( approximately 700-1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is approximately 135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp9(1-130)) containing the CARD domain of caspase-9-(1-130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp9(1-130), demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified apoptosome complex increased 6-8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.

Original languageEnglish
Pages (from-to)19665-82
Number of pages18
JournalThe Journal of biological chemistry
Issue number19
Publication statusPublished - 07 May 2004


  • Amino Acid Sequence
  • Apoptosis
  • Apoptotic Protease-Activating Factor 1
  • Blotting, Western
  • Caspase 3
  • Caspase 9
  • Caspases/metabolism
  • Cell Line
  • Centrifugation, Density Gradient
  • Cytochromes c/metabolism
  • Cytosol/metabolism
  • Dose-Response Relationship, Drug
  • Electrophoresis, Gel, Two-Dimensional
  • Glutathione Transferase/metabolism
  • Humans
  • Mitochondria/metabolism
  • Molecular Sequence Data
  • Peptides/chemistry
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Proteins/metabolism
  • Proteome
  • Recombinant Fusion Proteins/metabolism
  • Recombinant Proteins/chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Time Factors


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