title: CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13.
abstract: The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-of-function CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. fairlie@wehi.edu.au
authors.forename: W D
authors.initials: WD
authors.lastname: Fairlie
authors.forename: M A
authors.initials: MA
authors.lastname: Perugini
authors.forename: M
authors.initials: M
authors.lastname: Kvansakul
authors.forename: L
authors.initials: L
authors.lastname: Chen
authors.forename: D C S
authors.initials: DC
authors.lastname: Huang
authors.forename: P M
authors.initials: PM
authors.lastname: Colman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D051017:Apoptosis Regulatory Proteins; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; C504038:Ced-13 protein, C elegans; C089224:Ced-4 protein, C elegans; C087233:Ced-9 protein, C elegans; C112486:EGL-1 protein, C elegans; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins