Failure of Bcl-2 family members to interact with Apaf-1 in normal and apoptotic cells.
CED-9 blocks programmed cell death (apoptosis) in the nematode C. elegans by binding to and neutralizing CED-4, an essential activator of the aspartate-directed cysteine protease (caspase) CED-3. In mammals, the CED-9 homologs Bcl-2 and Bcl-xL also block apoptosis by interfering with the activation of CED-3-like caspases. However, it is unknown whether this occurs by binding to the CED-4 homolog Apaf-1. Whilst two groups previously detected an interaction between Bcl-xL and Apaf-1 in immunoprecipitates,1,2 another group found no interaction between Apaf-1 and any of ten individual members of the Bcl-2 family using the same experimental approach.3 In this study, we aimed to resolve this discrepancy by monitoring the binding of Apaf-1 to three Bcl-2 family members within cells. Using immunofluorescence and Western blot analysis, we show that whilst Apaf-1 is a predominantly cytoplasmic protein, Bcl-2, Bcl-xL and Bax mostly reside on nuclear/ER and mitochondrial membranes. This pattern of localization is maintained when the proteins are co-expressed in both normal and apoptotic cells, suggesting that Bcl-2, Bcl-xL or Bax do not significantly sequester cytoplasmic Apaf-1 to intracellular membranes. In addition, we confirm that Apaf-1 does not interact with Bcl-2 and Bcl-xL in immunoprecipitates. Based on these data, we propose that Apaf-1 is not a direct, physiological target of Bcl-2, Bcl-xL or Bax.
Conus
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S
Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland.
Rossé
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T
Borner
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C
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D002478:Cells, Cultured; D006801:Humans; D007668:Kidney / Q000166:cytology; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014162:Transfection
C506592:APAF1 protein, human; C506594:Apaf1 protein, rat; D053477:Apoptotic Protease-Activating Factor 1; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2