Failure of Bcl-2 family members to interact with Apaf-1 in normal and apoptotic cells

CED-9 blacks 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-x(L) 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-x(L) 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-x(L) 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-x(L) 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-x(L) in immunoprecipitates. Based on these data, we propose that Apaf-1 is not a direct, physiological target of Bcl-2, Bcl-x(L) or Bax.