Pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1) has a cytoplasmic localization distinct from Bcl-2 or Bcl-x(L)

How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the casp ase activator apoptosis protease-activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2-like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model , we generated monoclonal antibodies to Apaf-1 and used them to determine i ts localization within diverse cells by subcellular fractionation and confo cal laser scanning microscopy. Whereas Bcl-2 and Bcl-x(L) were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocaliz e with them, even when these pro-survival proteins were overexpressed or af ter apoptosis was induced. Immunogold electron microscopy confirmed that Ap af-1 was dispersed in the cytoplasm and not on mitochondria or other organe lles. After the death stimuli, Bcl-2 and Bcl-x(L) precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of lar ger Apaf-1 complexes, which are steps that presage apoptosis. However, neit her Bcl-2 nor Bcl-x, could prevent the in vitro activation of Apaf-1 induce d by the addition of exogenous cytochrome c. Hence, rather than sequesterin g Apaf-1 as proposed by the apoptosome model, Bcl-2-like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.