Pro-Apoptotic Apoptosis Protease–Activating Factor 1 (Apaf-1) Has a Cytoplasmic Localization Distinct from Bcl-2 or Bcl-XL

Abstract

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 caspase 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 its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-xL were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-xL precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-xL could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering 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.

Modifications and intracellular trafficking of FADD/MORT1 and caspase-8 after stimulation of T lymphocytes

“…37 When subcellular fractions of Jurkat (nonadherent) cells were analysed by immunoblotting with commercial antibodies, FADD was found exclusively in the cytosolic fraction (Figure 4a). The purity of the fractions was verified by probing blots with antibodies to cytoplasmic proteins, such as the Golgi complex resident protein b-COP (cytoplasmic fraction Figure 4a) and Apaf-1 38 (cytoplasmic fraction Figure 4b), the nuclear protein PARP (nuclear containing fraction Figure 4b) and caspase-2, found in both compartments (Figure 4a, b) as reported previously. 39 Further fractionation of the cytoplasmic fraction from the adherent cell line HeLa to generate light membrane (LM) and soluble fractions (S) showed that FADD was present in the soluble fraction similar to Apaf-1 and a small amount of FADD in the light membrane fraction but none in the HM fraction where the nuclei are found (Figure 4b).…”

Living with death: the evolution of the mitochondrial pathway of apoptosis in animals

“…Furthermore, despite early reports to the contrary 20,21 it is now accepted that mammalian Bcl-2 homologs do not functionally interact with APAF-1. [22][23][24] Given these significant differences, it is intriguing that the antiapoptotic functions of both Bcl-2 and CED-9 involve the mitochondria. Indeed, the functions of the mammalian Bcl-2 family members and those of CED-9/EGL-1 overlap in a number of interesting ways: despite sharing only around 22% homology with CED-9, expression of Bcl-2 inhibits apoptosis in C.elegans, 25 and Bcl-2 expression was found to rescue the excess of apoptosis found in CED-9 loss-of-function worms.…”

Bcl‐2 family proteins: The sentinels of the mitochondrial apoptosis pathway

“…The alternative view is based on the discovery that prosurvival C. elegans CED-9 prevents CED-3 caspase activation by blocking its adapter CED-4. Since Bcl-2 and its prosurvival relatives do not bind to Apaf-1 (mammalian homologue for CED-4) (17,18), mammalian Bcl-2-like molecules would have to interact with other adapters that activate ''initiator caspases'' other than caspase-9 (mammalian homologue for CED-3). Support for this idea comes from the observation that Bcl-2 over-expression inhibits apoptosis of haematopoietic cells in mice more potently than loss of Apaf-1 or caspase-9 (19).…”