Original Article

Pro-apoptotic Apoptosis Protease–activating Factor 1 (Apaf-1) Has a Cytoplasmic Localization Distinct from Bcl-2 or Bcl-x_L

Correspondence to: David C.S. Huang, The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Parkville, Victoria 3050, Australia. Tel:61-3-9345-2555 Fax:61-3-9347-0852 E-mail:huang_d{at}wehi.edu.au.

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-x_L 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-x_L 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-x_L 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.

Key Words: caspases, cell death, Bcl-2, mitochondria, subcellular localization

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