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 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.
Hausmann
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G
The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Parkville, Victoria 3050, Australia.
O'Reilly
L A
LA
van Driel
R
R
Beaumont
J G
JG
Strasser
A
A
Adams
J M
JM
Huang
D C
DC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D003574:Cytochrome c Group / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism* / Q000648:ultrastructure; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006801:Humans; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D011506:Proteins / Q000276:immunology / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051020:bcl-X Protein
C506592:APAF1 protein, human; D000911:Antibodies, Monoclonal; D053477:Apoptotic Protease-Activating Factor 1; C494811:BCL2L1 protein, human; D003574:Cytochrome c Group; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D020169:Caspases