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© The Rockefeller University Press, 0021-9525/2000//623 $5.00
The Journal of Cell Biology, Volume 149, Number 3, , 2000 623-634

Original Article

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



George Hausmanna, Lorraine A. O'Reillya, Rosemary van Drielb, Jennifer G. Beaumonta, Andreas Strassera, Jerry M. Adamsa, and David C.S. Huanga

a The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
b The Baker Medical Research Institute, Melbourne, Victoria 8008, Australia
The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Parkville, Victoria 3050, Australia.61-3-9347-085261-3-9345-2555

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-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.

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

© 2000 The Rockefeller University Press

Abbreviations used in this paper: Apaf, apoptosis protease–activating factor; CARD, caspase recruitment domain; CED, cell death abnormal; GFP, green fluorescent protein; HA, hemagglutinin.


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