Bcl-2 inhibits the release of an apoptosis-inducing factor (AIF) from mitochondria, thereby preventing apoptosis. AIF is a pre-formed, 50-kD protein located in the intermembrane space of mitochondria that, when released, induces nuclear apoptosis by causing chromatin condensation and DNA fragmentation. Bcl-2, a member of the Bcl-2 family of apoptosis-regulatory proteins, prevents the release of AIF by inhibiting mitochondrial permeability transition (PT), a process that leads to the collapse of the mitochondrial inner membrane potential (ΔΨm). However, Bcl-2 does not affect the formation of AIF or its action on the nucleus. AIF is inhibited by Z-VAD.fmk, an inhibitor of ICE-like proteases, suggesting a direct link between PT and protease activation during apoptosis. Bcl-2 hyperexpression in the outer mitochondrial membrane prevents AIF release but does not neutralize its apoptotic activity. This indicates that Bcl-2 prevents apoptosis by retaining AIF within mitochondria. The study also shows that AIF is a protease that activates pre-formed nuclear DNases, but does not cleave PARP or lamin in isolated nuclei. Bcl-2 does not interfere with AIF's action on nuclei or cells, suggesting that it acts upstream of AIF release. The findings suggest that Bcl-2 regulates apoptosis by controlling the release of AIF, which is a key factor in the apoptotic cascade. The study provides insights into the regulation of apoptosis by Bcl-2 and the role of AIF in the apoptotic process.Bcl-2 inhibits the release of an apoptosis-inducing factor (AIF) from mitochondria, thereby preventing apoptosis. AIF is a pre-formed, 50-kD protein located in the intermembrane space of mitochondria that, when released, induces nuclear apoptosis by causing chromatin condensation and DNA fragmentation. Bcl-2, a member of the Bcl-2 family of apoptosis-regulatory proteins, prevents the release of AIF by inhibiting mitochondrial permeability transition (PT), a process that leads to the collapse of the mitochondrial inner membrane potential (ΔΨm). However, Bcl-2 does not affect the formation of AIF or its action on the nucleus. AIF is inhibited by Z-VAD.fmk, an inhibitor of ICE-like proteases, suggesting a direct link between PT and protease activation during apoptosis. Bcl-2 hyperexpression in the outer mitochondrial membrane prevents AIF release but does not neutralize its apoptotic activity. This indicates that Bcl-2 prevents apoptosis by retaining AIF within mitochondria. The study also shows that AIF is a protease that activates pre-formed nuclear DNases, but does not cleave PARP or lamin in isolated nuclei. Bcl-2 does not interfere with AIF's action on nuclei or cells, suggesting that it acts upstream of AIF release. The findings suggest that Bcl-2 regulates apoptosis by controlling the release of AIF, which is a key factor in the apoptotic cascade. The study provides insights into the regulation of apoptosis by Bcl-2 and the role of AIF in the apoptotic process.