Mitochondria play a key part in the regulation of apoptosis (cell death). Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in the degradation phase of apoptosis. Here we report the identification and cloning of an apoptosis-inducing factor, AIF, which is sufficient to induce apoptosis of isolated nuclei. AIF is a flavoprotein of relative molecular mass 57,000 which shares homology with the bacterial oxidoreductases; it is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced. Recombinant AIF causes chromatin condensation in isolated nuclei and large-scale fragmentation of DNA. It induces purified mitochondria to release the apoptogenic proteins cytochrome c and caspase-9. Microinjection of AIF into the cytoplasm of intact cells induces condensation of chromatin, dissipation of the mitochondrial transmembrane potential, and exposure of phosphatidylserine in the plasma membrane. None of these effects is prevented by the wide-ranging caspase inhibitor known as Z-VAD.fmk. Overexpression of Bcl-2, which controls the opening of mitochondrial permeability transition pores, prevents the release of AIF from the mitochondrion but does not affect its apoptogenic activity. These results indicate that AIF is a mitochondrial effector of apoptotic cell death.

The proapoptotic Bax protein induces cell death by acting on mitochondria. Bax binds to the permeability transition pore complex (PTPC), a composite proteaceous channel that is involved in the regulation of mitochondrial membrane permeability. Immunodepletion of Bax from PTPC or purification of PTPC from Bax-deficient mice yielded a PTPC that could not permeabilize membranes in response to atractyloside, a proapoptotic ligand of the adenine nucleotide translocator (ANT). Bax and ANT coimmunoprecipitated and interacted in the yeast two-hybrid system. Ectopic expression of Bax induced cell death in wild-type but not in ANT-deficient yeast. Recombinant Bax and purified ANT, but neither of them alone, efficiently formed atractyloside-responsive channels in artificial membranes. Hence, the proapoptotic molecule Bax and the constitutive mitochondrial protein ANT cooperate within the PTPC to increase mitochondrial membrane permeability and to trigger cell death.

Early in programmed cell death (apoptosis), mitochondrial membrane permeability increases. This is at least in part due to opening of the permeability transition (PT) pore, a multiprotein complex built up at the contact site between the inner and the outer mitochondrial membranes. The PT pore has been previously implicated in clinically relevant massive cell death induced by toxins, anoxia, reactive oxygen species, and calcium overload. Here we show that PT pore complexes reconstituted in liposomes exhibit a functional behavior comparable with that of the natural PT pore present in intact mitochondria. The PT pore complex is regulated by thiol-reactive agents, calcium, cyclophilin D ligands (cyclosporin A and a nonimmunosuppressive cyclosporin A derivative), ligands of the adenine nucleotide translocator, apoptosis-related endoproteases (caspases), and Bcl-2–like proteins. Although calcium, prooxidants, and several recombinant caspases (caspases 1, 2, 3, 4, and 6) enhance the permeability of PT pore-containing liposomes, recombinant Bcl-2 or Bcl-XL augment the resistance of the reconstituted PT pore complex to pore opening. Mutated Bcl-2 proteins that have lost their cytoprotective potential also lose their PT modulatory capacity. In conclusion, the PT pore complex may constitute a crossroad of apoptosis regulation by caspases and members of the Bcl-2 family.