Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis.

SummaryIn a number of experimental systems, the early stage of the apoptotic process, i.e., the stage that precedes nuclear disintegration, is characterized by the breakdown of the inner mitochondrial transmembrane potential (Aqtm). This A~ m disruption is mediated by the opening of permeability transition (PT) pores and appears to be critical for the apoptotic cascade, since it is directly regulated by Bcl-2 and since mitochondria induced to undergo PT in vitro become capable of inducing nuclear chromatinolysis in a cell-free system ofapoptosis. Here, we addressed the question of which apoptotic events are secondary to mitochondrial PT. We tested the effect of a specific inhibitor of PT, bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, on a prototypic model of apoptosis: glucocorticoid-induced thymocyte death. In addition to abolishing the apoptotic A~,n disruption, BA prevents a number of phenomena linked to apoptosis: depletion ofnonoxidized glutathione, generation of reactive oxygen species, translocation of NFKB, exposure of phosphatidylserine residues on the outer plasma membrane, cytoplasmic vacuolization, chromatin condensation, and oligonucleosomal DNA fragmentation. BA is also an efficient inhibitor of p53-dependent thymocyte apoptosis induced by DNA damage. These data suggest that a number of apoptotic phenomena are secondary to PT. In addition, we present data indicating that apoptotic Aqr,, disruption is secondary to transcriptional events. These data connect the PT control point to the p53-and ICE/ Ced 3-regulated control points of apoptosis and place PT upstream of nuclear and plasma membrane features of PCD.

Although much emphasis has been laid on the role of caspase in cell death, recent data indicate that, in many instances, mammalian cell death is caspase-independent. Thus, in many examples of mammalian cell death the`decision' between death and life is upstream or independent of caspase activation. Similarly, it is unclear whether PCD of plants and fungi involves the activation of caspase-like enzymes, and no caspase-like gene has thus far been cloned in these phyla. Apoptosis inducing factor (AIF) is a new mammalian, caspaseindependent death effector which, upon apoptosis induction, translocates from its normal localization, the mitochondrial intermembrane space, to the nucleus. Once in the nucleus, AIF causes chromatin condensation and large scale DNA fragmentation to fragments of *50 kbp. The AIF cDNA from mouse and man codes for a protein which possesses three domains (i) an amino-terminal presequence which is removed upon import into the intermembrane space of mitochondria; (ii) a spacer sequence of approximately 27 amino acids; and (iii) a carboxyterminal 484 amino acid oxidoreductase domain with strong homology to oxidoreductases from other vertebrates (X. laevis), non-vertebrate animals (C.elegans, D. melanogaster), plants, fungi, eubacteria, and archaebacteria. Functionally important amino acids involved in the interaction with the prosthetic groups flavin adenine nucleotide and nicotinamide adenine nucleotide are strongly conserved between AIF and bacterial oxidoreductase. Several eukaryotes possess a similar domain organisation in their AIF homologs, making them candidates to be mitochondrial oxidoreductases as well as caspase-independent death effectors. The phylogenetic implications of these findings are discussed.Keywords: mitochondria; cytochrome c Abbreviations: AIF, apoptosis inducing factor; ANT, adenine nucleotide translocator; FAD,¯avin adenine dinucleotide; NAD, nicotinamide adenine nucleotide Phylogeny of apoptosis in the three metazoan kingdomsProgrammed cell death (PCD) is known to play a major role in the development and/or stress responses of all three metazoan kingdoms (Plantae, Animalia, and Fungi). Two opposite scenarios may account for the evolution of PCD. As a first possibility, PCD would have evolved during the metozoan radiation in each major evolutionary brench independently. In this case, shared characteristics of PCD across phyla such as partial chromatin fragmentation and condensation, increased generation of reactive oxygen species, or loss of plasma membrane asymmetry 1 would be understood as post mortem manifestations of entropic processes. As a second possibility, PCD would have evolved in a primitive, unicellular eukaryotic ancestor, before the separation of the three metozoan kingdoms. We prefer this latter hypothesis, because some unicellular eukaryotes (Trypanosoma cruzi, Trypanasoma brucei rhodesiense, Leishmania amazonensis, Tetrahymena thermophyla, Euglena gracilis, Schizosaccharomyces pombe) can undergo cell death with some features of PCD/apoptosis such ...