Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor

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1 Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor MARKUS LOEFFLER,* ERIC DAUGAS,*, SANTOS A. SUSIN,* NAOUFAL ZAMZAMI 1, DIDIER MÉTIVIER,* ANNA-LIISA NIEMINEN, GREG BROTHERS, JOSEF M. PENNINGER, AND GUIDO KROEMER*,1 *Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, F Villejuif, France; Assistance Publique-Hôpitaux de Paris, Service de Néphrologie B, Hôpital Tenon, F-75020, France; Case Western Reserve University, Department of Anatomy, School of Medicine, Cleveland, Ohio 44106, USA; and The Amgen Institute and Ontario Cancer Institute, Department of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario M5G 2C1, Canada ABSTRACT The complete AIF cdna comprising the amino-terminal mitochondrial localization sequence (MLS) and the oxidoreductase domain has been fused in its carboxyl terminus to enhanced green fluorescent protein (GFP), thereby engineering an AIF-GFP fusion protein that is selectively targeted to the mitochondrial intermembrane space. Upon induction of apoptosis, the AIF-GFP protein translocates together with cytochrome c (Cyt-c) to the extramitochondrial compartment. Microinjection of recombinant AIF leads to the release of AIF-GFP and Cyt-c-GFP, indicating that ectopic AIF can favor permeabilization of the outer mitochondrial membrane. These mitochondrial effects of AIF are caspase independent, whereas the Cyt-cmicroinjection induced translocation of AIF-GFP and Cyt-c-GFP is suppressed by the pan-caspase inhibitor Z-VAD.fmk. Upon prolonged culture, transfection-enforced overexpression of AIF results in spontaneous translocation of AIF-GFP from mitochondria, nuclear chromatin condensation, and cell death. These effects are caspase independent and do not rely on the oxidoreductase function of AIF. Spontaneous AIF-GFP translocation and subsequent nuclear apoptosis can be retarded by overexpression of a Bcl-2 protein selectively targeted to mitochondria, but not by a Bcl-2 protein targeted to the endoplasmic reticulum. Overexpression of a mutant AIF protein in which the MLS has been deleted (AIF 1 100) results in the primary cytosolic accumulation of AIF. AIF induced cell death is suppressed by neither Z-VAD.fmk or by Bcl-2. Thus, extramitochondrially targeted AIF is a dominant cell death inducer. Loeffler, M., Daugas, E., Susin, S. A., Zamzami, N., Métivier, D., Nieminen, A.-L., Brothers, G., Penninger, J. M., Kroemer, G. Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J. 15, (2001) Key Words: AIF apoptosis Bcl-2 caspases cytochrome c vitro, causes chromatin condensation and large-scale ( 50 kbp) DNA fragmentation. Under normal circumstances, transcription and translation of the nuclear AIF gene give rise to a 67 kda precursor molecule that carries a putative mitochondrial localization sequence (MLS) in its NH 2 terminus. Upon import into the mitochondrial intermembrane space, this 100 amino acid presequence is cleaved by a local peptidase, leading to generation of the mature AIF molecule ( 57 kda, AIF 1 100), which is confined to the mitochondrial intermembrane space (1 3). AIF is thus kept in check by rigorous compartmentalization, preventing its interaction with extramitochondrial targets (3 5). Upon induction of apoptosis, mitochondria release AIF and other soluble proteins from the intermembrane space, including cytochrome c (Cyt-c) (6 11). Cyt-c can interact with nonmitochondrial proteins, including Apaf-1, thereby provoking its oligomerization and/or a change in conformation that converts the Apaf-1/Cyt-c complex into a caspase-9 activator (12). In contrast to Cyt-c (which induces nuclear apoptosis via a cascade involving caspase-9, caspase-3, and caspaseactivated DNase, CAD) (12), AIF-induced changes are caspase independent (1 3). AIF possesses several nuclear localization sequences, allowing for its nuclear import. Inhibition of nuclear import prevents the local effects of AIF (1). One major difference between AIF-induced nuclear apoptosis and Cyt-c/ caspase/cad-induced nuclear apoptosis resides in the morphology of chromatin condensation. AIF causes a rather partial condensation without shrinkage ( stage I ) (1), whereas CAD can induce a more advanced condensation pattern (stage II) without (stage IIa) or with (stage IIb) formation of nuclear apoptotic bodies (13 15). Another difference resides in the degree of chromatin degradation induced by AIF and CAD. AIF triggers (caspase-independent) large-scale ( 50 kbp) DNA fragmentation (1, 2), whereas CAD causes a Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, when added to purified nuclei in 1 Correspondence: CNRS-UMR 1599, Institut Gustave Roussy, Pavillon de Recherche I, 39, rue Camille-Desmoulins, F Villejuif, France. kroemer@igr.fr /01/ FASEB

2 classical mono- or oligonucleosomal pattern of DNA fragmentation to multiples of 200 bp (12, 14, 15). It is conceivable that AIF and the Cyt-c/caspase/CAD pathways act in a complementary fashion to provide several molecular links between mitochondrial membrane permeabilization and nuclear apoptosis. The mitochondrial release of both AIF and Cyt-c is at least in part under the common control of members of the Bcl-2 family. Thus, the anti-apoptotic protein Bcl-2, which localizes to intracellular membranes including those of mitochondria, inhibits the translocation of both AIF and Cyt-c (3, 7, 16, 17). To further investigate the putative contribution of AIF to apoptosis, we generated AIF constructs fused to the green fluorescent protein (GFP), thus allowing for the continuous monitoring of the subcellular localization of AIF. As shown here, AIF constructs targeted to the mitochondrial intermembrane space rapidly redistribute to an extramitochondrial localization upon apoptosis induction. Transfection-enforced overexpression of such constructs can by itself induce apoptosis, via a process that involves spontaneous translocation of the proteins from mitochondria and is retarded by mitochondrion-targeted Bcl-2. A truncated AIF construct lacking the MLS exhibits a nonmitochondrial pattern of distribution and triggers caspase-independent apoptosis that is not inhibited by Bcl-2. Thus, extramitochondrially targeted AIF is a dominant cell death inducer. and 10% fetal calf serum. Cells were cultured in the presence of staurosporine (STS; 1 M; Sigma, St. Louis, Mo.), etoposide (20 M), ganglioside GD3 (200 M), doxorubicin (20 M), and/or Z-VAD.fmk (100 M; 30 min before STS) as indicated. If necessary, Z-VAD.fmk was added periodically in 24 h intervals. Apoptosis induction was monitored by incubation with the DNA-specific dye Hoechst (2 M). Nuclei with rippled contours and partial chromatin condensation were considered to represent stage I of chromatin condensation, nuclei with marked peripheral chromatin condensation as stage IIa, and cells with nuclear bodies as stage IIb. Transient transfection protocol For transient transfections, 10 7 COS or Rat-1 cells were trypsinized, centrifuged (300 g, 5 min), resuspended in 0.2 ml of complete medium, and mixed with 5 g of plasmid DNA. After 10 min of incubation on ice in a 0.4 cm cuvette (Bio-Rad, Hercules, Calif.), electroporation was performed at 960 F and 220V for ms (GenePulser II, Bio-Rad). Cells were immediately diluted with 10 ml of complete medium. For fluorescence microscopy, 10 5 cells/well were cultured on glass coverslips (100 m, 18 mm Ø, Polylabo, Strasbourg, France) in 12-well plates (Polylabo) and washed MATERIALS AND METHODS Cloning of AIF cdnas and construction of vectors AIF cdnas were cloned by a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid sequencing, using primers specific for the 5 UT and 3 UT of the published mouse AIF sequence (1). To generate the vector pcdna3.1 GFP, pcdna3.1 (Invitrogen, Carlsbad, Calif.) was cut and ligated with the KpnI/NotI fragment of pegfp (Clontech, Palo Alto, Calif.). PCR fragments coding for the different AIF sequences were generated using pcdna3.1 AIF or pcdna3.1 AIF exb as a template (1) and the following primers for the NH 2 terminus of the protein: 5 -ggg gta ccc cga aat gtt ccg gtg tg-3 (for full-length AIF), 5 -ggg gta ccc cat gtt agg act gtc-3 (for the AIF deletion mutant), and for the carboxyl terminus of all AIF constructs: 5 -ggg gta ccc ctt cat gaa tgt tga aga g-3.the introduced KpnI-sites were then used to ligate AIF into the KpnI-cut pcdna3.1 GFP. The Cyt-c-GFP construct has been described (18). PCR-based, site-directed mutagenesis of the AIF cdna was followed by verification of the entire AIF cdna sequence. Cell lines and apoptosis induction COS or Rat-1 cells stably transfected with a control vector (CMV), with a human Bcl-2/Act A fusion protein specifically targeted to mitochondria or a human Bcl-2/Cb5 fusion protein specifically targeted to the endoplasmic reticulum (19) were cultured in Dulbecco s modified Eagle s medium supplemented with L-glutamine, antibiotics, 2 mm pyruvate, Figure 1. Structure of the AIF gene and its splice variants. A) Genomic organization of the human AIF gene. The exons of AIF are indicated as boxes. Numbers on top of each box indicate the number of nucleotides corresponding to each exon (starting from base number 14734, EMBL accession no. Z81370 for exons I, Iia, and IIb; starting from base number 74963, EMBL accession no. Z81364 for all other exons). Numbers between the boxes indicate the length of the introns. Intron IIb has not been sequenced entirely. B) Amino acid sequence comparison of human and mouse AIF exon II splice-variants. Dashes indicate amino acid identity between the two species within the same exon whereas lined boxes indicate amino acid identity or similarity between the different AIF variants. The accession codes of these cdnas are AF and AF for mouse and human AIF, respectively, AL for the human AIF exon IIb variant. INDUCTION OF CELL DEATH BY AIF 759

3 with complete medium 3 h after transfection to eliminate cell debris. Microinjection experiments For microinjection, COS cells were transiently transfected with either AIF-GFP or Cyt-c-GFP and cultured overnight on glass coverslips. The setup for the injection itself was as follows: 25 M recombinant horse Cyt-c (Sigma) or 7.5 M recombinant AIF protein (purified as described in ref 1) were injected in PBS for 0.2 s under a pressure of 150 hpa, using a Microinjector equipment (Eppendorf, Hamburg, Germany). In some experiments, cells were preincubated with 100 M Z-VAD.fmk (Bachem, Basel, Switzerland) before microinjection. Then cells were culture for 3 h in the presence or absence of Z-VAD.fmk and fixed for fluorescence microscopy, as described below. Immunostaining protocols A rabbit antiserum generated against a mixture of 3 peptides derived from the mouse AIF amino acid sequence (residues , , ; ELISA titer ) was used (diluted 1/1000) on paraformaldehyde- (4% w:v) and picric acid-fixed (0.19% v:v) cells cultured on 100 m coverslips, and revealed with a goat anti-rabbit IgG conjugated to phycoerythrin (PE) (Southern Biotechnology, Birmingham, Ala.). Control experiments performed with the preimmune antiserum or in the presence of an excess (100 M) of the three immunogenic peptides confirmed that all detectable fluorescence was specific (not shown). Cytochrome c was detected by means of the mab 6H2.B4 (PharMingen, San Diego, Calif.), revealed by a goat anti-mouse IgG PE conjugate (Southern Biotechnology). Conventional and confocal laser scanning microscopy Conventional examination of samples was performed in a Leitz Labolux S microscope equipped with standard filters for FITC/GFP, PE, and Hoechst 33342, as well as a Leica camera. Confocal microscopy was performed on a Leica TC-SP (Leica Microsystems, Heidelberg, Germany) equipped with an ArKr laser mounted on an inverted Leica DM IFBE microscope with an NA oil objective. A minimum of 200 cells were monitored for these parameters for each data point. Cell-free system of apoptosis Purified HeLa nuclei were incubated with recombinant AIF protein (final concentration 1 g/ml; ref 1), preincubated in the absence or presence of 1 mm diphenyleneiodonium for 30 min) in CFS buffer for 90 min (20), and nuclear apoptosis was quantitated by staining with DNA-intercalating propidium iodide, followed by cytofluorometric determination of DNA content (20). RESULTS AND DISCUSSION Cloning of an alternatively spliced AIF isoform and generation of mitochondrially and extramitochondrially targeted AIF-GFP constructs RT-PCR cloning using primers for the extreme amino and carboxyl termini of the AIF coding sequence yielded an AIF cdna species (AIF-exB) that differed from the original AIF sequence (1; Fig. 1). These results, which were originally obtained with the murine AIF gene, were confirmed for human AIF, yielding two very similar sequence variants, suggesting that these sequence variations were not due to a cloning artifact (Fig. 1B). Comparison of the AIF and AIF-exB sequences with the human AIF genome sequence revealed that this difference was due to alternative exon usage (Fig. 1A) affecting part of the putative 100 Figure 2. Subcellular distribution of different GFP-fusion proteins in transient transfection experiments. COS cells were transfected transiently as described in Materials and Methods. After 24 h of culture, cells were examined by fluorescence microscopy for expression of the GFP transgenes. Apoptosis was induced by culture with 1 M staurosporine (STS) for 4 h. In the upper panel, transfection with GFP alone yields a diffuse cytosolic pattern, both in untreated and in STS-treated cells. Staining with the chromatin-specific dye Hoechst (2 M, 15 min; blue fluorescence) reveals the normal morphology of nuclear chromatin, whereas CMXRos (100 nm, 15 min; red fluorescence) yields a typical mitochondrial pattern in nontreated cells. The middle panel displays a series of different GFP fusion proteins: AIF-GFP, AIF-exB-GFP, AIF GFP, and Cyt-c-GFP, respectively. With the exception of AIF GFP, that lacks the mitochondrial localization sequence, all the fusion proteins show a typical mitochondrial distribution. However, on STS treatment (lower panel) all GFP fusion proteins exhibit a diffuse distribution, indicating that they have translocated from mitochondria to an ectopic (extramitochondrial) localization. 760 Vol. 15 March 2001 The FASEB Journal LOEFFLER

4 tained with GFP alone (Fig. 2). These observations confirm the existence of an MLS in the NH 2 terminus of AIF and indicate that the alternative exon 2 usage does not affect the mitochondrial import of AIF. Chronological relationship between AIF and Cyt-c release in STS-induced apoptosis Figure 3. Mitochondrio-nuclear translocation of AIF induced by various inducers of apoptosis. 24 h after transient transfection, COS cells were treated with etoposide (Etopo, 20 M, 4 h), doxorubicine (Doxo, 20 M, 4 h) or ganglioside GD3 (200 M, 12 h) and counterstained with Hoechst to determine the percentage of cells with mitochondrio-nuclear translocation by fluorescence microscopy: 5% in controls, 46 5% with Doxo, 73 8% with GD3 and 37 7% with Etopo (mean results se of three independent determinations). amino acid amino-terminal MLS. Both exons possessed significant homology in their carboxyl-terminal moiety (Fig. 1B). When GFP was fused to the carboxyl terminus of AIF (AIF-GFP) or AIF-exB (AIF-exB-GFP) to generate chimeric proteins, transient transfection of COS cells revealed that the GFP-dependent fluorescence was targeted to mitochondria (Fig. 2), as has been shown for a Cyt-c-GFP fusion construct (18; Fig. 2). In contrast, a truncated AIF-GFP fusion protein in which the aminoterminal MLS ( 1 100) was removed exhibited a diffuse cytoplasmic staining comparable to that ob- Upon addition of the apoptosis inducer STS, mitochondrially targeted AIF-GFP (and AIF-exB-GFP) translocate to an extramitochondrial localization (Fig. 2). Similar results are obtained with other apoptosis inducers including the proapoptotic second messenger ganglioside GD3 (21, 22) as well as two chemotherapeutic agents (doxorubicin and etoposide) (Fig. 3). The translocation of AIF-GFP can be observed in cells that still lack signs of fluorescence-detectable chromatin condensation (Fig. 3). Immunostaining of Cyt-c-GFP-transfected cells (green fluorescence) with an anti-aif antibody (revealed by a PE conjugate, red fluorescence) indicated that the translocation of AIF and Cyt-c occurs simultaneously after STS addition (Fig. 4). Similar results were obtained when AIF-GFP-transfected cells were stained with an anti-cyt-c antibody (not shown), thus excluding the possibility that the fusion with GFP might affect the kinetics of AIF or Cyt-c release. Staining of AIF-GFP (or Cyt-c) -transfected cells with anti-aif (or Cyt-c) antibodies yielded a similar distribution for the GFP fusion protein and the immunodetectable (endogenous transgene-encoded) protein (not shown; see ref 18), further arguing against the possibility that the GFP moiety might affect the kinetics of AIF redistribution. Cyt-c-GFP and AIF-GFP translocation was observed in all STS-treated cells having undergone incipient (stage I) or advanced (stage II) chromatin condensation, without (stage IIa) or with formation of nuclear apoptotic bodies (stage IIb) (Fig. 5A, B). In addition, a fraction of cells ( 40%) not having yet undergone Hoechst detectable chromatin condensation manifested the mitochondrial release of AIF- GFP or Cyt-c-GFP (Fig. 5B). The pan-caspase inhibitor Z-VAD.fmk failed to prevent STS-induced AIF-GFP or Cyt-c-GFP release, although it did affect the transition from stage I to stage II of chromatin condensation (Fig. 5B). In conclusion, in COS cells, Cyt-c and AIF are Figure 4. Chronological relationship between the mitochondrio-nuclear translocation of AIF and Cyt-c. 16 h after transient transfection of COS cells with Cyt-c-GFP, 1 M of staurosporine (STS) and/or Z-VAD.fmk (100 M, added 30 min before STS) were added for the indicated periods. After fixation and permeabilization, cells were immunostained with an anti- AIF antibody revealed by a PE conjugate and the percentage of cells having translocated the GFP fusion protein or the endogenous AIF protein was assessed by fluorescence microscopy. Data are representative of three independent experiments. INDUCTION OF CELL DEATH BY AIF 761

5 released simultaneously in a caspase-independent fashion before chromatin condensation occurs. Functional relationship between AIF and Cyt-c release To further investigate the putative functional relationship between the release of AIF and Cyt-c, COS cells transfected with either AIF-GFP or Cyt-c-GFP were microinjected with recombinant AIF or Cyt-c, followed by the determination of nuclear morphology and quantitation of the translocation of AIF-GFP or Cyt-c-GFP (Fig. 6). Microinjection of both AIF and Cyt-c resulted in the induction of nuclear apoptosis (Fig. 6A) and in the translocation of AIF-GFP and Cyt-c-GFP (Fig. 6B), indicating that the ectopic (nonmitochondrial) presence of both proteins suffices to cause permeabilization of the outer mitochondrial membrane. Both AIF and AIF had a similar effect on nuclear morphology (1) and mitochondria (not shown), indicating that these effects do not depend on the MLS. When introduced into the cytosol, AIF can trigger the release of mitochondrial AIF, as well as that of Cyt-c, in a reaction Figure 5. Effect of apoptosis induction by staurosporine (STS) with respect to the advancement of chromatin condensation. COS cells were transiently transfected with AIF-GFP or Cyt-c-GFP, respectively (24 h) and the apoptosis inducer STS (1 M) as well as the pan-caspase inhibitor Z-VAD.fmk (100 M) were added during the last 4hofculture, as indicated. Then cells were fixed and counterstained with Hoechst for the determination of the nuclear morphology. A) Fluorescence micrographs were taken at different stages of nuclear chromatin condensation; normal nuclear morphology (control) in the upper panel, stage I (with STS) in the second panel, stage II (with STS) in the third panel, and stage I (with STS Z-VAD.fmk). B) Histogram of the frequency of cells in the different stages of nuclear apoptosis observed in cells cultured in the presence or absence of STS and Z-VAD.fmk. The percentage of cells having translocated AIF-GFP or Cyt-c-GFP is indicated above each column. Results are pooled from 5 independent experiments, with at least 3 repetitions for each data point. 762 Vol. 15 March 2001 The FASEB Journal LOEFFLER

6 that is not affected by the pan-caspase inhibitor Z- VAD.fmk (Fig. 6B). AIF triggered nuclear apoptosis, both stage I and II. Z-VAD.fmk largely prevented the occurrence of stage II apoptosis, yet had no effect on stage I (Fig. 7), indicating that AIF-induced stage I chromatin condensation occurs in a caspase-independent fashion, whereas its advancement to stage II is caspase dependent, in accord with previous observations (3, 23). All Cyt-c induced effects were inhibited by Z-VAD.fmk at both nuclear (Fig. 6A) and mitochondrial levels (Fig. 6B), confirming that Cyt-c is acting through the activation of caspases. Altogether, these data suggest the existence of a feed-forward loop in which the two mitochondrial intermembrane proteins Cyt-c and AIF cause the release of further intermembrane proteins, either in a caspase-independent (AIF) or caspase-dependent (Cyt-c) fashion. Caspase-independent cell death induction by transfection-enforced AIF expression The results discussed above were obtained shortly (16 24 h) after transfection of COS cells with AIF-GFP or Cyt-c-GFP constructs, that is, at a stage at which these proteins are largely mitochondrial and 10% of the cells exhibit spontaneous translocation of AIF-GFP or Cyt-c-GFP to the rest of the cell (Fig. 7A). If cells were cultured for a longer period ( h), however, an increasing percentage of transfected cells lost the mitochondrial pattern of AIF-GFP, AIF-exB-GFP or Cyt-c- GFP and rather manifested a diffuse distribution of GFP fusion proteins (Fig. 7A). Cytofluorometric assessment of the percentage of GFP-positive cells revealed a progressive disappearance of viable cells expressing Figure 6. Nuclear and mitochondrial effects of AIF and Cyt-c microinjected into COS cells. AIF-GFP or Cyt-c-GFP transfected cells (16 h), were microinjected with PBS only (Ø), recombinant AIF, or Cyt-c and were then cultured for 3 h in the presence or absence of Z-VAD.fmk. As a negative control, cells were microinjected with PBS. A) The frequency of nuclear apoptosis was determined by Hoechst staining. B) Translocation of AIF-GFP or Cyt-c-GFP. Values represent mean percentages se from three experiments. At least 200 cells were microinjected in each experiment. AIF-GFP, AIF-exB-GFP, AIF GFP, or Cyt-c-GFP, compared with cells transfected with GFP only (Fig. 7B), suggesting that these proteins are actually lethal. Accordingly, 50% of the cells expressing AIF-GFP, AIF-exB-GFP, AIF GFP, or Cyt-c-GFP exhibited features of nuclear apoptosis 96 h after transfection (Fig. 7C). This effect was only partially inhibited by Z-VAD.fmk (Fig. 7C), which failed to prevent the appearance of condensed chromatin. However, Z-VAD.fmk (added each 24 h at a dose of 100 M) did prevent the progression of nuclear apoptosis from stage I to stage II (Fig. 7C), thus providing an internal control for its efficacy. An increase in the frequency of Z-VAD.fmk administrations or an increase in the dose of Z-VAD.fmk did not ameliorate the degree of inhibition (not shown), underscoring that the AIF-GFP constructs used in this study can induce a type of chromatin condensation and cell death that does not rely on the function of Z-VAD.fmk-inhibitable caspases. AIF induces apoptosis independently from its oxidoreductase activity Based on sequence comparisons with flavoproteins whose structure has been determined by X-crystallography, computer-assisted structural analysis (2), two conserved hexapeptide motifs critical for binding of nicotine adenine dinucleotide (NAD) and flavine adenine dinucleotide (FAD) were identified in the AIF protein: 303TVIGGG308 and 255CLIATG260, respectively. On theoretical grounds, mutations of these NAD/FAD-binding motifs by insertion of tri-alanine stretches (mutants 1 and 2, respectively) should abolish binding of the prosthetic groups required for electron transfer and hence abrogate the oxidoreductase activity of AIF. Transfection of COS cells with wild-type AIF- GFP and mutant AIF-GFP constructs induced a similar level of initially mitochondrial AIF-GFP expression, followed by spontaneous AIF-GFP translocation and apoptosis (Fig. 8A). These data suggest that the apoptogenic effect of AIF does not rely on its oxidoreductase activity. To confirm this hypothesis in another experimental system, recombinant AIF protein was preincubated with diphenyleneiodonium, an inhibitor of flavonoid-containing enzymes covalently reacting with FAD (24 26). Diphenyleneiodonium-pretreated AIF and untreated AIF had a similar capacity to induce DNA loss when added to purified HeLa nuclei (Fig. 8B), indicating that the nuclear effects of AIF do not require the presence of a redox-active reaction center. Mitochondrion-targeted Bcl-2 delays AIF-induced apoptosis unless AIF is targeted to an extramitochondrial localization Bcl-2 is a multifunctional inhibitor of apoptosis that has been suggested to act either on mitochondria or on the endoplasmic reticulum (ER) to confer cytoprotection (19, 27 29). We have taken advantage of Rat-1 cells expressing Bcl-2 fusion proteins targeted specifically to INDUCTION OF CELL DEATH BY AIF 763

7 Figure 7. Apoptosis induced by AIF-GFP or Cyt-c-GFP transfection into COS cells. Cells were transfected with the indicated GFP fusion construct and cultured for the indicated periods in the presence or absence of Z-VAD.fmk. Then the frequency of translocation events was determined among transfected (GFP-positive) cells (A). Alternatively, the percentage of GFP-positive cells among the entire populations was determined by cytofluorometry, while gating on the normal-sized, nonapoptotic population (B). Moreover, the advancement of nuclear chromatin condensation was determined by staining with Hoechst (C). Results are representative of five independent determinations. mitochondria or the ER (19) to evaluate its capacity to prevent cell death induced by transfection with AIF- GFP and Cyt-c-GFP constructs. ER-targeted Bcl-2 has no effect on the spontaneous translocation of AIF-GFP, AIF-exB-GFP, or Cyt-c-GFP (Fig. 9B) and does not affect the induction of nuclear chromatin condensation (Fig. 9A). In contrast, mitochondrion-targeted Bcl-2 does retard the translocation of AIF-GFP, AIF-exB-GFP, or Cyt-c-GFP (Fig. 9B) and concomitantly reduces the frequency of nuclear apoptosis (Fig. 9A). However, Bcl-2 has no effect on the frequency of apoptosis induced by extramitochondrially targeted AIF GFP (Fig. 9A). Bcl-2 only delays the translocation of AIF-GFP or AIF-exB-GFP, and no Bcl-2-mediated inhibition is found on prolonged culture (48 h in Rat-1 cells, Fig. 9). Altogether, these data underscore the functional importance of local mitochondrial effects for Bcl-2-mediated cytoprotection. CONCLUDING REMARKS The two dominant splice variants of AIF, which differ in the alternative use of exon 2a vs. exon 2b (Fig. 1), are both targeted to the mitochondrial intermembrane space, as shown by appropriate AIF-GFP fusion constructs (Fig. 2). To our knowledge, this is the first example of a splice variant affecting an MLS yet not affecting mitochondrial import. The alternative exon 2b (which is highly homologous to exon 2a) contains an uncharged yet relatively hydrophilic stretch of 23 amino acids, starting at residue 59, flanked by charged regions. This motif could be an alternative intermembrane space bipartite signal (30). Upon induction of apoptosis by STS (Fig. 2), doxorubicin, etoposide, or ganglioside GD3 (Fig. 3), both proteins (AIF-GFP and AIF-exB-GFP) translocate indistinguishably from mitochondria to the rest of the cell before signs of chromatin condensation are detectable (Fig. 3, Fig. 5B). The proapoptotic effect of AIF appears independent from its oxidoreductase activity (Fig. 8), similar to what has been reported for Cyt-c (31). It is thus tempting to speculate that AIF may have a dual function as an ubiquitous (32), perhaps vital oxidoreductase catalyzing electron transfer between cytochrome c and NAD (S. A. Susin and G. Kroemer, unpublished observation) in the intermembrane space and as an apoptosis effector/regulator molecule in its extramitochondrial localization. On the one hand, AIF may function as a death effect involved in caspase-independent apoptosis. On the 764 Vol. 15 March 2001 The FASEB Journal LOEFFLER

8 process of AIF release. Indeed, intermediate stages of AIF release (in which some AIF would be still retained in mitochondria) has rarely been ( 1% of cells, not shown) observed by confocal microscopy, suggesting that the translocation of AIF from mitochondria to the rest of the cell is a rapid event occurring in an all-or-nothing fashion. Upon prolonged culture, the transfection-enforced overexpression of AIF or AIF-exB led to a gradual increase of the frequency of cells in which AIF diffusely distributed to the cytosol (Fig. 7A), correlating with nuclear apoptosis (Fig. 7C) and cell death (Fig. 7B). On theoretical grounds, this could be due to a saturation of the mitochondrial protein import pathway by accumulating AIF protein or, alternatively, due to local AIF- Figure 8. Apoptogenic effect of AIF after mutation of NAD/ FAD-binding hexapeptide motifs or chemical neutralization of FAD. A) Effect of AIF mutations on its apoptogenic potential in intact cells. Wild type or mutant AIF-GFP constructs (mut 1: 303TVIGGG TAAAGG308; mut 2: 255CLIATG CLIAAA260) were transfected into COS cells, followed by determination of the frequency of cells having translocated AIF or undergone nuclear apoptosis. B) Effect of diphenyleneiodonium (DPI) on AIF activity as determined in a cell-free system. Purified nuclei were incubated with AIF (final concentration 1 g/ml) and/or DPI (final concentration 1 mm; preincubated for 30 min with AIF) for 90 min, followed by staining with the DNA intercalating dye propidium iodine and cytofluorometric assessment of the DNA content. Numbers in circles indicate the frequency of hypoploid nuclei. This experiment has been repeated twice, yielding similar results. other hand, AIF may function, at least in some pathways, as an upstream regulator of Cyt-c release and caspase activation. In COS cells, the translocation of AIF (endogenous or AIF-GFP) and Cyt-c (endogenous or Cyt-c-GFP) occurs in a caspaseindependent (that is, Z-VAD.fmk noninhibited) fashion (Fig. 5A). Extramitochondrial AIF can trigger the mitochondrial release of AIF-GFP and that of Cyt-c- GFP, and this effect does not rely on caspase activation (Fig. 6), emphasizing the possibility that AIF participates in a positive amplification loop in which partial release of AIF triggers the release of further AIF. Such a feed-forward system would accelerate the Figure 9. Apoptosis induced by AIF-GFP or Cyt-c-GFP transfection in Rat-1 cells overexpressing Bcl-2 targeted to mitochondria or to the endoplasmic reticulum. 12 h and 48 h after transient transfection with GFP(1), AIF-GFP (2), AIF-exB-GFP (3), AIF GFP (4), or Cyt-c-GFP (5), cells were fixed and counterstained with Hoechst As indicated, all experiments were performed with three cell lines: a control line transfected with a CMV vector only (control), a cell line overexpression mitochondrially targeted Bcl-2 (Bcl-2 MITO )or a cell line expressing a Bcl-2 protein targeted to the endoplasmic reticulum (Bcl-2 ER ). A) Quantitation of chromatin condensation B) Quantitation of the frequency of cells exhibiting a diffuse, nonmitochondrial distribution of GFP. Data are mean values ( se) of 3 independent experiments. INDUCTION OF CELL DEATH BY AIF 765

9 mediated damage of the mitochondrial outer membrane, leading to the release of AIF that previously has been imported into mitochondria. We favor this latter possibility, based on three observations: 1) the intensity of AIF-GFP-dependent fluorescence did not increase in COS cells between 48 and 144 h after transfection, although the percentage of cells exhibiting translocation greatly increased (Fig. 7A); 2) mitochondrial vs. diffuse staining patterns obtained with AIF-GFP appeared to be mutually exclusive; and 3) Bcl-2 overexpression (which stabilizes mitochondrial membranes, yet has no reported effect on the import of proteins; see ref 29, 33, 34) retarded the accumulation of AIF in the cytosol (Fig. 9B). When AIF is present ectopically in the extramitochondrial compartment, it induces chromatin condensation, the first stage of which appears to be caspase independent. This has been demonstrated in three different experimental settings: 1) by microinjection of the recombinant AIF protein (Fig. 6A), 2) by transfection of cells with mitochondrially targeted AIF which spontaneously translocates after prolonged culture (Fig. 7C, Fig. 9A), and 3) by transfection of cells with an AIF mutant lacking the MLS ( 1 100) (Figs. 2, 7B, C, 9). AIF causes apoptosis in a fashion that is not affected by Z-VAD.fmk (Fig. 7C) nor by Bcl-2 (Fig. 9), presumably by directly entering the nucleus. Altogether, these data confirm in a genetic system that AIF can cause apoptosis in a caspase-independent fashion. Furthermore, they demonstrate the feasibility of engineering a dominant apoptosis inducer (AIF ) that overcomes Bcl-2-mediated apoptosis inhibition. Such dominant apoptosis inducers could prove useful in the gene therapy-mediated ablation of cancer cells overexpressing caspase inhibitors and/or anti-apoptotic Bcl-2-like proteins. 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