The apoptosis mediator mDAP-3 is a novel member of a conserved family of mitochondrial proteins

The process of cellular suicide known as apoptosis or programmed cell death is fundamental to the development and maintenance of all multicellular organisms (Thompson, 1995). Cells undergoing apoptosis show cell shrinkage, membrane blebbing, and condensation and fragmentation of nuclear chromatin (Kerr et al., 1972). Disregulation of this process results in various pathologies including several autoimmune and neurodegenerative diseases and cancer.

Over the last year overwhelming evidence was presented, that mitochondria act as the central cellular organelles for the control of life and death of a cell. Once thought to only generate energy, they are now considered to act as important ‘decision centers’ involved in programmed cell death (Martinou, 1999). They harbor proteins that trigger apoptosis upon release into the cytosol (Alnemri, 1999). One of these proteins, cytochrome c, shuttles electrons between protein complexes of the respiratory electron transport chain. After release from the mitochondria, cytochrome c participates in the activation of caspases. A flavoprotein known as apoptosis inducing factor (AIF) is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced. 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. Like cytochrome c, AIF is likely to be a phylogenetically old, bifunctional protein with an electron acceptor/donor function and a second, independent apoptogenic role (Susin et al., 1999).

ANT-1 is another member of mitochondrial proteins involved in apoptosis induction. In a recent study it could be shown that ANT-1 overexpression led to all features of apoptosis (Bauer et al., 1999). ANT-1 is a component of the mitochondrial permeability transition complex, a protein aggregate connecting the inner with the outer mitochondrial membrane that has recently been implicated in apoptosis.

The apoptosis mediator, hDAP-3, was isolated through a functional selection approach in HeLa cells (Kissil et al., 1995). Expression of anti-sense hDAP-3 mRNA protected cells from apoptosis induced by activation of Fas- and tumor necrosis factor α (TNF-α)-receptors. Thus hDAP-3 is implicated as a positive mediator of these death-inducing stimuli (Kissil et al., 1999).

In the present study we report the identification, molecular cloning and initial characterization of a murine protein termed mDAP-3. mDAP-3 was identified by an approach to isolate markers of murine nephrogenesis, based on a modified differential display polymerase chain reaction (Kretzler et al., 1996). mDAP-3 is a putative orthologue to the human DAP-3. Orthotopic overexpression of a mDAP-3/EGFP fusion protein induces apoptosis. Fluorescence microscopy revealed a mitochondrial distribution of the fusion protein. The native mDAP-3 could be localized to the mitochondrial matrix according to its release after digitonin and carbonate treatment from isolated mitochondria.

mDAP-3 showed a significant sequence similarity to a putative yeast orthologue (YGL129c), here refered to as yDAP-3. A yDAP-3 defective yeast strain progressively lost mitochondrial DNA. The loss of mitochondrial DNA was significantly mitigated upon expression of the mammalian mDAP-3.

To clone full length mDAP-3, the EMBL mouse EST database was searched using BLAST (Altschul et al., 1990). A 578 bp mouse EST clone (MM4446) with a 99% sequence identity in a 100 bp overlap with MERM-3 was identified. This sequence was used to screen 3×10⁶ clones of a λ-ZAP cDNA library of E17.5 (embryonic day 17.5 post conception) mouse kidneys (Valentini et al., 1997) as described earlier (Church and Gilbert, 1984). The two longest of seven independent clones were bi-directionally sequenced by primer walking. The 5′ sequence of the mDAP-3 cDNA was verified by 5′-Marathon PCR (Clonetech, Heidelberg, Germany) in three independent bi-directionally sequenced clones, giving a full length cDNA of 1,656 bp.

The Heidelberg Unix Sequence Analysis Resource (HUSAR) was used to assemble the full-length consensus sequence of the mDAP-3 cDNA. For database searches the BLASTN program was applied at the nucleotide and the BLASTP program at the protein level. The PROTEIN and PROSITE programs were used to analyze the deduced mDAP-3 protein and potential motives, respectively. The PSORT-program (http://psort.ims.u-tokyo.ac.jp/) identified the putative intracellular localization.

Single-stranded radiolabeled riboprobes for mDAP-3 were prepared by in vitro transcription of a 286 bp PCR fragment, corresponding to position 334 to 619 of the mDAP-3 consensus sequence and hybridized as described earlier (Angerer and Angerer, 1992; Simmons et al., 1989). The ³⁵S-labeled antisense and sense RNA transcripts served as hybridization probe and control, respectively. After hybridization and development, the tissue was counterstained with Harris hematoxylin and eosin and examined on a microscope with dark and bright field optics.

For northern blot analysis total RNA was isolated from adult mouse tissue by guanidine thiocyanate/phenol/chloroform extraction (Chomczynski and Sacchi, 1987). After separation and immobilization of 20 μg total RNA on nylon membranes, hybridization was performed with the 578 bp long mDAP-3 riboprobe also used for library screening. For detection of signal the Storm Phosphorimaging System (Molecular Dynamics, Krefeld, Germany) was used. To assess the relative quantity of mRNA per lane, the organ screen blot was stripped and re-probed with a mouse β-actin probe.

Mouse tubular cells (MTC) and mouse mesangial cells (MMC) were cultured as described earlier (Haverty et al., 1988; Wolf et al., 1992). Jurkat cells were supplied by ATCC and cultured in RPMI with 10% fetal calf serum and 1% penicillin/streptomycin. For transient transfections, cells were harvested with trypsin/EDTA (0.05% trypsin, 0.53 mM EDTA 4Na; Life Technologies, Inc., Karlsruhe, Germany), resuspended in PBS to a concentration of 2.5×10⁶/ml and electroporated at 250 V and 960 μF with 1-10 μg of plasmid DNA. Following transfection cells were cultured under normal conditions for 24-48 hours prior to further analysis.

MMC and MTC were harvested in a lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholat, 150 mM NaCl, 1 mM EGTA, 1 mM Na₃VO₄, 1 μg/ml pepstatin, 1 μg/ml leupeptin). Extracted proteins or isolated mitochondria (see below) were boiled in loading buffer for 10 minutes, resolved by SDS-PAGE under reducing conditions and transferred to an Immobilon-P membrane (Millipore, Eschborn, Germany). Blots were probed with the respective primary antibodies and immune complexes were visualized using enhanced chemoluminescence (ECL-kit, Amersham International, Braunschweig, Germany).

The following antibodies were used: DAP-3 (Transduction Laboratories, Lexington, KY); cytochrome c (Santa Cruz Biotechnology Inc., Heidelberg, Germany). HSP60 (heat shock protein 60) and AAC (ADP/ATP carrier) antibodies were kind gifts from W. Neupert (Physiologic Chemistry, University of Munich, Germany).

mDAP-3-enhanced green fluorescence fusion protein (mDAP-3/EGFP) was obtained by cloning a PCR fragment containing nucleotides 1-1342 of mDAP-3 into the BamHI/SalI site of the vector pEGFP-N1 (Clonetech, Heidelberg, Germany). The mDAP-3 stop codon at position 1342-1345 was changed from the nucleotides TAA that code for the translation stop to the nucleotides TCA which code for the amino acid serine. The mDAP-3 coding region is then followed by 6 (7 including the changed stop codon) linker codons originating from the pEGFP-N1 vector multiple cloning site and the EGFP ATG translation start codon, that marks the beginning of the EGFP open reading frame.

A mDAP-3 P-loop mutant was obtained by site directed mutagenesis of amino acid 127 from lysine to arginine with the Quick Change™ Site-Directed Mutagenesis Kit as recommended by the manufacturer (Stratagene, Amsterdam, The Netherlands).

For the expression of mDAP-3 in yeast, a PCR fragment containing nucleotides 151 to 1345 of the mDAP-3 cDNA was subcloned into the EcoRI/SalI site of the yeast expression vector pYX 123 (Novagen, Madison, USA).

MMCs were transfected by electroporation with 9 μg of the indicated plasmids (pEGFP-N1 or mDAP-3/EGFP) together with 1 μg of a plasmid coding for farnesylated green fluorescent protein (fGFP). fGFP as control was required do to leaching of the pEGFP-N1 after ethanol fixation. For FACScan analysis cells were detached 48 hours after transfection with trypsin/EDTA, washed with PBS, and fixed in 50% ethanol for at least 30 minutes at room temperature. After washing with PBS, cells were treated with RNase A (50 μg/ml) for 30 minutes at room temperature and stained with propidium iodide (PI) for 20 minutes on ice. Cells were subjected to flow cytometry analysis with a FACS-Calibur (Becton Dickinson, Mountain View, CA), where green fluorescent protein (GFP)-positive cells were gated by their high fluorescent intensity (FL1). For each sample, 10,000 GFP-positive cells were collected and cell-cycle distribution was analyzed according to DNA content using PI staining. The results represent three independent transfections. Jurkat cells were stimulated with 50 ng/ml anti-human Fas antibody (Biomol, Hamburg, Germany) for up to 5 hours and prepared for FACScan analysis as described above. Control cells received carrier (PBS) only.

To assess mitochondrial and lysosomal morphology, fluorescent dyes were utilized according to the manufacturer’s suggestions. In brief: MTCs or MMCs were grown on glass coverslips for 24 hours after transient transfection with mDAP-3/EGFP and then stained with the appropriate cell culture medium containing either Mito-Tracker-Red CMXRos™ or Lyso-Tracker-Red™ (Molecular Probes, Leiden, The Netherlands) at a concentration of 100 nM for 40 minutes at standard cell culture conditions. Subsequently cells were rinsed 3 times with PBS, fixed with 4% paraformaldehyde and embedded in Mowiol solution (Calbiochem-Novabiochem, Bad Soden, Germany) and visualized by fluorescence microscopy. For confocal laser scanning microscopy cells were analyzed after fixation with 4% paraformaldehyde in PBS.

Using an established protocol for isolating mitochondria by differential centrifugation (Werner and Neupert, 1972), mitochondria were isolated from adult rat liver. Lactate dehydrogenase, malate dehydrogenase and glucose 6-phosphat dehydrogenase measurements were used to confirm effective cell fractionation.

Mitochondrial and cytosolic fractions were obtained by differential centrifugation of MTCs and Jurkat cells. In brief, cells were harvested and washed in PBS, and resuspended in 5 mM HEPES, 2 mM MgCl₂, 75 mM NaCl, 5 mM EDTA and protease inhibitors. Cells were broken by multiple passages through a 25GA1 0.5×25 needle. The suspension was centrifuged at 2,000 g at 4°C for 5 minutes to remove unbroken cells. The supernatant was centrifuged at 15,000 g at 4°C for 20 minutes. The resulting supernatant (cytosol) and pellet (mitochondria rich pellet) were analyzed by SDS/PAGE immunoblotting (30 μg per lane).

Isolated mitochondria were incubated with increasing digitonin concentration, from 0% to 0.4% (see Fig. 5) for 2 minutes on ice. After incubation, the mitochondria were pelleted as above, resuspended in loading buffer, and supernatant and resuspended mitochondria were submitted to western blot analysis.

Isolated mitochondria (0.2 mg) were pelleted at 7,000 g for 10 minutes, resuspended in 1 ml 0.1 M Na₂ HCO₃, and incubated at 4°C for 30 minutes. The samples were split and one half was directly trichloracetate (TCA) precipitated with 0.5 ml 70% TCA, representing the total protein content. The second half was centrifuged for 60 minutes at 4°C and 200,000 g. The pellet containing integral membrane proteins was directly resolved in loading buffer. The supernatant containing the soluble proteins and membrane associated proteins was trichloracetate precipitated. All three fractions were then submitted to western blot analysis.

Standard techniques were used for the growth and manipulation of yeast strains (Guthrie and Fink, 1991). A yDAP-3 (YGL129c) null strain was a kind gift from E. Cerdan (La Coruña, Spain; Tizon et al., 1999). This mutant did not contain mitochondrial DNA and was backcrossed with a YPH500 wild-type strain (Sikorski and Hieter, 1989). Sporulation revealed yDAP-3 null mutants containing mitochondrial DNA. These strains were used for the experiments shown. To test the stability of the mitochondrial genome, yeast cultures were grown for various times in liquid glucose medium prior to crossing with a tester strain lacking mitochondrial DNA. Respiration competence of the resulting diploids was tested on glycerol plates.

The MERM-3 (for mouse embryonic renal marker 3) cDNA fragment had been initially identified through a modified differential display polymerase chain reaction (Kretzler et al., 1996). Semiquantitative RT-PCR was used to reconfirm the MERM-3 expression pattern and to analyze MERM-3 mRNA levels during in vivo and in vitro nephrogenesis. MERM-3 was found to be upregulated during embryogenesis (Kretzler et al., 1996). Northern blot analysis of either adult or E17.5 mouse kidney mRNA hybridized with a 578 bp riboprobe gave a specific signal for mDAP-3 indicating a length of about 1.7 kb for the mDAP-3 mRNA (data not shown). The same probe was used to screen an E17.5 days p.c. mouse kidney cDNA library and a full-length cDNA of 1656 bp was obtained. These sequence data have been submitted to the EMBL database under accession number: AJ250375. The mDAP-3 cDNA sequence contains a single open reading frame (ORF) coding for a potential protein of 391 amino acids with a calculated molecular mass of about 45 kDa. No potential transmembrane regions were identified by hydrophobicity blot.

The mDAP-3 sequence contains a typical P-Loop motive, that fits to the consensus sequence (G)XXXXGK(S/T) at positions 121-128, which implies that mDAP-3 is a potential ATP/GTP-binding protein. In addition mDAP-3 contains multiple putative PKC- and CK2-phosphorylation sites. The N terminus of mDAP-3 is positively charged and could serve as a mitochondrial targeting sequence (Nakai and Horton, 1999).

In situ hybridization with mDAP-3 specific riboprobes showed an abundant expression in mouse embryo (Fig. 1A). The corresponding sense probe gave no specific signal (Fig. 1A, inset). Strong signal was detected in the developing epithelial compartments, particularly of the oropharynx, the gastrointestinal tract, dermis and the urogenital system. The central nervous system, the cerebral cortex and the trigeminal nerve showed prominent expression. Maximal expression was found in the derivatives of the pharyngeal pouch (Glandulae thyreoidea and subingualis) with highest mRNA levels in the olfactorial epithelium.

Northern blot analysis of 20 μg total RNA from 11 organs of adult mice showed mDAP-3 mRNA expression in the kidney, heart, liver, thymus, muscle, spleen, intestine and stomach, with an expression maximum in the testis (Fig. 1B).

Minimal to absent expression was seen in ovary and lung, respectively.

By searching the EMBL protein database with a BLAST algorithm using the full length mDAP-3 cDNA as the query, the DNA sequence for the hDAP-3 was retrieved. A nucleic acid comparison of mDAP-3 and hDAP-3 showed an 82% identity over 1299 base pairs. The amino acid identity between the 391 amino acid mDAP-3 protein and the 398 amino acid hDAP-3 protein is 82% (Fig. 2A). Alignment of the C. elegans cec14a_4.2 with mDAP-3 shows 33% identity between the proteins (Fig. 2A). In addition, in a search of the yeast genome, an open reading frame (YGL129c) with a 17% identity with mDAP-3 (including the P-loop region) was found and is referred to as yDAP-3 (Fig. 2A). Screening the mouse EST database at the NCBI with the human DAP-3 sequence revealed 59 matching murine ESTs (P<0.01). Using the consensus sequence generated from those ESTs a mDAP-3 identical ORF could be deduced, further supporting the notion of mDAP-3 being the murine orthologue of hDAP-3.

By searching the EMBL protein database using only the mDAP-3 P-loop sequence as a query, two additional proteins were retrieved. C.elegans protein Ced-4 (cell death gene 4) and the mammalian orthologue to Ced-4, Apaf-1. An alignment of the P-loop sequences of these proteins (Fig. 2B) shows that the homology exceeds the consensus sequence defining a P-loop motive XXGXXXXGK(S/T), giving a consensus sequence of LXGXXGXGK(S/T).

To test whether mDAP-3 has an effect on cell survival, MMCs were transiently transfected with a mDAP-3/EGFP fusion protein or the empty expression vector.

Expression of the mDAP-3/EGFP fusion protein was verified by western blot analysis using an anti-hDAP-3 antibody. Two distinct bands were detected, the lower 45 kDa signal represents the endogenously expressed mDAP-3 and the 75 kDa band corresponds to the mDAP-3/EGFP fusion protein (Fig. 3A), confirming transient expression of mDAP-3 and cross-reactivity of the hDAP-3 antibody.

The mDAP-3/EGFP overexpression experiments were designed as co-transfections with a vector expressing a membrane localized form of GFP also used in controls. The cells were fixed with ethanol 48 hours after transfection, stained with PI and analyzed by FACScan for their DNA content and the overall cell survival. Apoptosis was assessed by determing the number of cells within the GFP positive gated population with a sub G₁ DNA content, indicative of DNA fragmentation.

mDAP-3/EGFP fusion protein overexpression increased the number of MMCs displaying a sub-G₁ DNA content from a background of 11.9±2.8% to 27.6±2.6% apoptotic cells (n=3) (Fig. 3B). The cell cycle distribution of the viable cells did not change significantly. Thus mDAP-3 expression in MMCs induced an increase in the number of apoptotic cells.

To further dissect the pro-apoptotic effect of DAP-3, transient transfection of wild type mDAP-3 and a construct with a point mutation in the P-loop (lysine 127 changed to arginine 127) were analyzed. P-loop mutation resulted in an abolishment of the proapoptotic effect of mDAP-3 (wild-type mDAP-3: 25%, P-loop mutant mDAP-3: 4.1%, CON 8.6% sub-G₁ DNA content). In the mDAP-3 transfected cells cytochrome c could be detected in the cytoplasm by cell fractionation assays (Fig. 3C). In the P-loop mutants and controls no cytochrome c release could be shown (Fig. 3C). Furthermore, mDAP-3 remained intra-mitochondrial after apoptosis induction (Fig. 3C). These data indicate the functional requirement of the P-loop for mDAP-3 mediated apoptosis, and locate DAP-3 upstream of cytochrome c release during cell death.

To determine the intracellular localization of mDAP-3, the mDAP-3/EGFP expression plasmid was employed. Examining transiently transfected MTCs and MMCs (data not shown) by fluorescence light microscopy allowed detection of a punctate cytoplasmic signal (Fig. 4B). This pattern was distinctly different from the even and diffuse fluorescence derived from the expression of the pEGFP-N1 vector alone (Fig. 4A).

To further analyze the punctate intracellular localization of mDAP-3, double labeling studies with the mDAP-3/EGFP fusion protein and cell organelle specific dyes, Mito-Tracker-Red CMXRos™ for mitochondria or Lyso-Tracker-Red™ for lysosomes, were performed. By light microscopy the mDAP-3/EGFP fusion protein colocalized with the mitochondrial dye (Fig. 4C+D) but not with the lysosomal marker (Fig. 4E-F). These results were confirmed by confocal scanning laser microscopy (Fig. 4G-I). Merging the fluorescence of the mitochondrial dye and the mDAP-3/EGFP fusion protein revealed a significant overlap of the signals, consistent with a mitochondrial localization of the mDAP-3/EGFP fusion protein.

Cell fractionation assays were performed to examine the subcellular distribution of mDAP-3 biochemically.

Mitochondrial, endoplasmatic reticulum and cytosol fractions were isolated from rat liver and subsequently tested for their mDAP-3 content by western blot analysis. The fractionation was controlled by measuring compartment specific enzyme activity (data not shown). mDAP-3 protein was only detectable in the mitochondrial fraction consistent with the mDAP-3/EGFP data (Fig. 5A).

Proteinase K treatment of the mitochondrial fraction did not change the amount of mDAP-3, indicating an intramitochondrial localization of mDAP-3 (Fig. 5B).

To further dissect intramitochondrial localization of mDAP-3, sequential disruption of mitochondrial membranes with digitonin was performed. Digitonin treatment lead to a concentration dependant release of mDAP-3. As controls, cytochrome c, localized in the intermembrane space of mitochondria, ADP/ATP carrier (AAC), an integral protein of the inner mitochondrial membrane, and HSP60, localized to the mitochondrial matrix, were used.

As shown in Fig. 5C, mDAP-3 was released late from the mitochondria at 0.3% digitonin, as was AAC (0.3%) and HSP60 (0.2%). Cytochrome c was already detectable in the supernatant after treatment with 0.075% digitonin. These data are consistent with a mDAP-3 localization in the mitochondrial matrix similar to HSP60 or in the inner mitochondrial membrane as the marker AAC. Thus, mDAP-3 is not localized in the intermembrane space as indicated by the different pattern of release by digitonin in comparison to cytochrome c (Fig. 5C).

The mDAP-3 amino acid sequence does not contain hydrophobic stretches. To further analyze the submitochondrial localization carbonate treatment was used which allows to distinguish between soluble and integral membrane proteins. mDAP-3 was found in the soluble fraction, as was cytochrome c and HSP60. AAC was detected in the membrane fraction as expected (Fig. 5D).

Jurkat cells were stimulated to undergo apoptosis with an agonistic anti-Fas antibody and harvested at 3 hours (45% apoptotic cells) and 5 hours (54% apoptotic cells) after stimulation. Subsequent differential centrifugation separated cytosolic and mitochondrial fractions which were then analyzed for their DAP-3, HSP60, AAC and cytochrome c content by SDS/PAGE immunoblotting. DAP-3 as well as the two other mitochondrial matrix or intramembrane proteins HSP60 and AAC could only be detected in the mitochondrial fraction (Fig. 5E). In contrast, cytochrome c could be seen in the cytosol of cells undergoing apoptosis (Fig. 5E).

In summary, these data identify DAP-3 as a mitochondrial matrix protein excerting it’s proapoptotic effect inside the mitochondrion.

Since the DAP-3 family is conserved among eukaryotes and has orthologues in non-apoptotic organisms such as yeast, DAP-3 proteins obviously perform an additional role besides the mediation of apoptosis. The localization of DAP-3 in mitochondria lead us to test whether DAP-3 orthologues are required for the biogenesis of this organelle. S. cerevisiae appeared to us to be the best suited system to test this non-apoptotic function since the absence of apoptosis in yeast simplifies the interpretation. Disruption of the yDAP-3 gene did not lead to a reduced growth on non-fermentable carbon sources such as glycerol or lactate. This indicates that mitochondrial respiration is not dependent on yDAP-3 function. However, in the absence of yDAP-3, yeast cells were significantly defective in the maintenance of the mitochondrial genome (46% in ΔDAP-3 vs 3% in wild type) (Fig. 6A). Although the molecular role of yDAP-3 remains unclear, this indicates a role of yDAP-3 in mitochondrial biogenesis. To test whether mammalian DAP-3 orthologues perform a similar role as yDAP-3, we expressed mDAP-3 in yeast. mDAP-3 was efficiently processed to its mature size indicating import of the mammalian orthologue into yeast mitochondria (Fig. 6C). The tendency to loose mitochondrial DNA in yDAP-3 null mutant was significantly mitigated upon expression of the mouse DAP-3 orthologue (Fig. 6B) and thus, the function of DAP-3 homologues in the biogenesis of mitochondria seems to be conserved among eukaryotes.

Here we report the isolation and initial molecular characterization of the mDAP-3 full-length cDNA. Northern blot organ screen and in situ hybridization showed a widespread expression with induction in metabolic active tissues. The abundant expression and high evolutionary conservation of mDAP-3 is consistent with the involvement of this molecule in basal cell activities. Given its mitochondrial localization, it can be speculated that mDAP-3 is involved in oxidative metabolism and that the expression induction seen in the in situ hybridization in specific compartments with high mitotic activity mirrors the increased metabolic demand of these cells.

The 1.7 kb mRNA of mDAP-3 contains a single ORF coding for a 45 kDa protein that shares an identity of 82% to the human DAP-3 and 33% identity to the recently identified C. elegans DAP-3. hDAP-3 is required for mediation of TNF-α- and Fas induced cell death. A nucleotide binding motive (P-loop) defective mutation of hDAP-3 was unable to induce apoptosis (Kissil et al., 1999). Using a panel of N- and C-terminal truncations preliminary results indicate a requirement of the initial 100 N-terminal amino acids for mitochondrial targeting and/or apoptosis induction by mDAP-3. A P-loop point mutated mDAP-3 is localized to the mitochondria (data not shown), but did not induce apoptosis, consistent with ATP processing by DAP-3 in the mitochondrial matrix being relevant for its proapoptotic activity.

hDAP-3 mediated apoptosis was shown to be suppressed by caspase inhibitors. Dominant negative forms of either FADD/MORT-1 or caspase-8 could not inhibit hDAP-3 induced cell death (Kissil et al., 1999). DAP-3 overexpression resulted in a P-loop dependant release of cytochrome c from mitochondria. These results place molecules of the DAP-3 family downstream of caspase-8 and upstream of cytochrome c and effector caspases, exactly where the decision center between ‘life and death’, the mitochondrion, channels into the pathway.

Amino acid sequence analysis of mDAP-3 with the program PSORT reveals a putative mitochondrial localization of mDAP-3. Similar results were obtained for hDAP-3 and yDAP-3. This subcellular localization of mDAP-3 to the mitochondria was further supported by three independent approaches. First, the mDAP-3/EGFP fusion protein showed a punctate mitochondrial pattern of mDAP-3 distribution by fluorescence microscopy. Second, the mDAP-3/EGFP fusion protein co-localized with a mitochondrial staining demonstrated by confocal microscopy. Third, liver cell fractionation analysis confirmed the mitochondrial localization of mDAP-3 biochemically. Furthermore, proteinase K and digitonin treatment of isolated mitochondria confirmed the intramitochondrial localization of mDAP-3. Carbonate treatment experiments showed, as expected from the sequence prediction, no integral association of mDAP-3 with mitochondrial membranes. Our data therefore localize mDAP-3 to the mitochondrial matrix. An association with the inner membrane via binding to integral membrane proteins remains possible. Sonication of isolated mitochondria, which ruptures the membranes, and subsequent western blot analysis of membrane- and soluble-fractions revealed an equal distribution of mDAP-3 between these two fractions (data not shown), suggesting an interaction of mDAP-3 with integral membrane proteins of the inner mitochondrial membrane.

The regulation of apoptosis by mitochondria is a matter of intense research (Alnemri, 1999; Martinou, 1999; Susin et al., 1998). Several evolutionary conserved mitochondrial proteins essential for oxidative phosphorylation play an additional proapoptotic role. These mitochondrial factors implicated in apoptotic function have been localized to either the outer or inner mitochondrial membrane, or the intermembrane space.

Cytochrome c, a highly conserved electron shuttle in the mitochondrial intermembrane space, was the first molecule identified to be a key player in caspase activation after release from mitochondria. Another electron carrier molecule, the flavoprotein AIF appears to be involved in apoptosis induction after translocation to the nucleus (Susin et al., 1999). These two phylogenetically old proteins play an intramitochondrial electron acceptor/donor function and a second, independent apoptogenic role in higher eukaryotes after release from mitochondria. DAP-3, in contrast to cytochrome c, retained its intramitochondrial localization during Fas induced cell death, indicating an intramitochondrial mode of action during apoptosis.

ANT-1, a highly expressed protein of the mitochondrial permeability transition complex, which is under physiological conditions a strictly specific ADP/ATP antiporter, can induce apoptosis after overexpression or by stimulation through a secondary signal (Bauer et al., 1999). This indicates an inhibition of ANT-1s proapoptotic function by other proteins of the permeability transition pore. For inhibition of apoptosis a delicate stoichometric balance between the various components of the transition pore appears to be required. A similar inhibition of the proapoptotic function of mDAP-3 by interacting molecules in non-apoptotic cells can be hypothesized, since mDAP-3 is also highly expressed in normal mitochondria.

As for cytochrome c the presence of DAP-3 homologues in non-apoptotic eukaryotes such as yeast indicates a function besides the regulation of apoptosis. The characterization of this function will be important to understand the molecular role for mitochondrial metabolism of this conserved protein family.

mDAP-3 showed significant amino acid identity to the yeast molecule YGL129c, here referred to as yDAP-3. This molecule contains the same conserved P-loop sequence as mDAP-3/hDAP-3/Apaf-1/CED-4. yDAP-3 mutants were reported to be unable to grow on glycerol as their sole carbon source (Tizon et al., 1999), indicating a yeast phenotype typically seen with disturbance of mitochondria (Lawson and Douglas, 1988).

However, our data indicate, that yDAP-3 is not directly required for respiration, but in the absence of yDAP-3 yeast cells tend to loose the mitochondrial genome. A direct role of yDAP-3 in mitochondrial DNA replication seems unlikely since the DNA loss was only moderate although significant. Loss of mitochondrial DNA was observed in yeast mutants defective in a variety of mitochondrial processes. For example mitochondrial morphology mutants often contain an unstable mitochondrial genome (Rapaport et al., 1998). However morphology of mitochondria in the yeast ΔDAP-3 strain did not show an altered morphology (data not shown). The molecular function of yDAP-3 in mitochondrial biogenesis seems to be conserved among the different members of the DAP-3 family, since the yeast mutant could be partially complemented by the mammalian mDAP-3 orthologue. Interestingly, the disruption of a yeast homologue to the ANT-1 transporter, RIM-2, also resulted in a loss of mitochondrial DNA (Van Dyck et al., 1995). These data might indicate a common, but so far unknown role of the ANT-1 and DAP-3 proteins for mitochondrial biogenesis and apoptosis induction. The yeast system should allow the identification of this function in the near future.

We thank W. Neupert, P. Nelson, U. Heinzmann, S.S Bolz and C.N. David for valuable discussions, Dr E. Cerdan for providing a yDAP-3 null strain and G. Fuhrer and I. Edenhofer for excellent technical assistance. This work was supported in part by DFG Kr 1492/2-1/2, Sander 96.016.1 and DFG Graduiertenkolleg GK438.