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Burnham Institute for Medical Research, La Jolla, California, USA
5Correspondence: Burnham Institute for Medical Research, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: reedoffice{at}burnham.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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day 9.5 in utero. The dap3–/– embryos and placentas were markedly shrunken. Embryos had arrested development, displaying severe growth restriction and lack of axial turning. Transmission electron microscopy analysis revealed abnormal, shrunken mitochondria with swollen crystae in dap3–/– embryos. Levels of cytochrome c oxidase-I, a protein encoded in the mitochondrial genome, were reduced in dap3–/– embryos, consistent with a role for DAP3 in intramitochondrial protein synthesis. A requirement for DAP3 in mitochondrial respiration was also revealed by oxygen consumption measurements using cultured cells treated with DAP3-specific small interfering RNA (siRNA). Studies of cultured cells from dap3–/– embryos confirmed a role in apoptosis induced by stimuli that trigger the extrinsic (TNF
, TRAIL, anti-Fas antibody) but not intrinsic (mitochondrial) cell death pathway. Thus, DAP3 joins a growing list of bifunctional proteins that play roles in normal mitochondrial physiology and in apoptosis.—Kim, H-R, Chae, H-J., Thomas, M., Miyazaki, T., Monosov, A., Monosov, E., Krajewska, M., Krajewski, S., Reed, J. C. Mammalian dap3 is an essential gene required for mitochondrial homeostasis in vivo and contributing to the extrinsic pathway for apoptosis.
Key Words: mitochondrial respiration • DAP3 protein • genotypic analysis • ß-actin • GTP binding protein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The DAP3 protein of mammals is predicted to have an N-terminal leader peptide typical of nuclear genome-encoded proteins that are imported into mitochondria; fusion with this N-terminal leader peptide directs heterologous proteins into mitochondria (2)
. Mutagenesis experiments also suggest that deleting this domain results in failed mitochondrial import of DAP3 in mammalian cells. Mass spectrometry analysis of isolated mitochondrial ribosomes from yeast identified the apparent yeast ortholog of DAP3 (YGL129C) (yDAP3) among the 15 proteins associated with this macromolecular assembly (3)
. Investigation of yDAP3-deficient yeast (YGL129C) (yDAP3) revealed a requirement for mitochondrial DNA synthesis and mitochondrial respiration, with yDAP3-deficient yeast cells significantly defective in maintenance of the mitochondrial genome. Furthermore, the tendency to lose mitochondrial DNA in yDAP3 null mutants was significantly mitigated upon expression of the mouse DAP3 ortholog, thus demonstrating evolutionarily conserved function of DAP3 homologues among eukaryotes (4)
. In addition to mitochondrial ribosome function, a role has been proposed for mammalian DAP3 in mitochondrial fission (5)
. DAP3 protein stability appears to depend on preservation of the mitochondrial genome, as shown by experiments with rho-zero cells (6)
.
Although a role for DAP3 in mitochondrial physiology has begun to emerge, the gene encoding this protein was first recognized for its role in apoptosis regulation, where antisense-mediated reductions in its expression in human tumor cell lines were associated with resistance to apoptosis induced by certain cytokines (7)
. In addition, expression of mutants of DAP3 protein can also have an apparent dominant-negative effect, suppressing apoptosis induced via tumor necrosis factor (TNF) family death receptors. Mitochondria are recognized for their important role in apoptosis, and ample precedence exists for mitochondrial proteins participating in cell death regulation (8
, 9)
. However, previous attempts to explore the role of DAP3 in apoptosis regulation in human tumor cell lines by use of antisense and dominant-negative experiments have generally failed to demonstrate a requirement for DAP3 in cell death pathways where mitochondria participate, showing instead a role for this protein in a mitochondria-independent pathway for apoptosis induced by TNF family members (10
, 11)
. Moreover, it has been shown that some portion of the DAP3 protein in cells can be found in the cytosol associating with components of TNF family death receptor complexes, particularly the adapter protein FADD (11
, 12)
. While evidence of a pool of cytosolic DAP3 in some types of intact cells has been obtained by immunofluorescence and subcellular fractionation (12)
, some experiments have suggested that DAP3 only associates with FADD after experimental lysis of cells, causing release of DAP3 from mitochondria (13)
. Nevertheless, DAP3 is reportedly phosphorylated in an Akt-dependent manner, correlating with suppression of DAP3-facilitated apoptosis (14)
, thus suggesting a cytosolic role for this interesting GTP binding protein.
To explore the role of the DAP3 protein in vivo, we generated mice with homozygous disruption of the dap3 gene. Our findings reveal that dap3 gene ablation results in an embryonic lethal phenotype, associated with aberrant mitochondria morphology and reduced production of proteins encoded by the mitochondrial genome. We also show that cultured cells in which DAP3 protein expression has been ablated have defective respiration. However, cells cultured from dap3–/– embryos also display a reduction in sensitivity to apoptosis induced via TNF family death receptors. Thus, we hypothesize that the predominant role for the DAP3 protein in vivo concerns maintenance of mitochondrial function, but that an additional role for DAP3 as a facilitator of certain apoptosis pathways also exists. As such, DAP3 joins a growing list of mitochondrial proteins (e.g., cytochrome c, AIF, endonuclease G, Bit) that have dual functions in both mitochondrial physiology and cell death regulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The chimeric mice were bred to C57BL/6J mice for several (>10) generations. All mice analyzed were of mixed genetic background (129/SvEvBrd and C57BL/6J).
PCR genotyping and Southern blot
Mouse tail genomic DNA was isolated by phenol-chloroform extraction after cell lysis with Triton X-100 and digestion with proteinase K. For some experiments, DNA was isolated from muscle tissue using QIAamp tissue kits (Qiagen, Chatsworth, CA, USA). Exons 1 and 2 of DAP3 were amplified from genomic DNA. The forward primer used for exon 1 was 5'-ATGAGATGCTGGTAGTAAAAATTAGCTAACC-3'; the reverse primer for intron 2 was 5'-GTTCTGGAGATGAATGGAACGC-3'. The reverse primer used for the retroviral targeting vector was 5'-AAATGGCGTTACTTAAGCTAGCTTGC-3'. The PCR reaction cycle for both sets of primers was 94°C for 5 min, 94°C for 30 s, and 60°C for 60 s, for 35 cycles, with a final extension at 72°C for 10 min. The PCR product of DAP3 wild-type (WT) gene is 350 bp in length; that of heterozygote is 350 and 200 bp and that of knockout cells is 200 bp.
For Southern blot, mouse embryo genomic DNA was isolated by digesting cells in lysis buffer (100 mM Tris-Cl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100 µg/ml proteinase K) for 5 h at 37°C and spooling genomic DNA after adding an equal volume of isopropyl alcohol. The DNA was digested overnight with Bgl-I and HindIII endonucleases and size-fractionated by agarous gel electrophoresis (0.8%). Before transfer, gels were soaked in denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 1 h, followed by a 1 h wash in pH neutralization solution (1.5 M NaCl, 1 M Tris-HCl at pH 7.4). The gels were then rinsed with 20x saline sodium citrate (SSC) (3 M NaCl, 300 mM trisodium citrate at pH 7.0) for 10–15 min and transferred to Hybond N membranes overnight using the Turboblotter system (Scleicher and Schuell, Keene, NH, USA) according to the manufacturer’s protocol. After rinsing with 6x SSC, the membranes were UV cross-linked (Stratalinker ®; Stratagene, San Diego, CA, USA). A probe covering the first intron of DAP3 (200 bp) was random primed or nick translated in the presence of 32P--cytidine triphosphate, and unincorporated nucleotides were removed by gel filtration (Biospin P30; Bio-Rad, Hercules, CA, USA). The probe was phenol-chloroform-extracted and boiled before addition to the hybridization solution. After 12 h of hybridization, the membranes were washed twice with 2x SSC, 0.1% SDS at 42°C and 30°C, respectively, and once with 0.2x SSC, 1% SDS at 27°C. The Hybond membranes were exposed to X-ray film with intensifying screens for 2–5 days.
For Southern blot analysis of the mitochondrial genome, total (low and high molecular weight) DNA was isolated and digested with SacII endonuclease. After gel electrophoresis and blotting as described above, the resulting blots were hybridized with a 32P-labeled, nick-translated rat 12s mitochondrial ribosome gene (gift from J. M. Cuezva).
Tissue isolation and processing
Embryos were dissected from deciduas using a surgical microscope. Whole embryos were photographed using either a phase-contrast microscope (Nikon Optiphot 66 with bright-field) or a stereomicroscope (wild M38 with MI-150 high-intensity illuminators; Dolan-Jenner Industries, Boxborough, MA, USA), both equipped with SPOT 3.1 digital cameras (Diagnostics Instruments, Inc., Sterling Heights, MI, USA). Embryonic stage was estimated by timed pregnancies and somite counts. The embryos were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E).
Embryo/tissue harvesting, isolation, and processing
Timed pregnancies of heterozygous females were scheduled for embryo harvesting at 5.5 to 14.5 days gestation. At the earliest times (E5.5–8.5), mothers were perfused with PBS/Z-Fix solution (zinc-buffered formalin; Anatech Inc., Battle Creek, MI, USA) and the entire uteri were removed, fixed, and embedded in paraffin for histological investigation. For all remaining stages, the uteri with embryos were dissected under deep anesthesia and placed in Petri dishes containing tissue culture medium (Dulbecco with 10% FBS). Further preparation included embryo removal from deciduas under a stereomicroscope. Yolk sac vascularization status was registered. Whole, isolated embryos were photographed using either a stereomicroscope or a bright-field phase-contrast microscope. Embryonic stage for timed pregnancies at 9.5–12.5 postconception was estimated by somite counts. The embryos were postfixed in Z-Fix, embedded in paraffin, sectioned, and stained with H&E or Masson’s trichrome.
Transmission electron microscopy analysis
Whole E9.5 embryos were fixed with Karnovsky standard 2% formalin and 2% glutaraldehyde in 0.1 M cacodylate buffer solution (pH 7.4) (16)
at room temperature, washed, and postfixed in 1% osmium tetroxide in the same buffer. Samples were dehydrated in ethanol, then embedded in Eponate 12 epoxy resin (Ted Pella Inc., Redding, CA, USA) and sectioned on Reichert-Jung Ultracut microtome. Semithin sagittal sections of whole embryos were stained with Toluidine blue to locate and mark the heart regions. The tissue was then trimmed around areas of interest for ultrathin sectioning. The ultrathin sections were stained with uranyl acetate, followed by lead citrate, and imaged using a Hitachi H-600 electron microscope at 75 kV.
For morphometric analysis, electron microscopy (EM) micrographs of ultrathin sections were scanned at a resolution of 2000 ppi using a DuoScan T2500 (Agfa Corp., Ridgefield Park, NJ, USA). The digitized images were subjected to morphometric analysis using Image Pro Plus, v.4.5 (Media Cybernetics, Silver Spring, MD, USA). Measurements of cell areas and areas of mitochondria of WT (dap3+/+) and knockout (dap3–/–) cells were exported into MS Excel 2002. Statistical significance of data was determined by an unpaired t test.
Immunoblot analysis
Protein samples (50 µg) were mixed with an equal volume of 2x SDS sample buffer, boiled for 5 min, then subjected to SDS-PAGE (10 or 12.5% gels). After electrophoresis, proteins were transferred to nitrocellulose membranes by a semidry electrophoretic transfer method. The membranes were blocked in a Tris-buffered saline (TBS) containing 10 mM Tris (pH 7.4), 150 mM NaCl) 5% dry milk (1 h), then rinsed and incubated with primary antibodies 1:100 v/v, rabbit anti-DAP3 (IgG, #IMG-295, Imagenex Technology Corp., Port Coquitlam, BC, Canada) or -HSP60 (clone LK-1, IG1, #SPA-806; Stressgen Bioreagents Corp., Ann Arbor, MI, USA) in TBS (10 mM Tris [pH 7.4], 150 mM NaCl) overnight at 4°C. Membranes were washed four times in TBS, then 0.1 µg/ml peroxidase-labeled mouse or rabbit secondary antibody (Ab) was added for 1 h. After four washes in TBS, bands were visualized by enhanced chemiluminescence with exposure to X-ray film. After photography, the same blot was stripped frequently for 15 min in 0.2 M glycine, pH 2.5, preblocked, and immunolabeled with additional antibodies as above.
Mouse embryo cell cultures
Embryos from pregnant mice at day 9 postcoitus were sacrificed. Both uterine horns containing the fetuses were taken and the deciduas were separated away. Yolk sacs with amnion were collected for genotyping. Cell suspensions from whole fetuses were generated in Dulbecco’s modified Eagle medium (DMEM) -F12 medium (Life Technologies, Carlsbad, CA, USA) by mincing tissue with 30 gauge needles, followed by partial digestion in trypsin-EDTA (Life Technologies) supplemented with collagenase (Sigma, St. Louis, MO, USA; C-9891) at 0.5% w/v trypsin and 20 µg/ml collagenase final concentrations. The cell suspension was plated on fibronectin (Invitrogen, Carlsbad, CA, USA) -coated (15 µg/ml) 12-well dishes in DMEM-F12 containing 15% FBS, 2% penicillin/streptomycin, 50 µg/ml L-uridine (Sigma), 0.1 mM nonessential amino acids (Invitrogen), and 1 mM sodium pyruvate (Sigma). Cells were cultured at 37°C in 95% air/5% CO2.
HeLa cell culture and siRNA transfections
HeLa human cervical carcinoma cells were maintained in DMEM medium supplemented with 10% heat-inactivated FBS, penicillin G (100 IU/ml), streptomycin (100 µg/ml), and L-glutamate (2 mM) and cultured at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. Exponentially growing HeLa cells were seeded at 106 cells per 6-well plate or at 107 cells per 10 mm dish, then cells were exposed to various agents as indicated below. Twenty-one nucleotide pair, double-stranded RNA were chemically synthesized, deprotected, and purified by Dharmacon Research Inc. (Lafayette, CO, USA). The sequence of the sense strand of the DAP3 siRNA is 5'-CCAGGTTCCAGTTGAGAGT-3'. A mutant DAP3 siRNA of sequence 5'-CCGAGTTCCAGTTGAGAGT-3' was used as a negative control. Transfection of siRNA duplexes was performed using oligofectamine (Invitrogen). HeLa cells in 0.2 µl medium in 24-well plates at 30–50% confluence were transfected with 2 µl oligofectamine and 3 µl of siRNA containing 60 pmol dsRNA. Cells were incubated in DMEM without serum or antibiotics at 30°C for 5 h, then replaced with DMEM containing 10% FBS and antibiotics. At 2 days post-transfection, cells were lysed and immunoblotted with DAP3 Ab.
Oxygen consumption measurements
Cellular oxygen consumption rates were measured as described previously (17)
. Aliquots of cells were placed into a respirometer fitted with a polarographic oxygen electrode. The electrode was calibrated with a humidified gas mixture containing various oxygen levels. Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma) (5 µM) or Gramicidin D (Sigma) (1 µg/ml) was added directly to the cell suspensions in the respirometer and the resultant rate of oxygen consumption was measured.
Viability assays
Mouse embryonic cells were seeded at 20,000 cells per well in 96-well dishes and allowed to attach overnight. Cells were then treated with cycloheximide (Sigma) at 10 µg/ml and agonistic Fas Ab (clone Jo2, BD PharMingen, San Diego, CA, USA), mouse recombinant TNF (R&D Systems, Minneapolis, MN, USA), or human TRAIL (BIOMOL) at various concentrations. Alternatively, cells were treated with staurosporine or etoposide (both from Sigma) at increasing concentrations without cycloheximide. Cells were treated overnight, then assayed by MTS (Promega, Madison, WI, USA). Percentage viability was calculated from optical density at 490 nm readings taken with a Powerwave x340 ELISA plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) by normalizing readings to control values from untreated cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A), a line of heterozygous mice was produced that contained one targeted and one normal dap3 allele. A PCR was designed using amplification primers flanking the insertion site in dap3 and used for genotyping mice and embryos (Fig. 1B
). Southern blot analysis also confirmed the presence of the proviral insertion in intron 1 (Fig. 1B
).
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Homozygous targeting of dap3 is embryonic lethal
Attempts to breed dap3± mice to homozygosity never resulted in live offspring among >50 successful dap3± matings, suggesting that homozygous dap3 gene ablation is embryonic lethal. Inspection of embryos from timed pregnancies, in combination with PCR-based genotyping, indicated that dap3–/– embryos fail to progress beyond E9.5. A comparison of E9.5 embryos of dap3+/+ and dap3–/– embryos revealed that the dap3–/– embryos were markedly shrunken and poorly developed (Fig. 2
A, B). Though rostral-caudal relations were preserved, these embryos lacked axial turning and appeared to be highly anemic. Histological examination showed that although the placenta of dap3–/– embryos was smaller, it nevertheless appeared to be properly formed initially, but deteriorated later in some embryos (Fig. 2C
). In contrast, heterozygous dap3± embryos developed normally (not shown).
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Defective mitochondria in dap3–/– embryos
To further evaluate the defects in dap3–/– embryos, we performed transmission electron microscopy (TEM) analysis. Unlike dap3+/+ and dap3± embryos, cells within dap3–/– embryos contained mitochondria with abnormal morphology in all tissues examined. Two types of abnormal mitochondria were observed, including mitochondria with overall normal size and shape, but with swollen cristae and mitochondria that were shrunken with increased electron density (Fig. 3
). In contrast, no abnormalities were noted in other organelles (Golgi, ER, nuclear envelope, lysosomes) of dap3–/– embryos by EM analysis (Fig. 3
and data not shown).
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Morphometric analysis of mitochondria of dap3+/+ and dap3–/– embryos revealed that the average size of mitochondria was 2.0-fold smaller (P=0.0002) in dap3–/– embryos at E9.5 than dap3+/+ embryos (Fig. 4
). In contrast, no statistically significant difference was detected in the average number of mitochondria per cell (Fig. 4)
or overall size of cells of dap3–/– embryos as judged from surface area measurements using the same TEM data (not shown). The dap3+/+ and dap3± embryos were indistinguishable (not shown).
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Characterization of mitochondria in DAP3-deficient cells
The altered size and density of dap3–/– mitochondria made it difficult to obtain high-quality isolation of these organelles. We therefore sought alternative ways of characterizing some of the properties of mitochondria in dap3–/– embryos. Immunoblot analysis confirmed that dap3–/– embryos fail to produce the DAP3 protein (Fig. 5
A). The absence of DAP3 protein was also confirmed by immunohistochemical analysis of dap3–/– embryos (not shown). Equal loading of protein samples was confirmed by probing the same blots with antibodies recognizing cytosolic ß-actin and mitochondrial Hsp60, a resident mitochondrial protein encoded in the nuclear genome. Note that all of the detectable Hsp60 migrated as the mature form of the protein, indicative of removal of the N-terminal leader peptide during import into mitochondria. Moreover, immunostaining of dap3–/– embryos revealed punctuate localization of Hsp60 protein in cytosol of cells, typical of mitochondria-targeted proteins (not shown). However, in dap3–/– embryos we observed reduced levels of cytochrome c oxidase-I (COI), a protein encoded in the mitochondrial genome that is produced within these organelles by transcription and translation from the mitochondrial gene expression machinery (Fig. 5A
). Scanning densitometry analysis of blots suggested a decrease in COI protein levels of approximately half compared with dap3+/+ embryos after normalization for Hsp60 or ß-actin. The defect in COI expression was not secondary to fewer mitochondria genomes on average, as determined by quantitative Southern blot analysis of low molecular weight DNA (Fig. 5B
).
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Mammalian DAP3 is required for oxidative respiration
To ascertain the respiratory competency of DAP3-deficient cells, we used an oxygen electrode to measure oxygen consumption in intact cells. Because we were unable to grow sufficient numbers of dap3–/– cells from embryos to accomplish these O2 consumption measurements, we resorted to use of established cell lines in which DAP3 expression was ablated by transfection of small interfering RNA (siRNA). As shown in Fig. 6
A, transfection of DAP3 siRNA oligonucleotides into HeLa cells resulted in essentially complete loss of DAP3 protein production, while a mismatched control RNA duplex did not. Oxygen consumption by DAP3-deficient HeLa cells was reduced 5-fold compared with control cells (Fig. 6B
), indicating that DAP3 protein is required for mitochondrial respiration.
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Defective apoptosis in DAP3-deficient cells
It has been reported that antisense-mediated reductions in DAP3 reduce the sensitivity of tumor cell lines to apoptosis induction by TNF family death receptors and ligands, including Fas and TNF. We therefore, examined the sensitivity of mouse embryonic cells derived from dap3+/+ vs. dap3–/– embryos to apoptosis-inducing agents. Cells cultured from dap3–/– embryos grew very little in culture. Despite the use of media adapted for culturing rho-zero cells with mitochondrial defects, dap3–/– cells from E9.5 embryos were capable of surviving but did not propagate significantly in culture. Attempts to immortalize dap3–/– cells by infection with recombinant retroviruses encoding SV40 large T antigen were also unsuccessful.
We therefore explanted embryonic dap3–/–, dap3±, and dap3+/+ cells into culture on 24-well plates and used them within 24 h for cell death assays. When challenged with staurosporine (STS), a broad-spectrum kinase inhibitor that induces apoptosis via the intrinsic pathway, little difference in apoptosis sensitivity was noted in comparisons of dap3–/– and dap3+/+ MEFs (Fig. 7
). Similarly, a challenge with VP16, a DNA-damaging agent, revealed no significant difference in cell death induced in cultures of dap3+/+, dap3±, or dap3–/– cells (not shown). In contrast, dap3–/– cells demonstrated reduced sensitivity to cell death induced by stimulation of TNF family death receptors using TNF, TRAIL, or anti-Fas Ab in combination with cycloheximide. Cells from dap3± heterozygous embryos exhibited intermediate sensitivity (Fig. 7)
. Similar results were obtained in experiments where the percentages of apoptotic cells were evaluated by staining with FITC-annexin V and propidium iodide (not shown). We conclude therefore that loss of dap3 expression is associated with reduced sensitivity of mouse embryonic cells to apoptosis induced via TNF family death receptors, consistent with prior results obtained by use of antisense methods.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E9.5. Mitochondria in these developing embryos show morphological defects, including reduced size and swollen crystae, whereas other organelles appear to be intact. These observations are consistent with prior data indicating that DAP3 is imported into mitochondria, where it associates with mitochondrial ribosomes. It has been speculated that DAP3 functions akin to other GTP binding proteins involved in ribosome-mediated translation of mRNAs, including Ski7p of Saccharomyces cereviasiae and EF-Tu of Homo sapiens (18
, 19)
. One of the GTP binding proteins, EF-Tu (elongation factor Tu) in the GTP-bound conformation forms a high-affinity complex with aminoacyl-tRNA that binds to the ribosomal A site. In addition, the Ski7p’s C-terminal region, which has homology to the GTPase domains of EF1A (the translation elongation factor), can bind the empty A site on the ribosome stalled at the 3' end of RNAs. As a result of GTP hydrolysis, EF-Tu changes conformation from the GTP to the GDP form, where it loses the affinity for aa-tRNA. Consistent with a potential role for DAP3 in mitochondrial protein synthesis, we observed that levels of a protein encoded in the mitochondrial genome (COI) are reduced in DAP3-deficient tissues.
Several proteins encoded in the mitochondrial genome are required for respiration, and thus we predicted that DAP3-deficient tissues would show defects in oxygen consumption. Due to the inability to obtain sufficient numbers of intact mitochondria from dap3–/– embryos, we addressed this question by using siRNA to reduce DAP3 protein production in cultured intact cells. Indeed, the loss of DAP3 expression resulted in significant impairment of respiration, consistent with previous data from yeast where disruption of yDAP3 (YGL129c) renders cells unable to grow on substrates that require aerobic metabolism (20)
.
A curious property of DAP3 is that it not only plays important roles in mitochondrial physiology, but also participates in apoptosis. DAP3 was first identified in an antisense screen for genes required for cytokine-induced apoptosis (7)
and was shown to be involved in cell death pathways activated by TNF family cytokine receptors (e.g., Fas; TRAIL-Rs; TNFR1) but not pathways induced by DNA-damaging agents or the broad-spectrum kinase inhibitor STS (10)
. DNA-damaging agents and STS are known to induce cell death through a mitochondria-dependent pathway involving release of cytochrome c from these organelles (21
, 22)
, whereas TNF family death receptors can induce apoptosis through either mitochondria-independent or -dependent mechanisms, depending on the type of cell examined (23
, 24)
. Previously, we showed that a cytosolic pool of DAP3 exists in many cell lines, in addition to the mitochondrial pool of DAP3 (12)
, and demonstrated that DAP3 protein associates with TNF family death receptor complexes, where we speculate that it operates as a chaperone to facilitate assembly of the caspase-activating complex known as the "death-inducing signaling complex" (11)
. Here we extended these studies showing that cells from dap3–/– embryos undergo significantly less apoptosis when stimulated with agonists of TNF family death receptors compared with dap3+/+ cells. Heterozygous dap3± cells showed an intermediate reduction in sensitivity to cell death induction by TNF family death receptors.
It has been suggested that DAP3 regulates mitochondrial fission (5)
. A role for mitochondrial fission in cell death has emerged recently, with many apoptosis-inducing agents triggering this phenomenon and several proteins implicated in mitochondria fusion/fission showing an ability to modify the kinetics of cell death in vitro (reviewed in ref. 25
). At present, it is unclear whether the effects of DAP3 on mitochondrial fission represent a direct (proximal) effect of this GTP binding protein on some aspect of the fission mechanism vs. an indirect (distal) reflection of its role in regulating apoptosis.
Though many questions remain to be resolved about the roles of DAP3 in both mitochondrial physiology and apoptosis, the findings reported here provide further evidence that DAP3 is a bifunctional protein and, as such, is similar to several other mitochondrial proteins that have been reported to have dual functions in both mitochondrial biology and cell death regulation. For example, cytochrome c is required for electron transfer from complex III to IV in mitochondria, but also is an essential activator of Apaf1, a caspase-activating protein (26
, 27)
. HtrA2 is a mitochondrial serine protease homologous to the bacterial DegP/Omi protein (28)
, which also binds antiapoptotic IAP family proteins when released into the cytosol, freeing caspases to induce apoptosis (29
, 30)
. Bit (brain immunoglobulin-like molecule with tyrosine-based activation motifs) is a mitochondrial peptidyl-tRNA hydrolase that also modulates the activity of transcription factors controlling expression of antiapoptotic gene bcl-2 when released into the cytosol (31)
. AIF is a mitochondrial flavoprotein of unknown function that is released into cytosol and enters the nucleus to promote genome digestion during apoptosis (32)
. Endonuclease G is a mitochondrial protein involved in mitochondrial DNA replication that is released into the cytosol during apoptosis and induces nuclear DNA fragmentation (33)
. These mitochondrial proteins are all encoded in the nuclear genome, undergo import into mitochondria, and sort to the intermembrane space between the inner and outer membranes of these organelles, becoming released into the cytosol during apoptosis. In contrast, DAP3 is sorted to the matrix of mitochondria and does not appear to be released into cytosol during apoptosis (5
, 13)
. Under normal conditions, however, a pool of nonmitochondrial DAP3 appears to be resident in cells, the amount varying among cell lines examined (12)
. The cytosolic activity of DAP3 is known to be regulated by phosphorylation, where Akt (PKB) -mediated phosphorylation of DAP3 inhibits its proapoptotic role in the context of signaling by TNF family death receptors (14)
. Thus, the participation of DAP3 in apoptosis may be regulated at several levels, including 1) gene expression; 2) protein localization (sequestration in mitochondria); and 3) phosphorylation. Additional studies of this interesting GTP binding protein may help provide further insight into its biochemical mechanisms and DAP’s dual functions in mitochondrial physiology and cell death regulation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Department of Dental Pharmacology, School of Dentistry, Wonkwang University, Iksan, Chonbuk, South Korea.
3 Present address: Department of Pharmacology, School of Medicine, Chonbuk National University, Jeonju, Chonbuk, South Korea.
4 Present address: Department of Bioresources, Hokkaido University, Research Center for Zoonoisis Control, North-21, West-11, Kita-ku, Sapporo 001-0021, Japan.
Received for publication April 19, 2006. Accepted for publication August 7, 2006.
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REFERENCES |
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-induced cell death. J. Biol. Chem. 270,27932-27936 and Fas-induced cell death. EMBO J. 18,353-362[CrossRef][Medline]This article has been cited by other articles:
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  J. L. Miller, H. Koc, and E. C. Koc Identification of phosphorylation sites in mammalian mitochondrial ribosomal protein DAP3 Protein Sci., February 1, 2008; 17(2): 251 - 260. [Abstract] [Full Text] [PDF]
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700 bp containing 1st exon and the 1st intron proximal to the insertion site of the retroviral vector). Molecular weight markers are indicated in kilobase pairs. Data are representative of at least 3 mice analyzed for each phenotype.
