Atad3 Function Is Essential for Early Post-Implantation Development in the Mouse

The mitochondrial AAA+-ATPase ATAD3 is implicated in the regulation of mitochondrial and ER dynamics and was shown to be necessary for larval development in Caenorhabditis elegans. In order to elucidate the relevance of ATAD3 for mammalian development, the phenotype of an Atad3 deficient mouse line was analyzed. Atad3 deficient embryos die around embryonic day E7.5 due to growth retardation and a defective development of the trophoblast lineage immediately after implantation into the uterus. This indicates an essential function of Atad3 for the progression of the first steps of post-implantation development at a time point when mitochondrial biogenesis and ATP production by oxidative phosphorylation are required. Therefore, murine Atad3 plays an important role in the biogenesis of mitochondria in trophoblast stem cells and in differentiating trophoblasts. At the biochemical level, we report here that ATAD3 is present in five native mitochondrial protein complexes of different sizes, indicating complex roles of the protein in mitochondrial architecture and function.


Introduction
ATAD3 belongs to the ancient family of AAA+-ATPases (ATPases associated with a wide variety of cellular activities) [1]. The protein structure of ATAD3 is characterized by two Nterminal coiled-coil domains, a central trans-membrane segment and a conserved C-terminal ATPase domain of the AAA+-type with an ATP-binding (Walker A motif) and a catalytic ATPase domain (Walker B motif) [2]. AAA+-ATPases are proposed to be chaperones or proteases and are involved in a variety of cellular processes e.g. cell cycle regulation, biogenesis of cell organelles and dis/assembly of protein complexes [3][4][5].
Except for humans, only one ATAD3 gene locus is present in most species. During the development of the human lineage three genes, ATAD3A, ATAD3B [2] and ATAD3C [6], have likely been evolved by replication of a single precursor gene, as these three genes form a tandem array on chromosome 1.
A localization of ATAD3 to mitochondria was shown in several studies [2,7,8]. Analysis of ATAD3A topology in mitochondria by employing trypsin digestion experiments showed that the Cterminal AAA+-ATPase domain is located in the matrix, whereas a central trans-membrane segment anchors the protein in the inner membrane. The N-terminal domain interacts with the outer membrane [2]. It remains unclear, however, whether the Nterminus is spanning through the outer membrane into the cytosol. Nevertheless, an oligomerization of ATAD3A monomers has been proposed [2] which is supported by findings showing that other AAA+-proteins are assembling as hexameric rings [4,9,10].
It was proposed that ATAD3 is involved in the control of mitochondrial dynamics [2,7]. Mitochondrial dynamics is medi-ated by fission and fusion of mitochondria, which are important for cell viability [11][12][13][14]. Also, the mitochondrial network gets fragmented during apoptosis resulting in smaller and more numerous mitochondria [15][16][17][18]. Down-regulation of ATAD-3 in Caenorhabditis elegans and in cultured human cells gave opposite effects on the mitochondrial network, respectively. Following RNAi of ATAD-3 in Caenorhabditis elegans, the mitochondria appeared thinner and slightly disorganized and the mitochondrial network was more filamentous [19]. In contrast, RNAi of ATAD3 in HeLa and lung cancer cells showed increased mitochondrial fragmentation and additionally a decreased co-localization of mitochondria and endoplasmatic reticulum (ER) [2,6]. Mitochondrial fragmentation was also observed following overexpression of a Walker A deficient version of ATAD3A [2,7]. Thus, ATP-bound ATAD3A might be required for the maintenance of mitochondrial integrity in mammalian cells [2].
Co-immunoprecipitation and two-dimensional immuno-blotting of mitochondria-associated membrane fractions revealed bindings of ATAD3A to the mitochondrial fission protein dynamin-related protein 1 (DRP1) and to the mitochondrial fusion proteins mitofusin-2 (MFN2) and optic atrophy 1 (OPA1) [6]. DRP1 is a GTPase that is normally located to the cytoplasm, but is recruited to the mitochondria by binding to its receptor Fis1, which resides in the outer mitochondrial membrane [20,21]. DRP1 mediates fission of mitochondria by formation of a homomultimeric complex [22][23][24]. MFN2 and the structurally related mitofusin-1 (MFN1) are GTPases, which are located in the outer mitochondrial membrane [25][26][27][28]. A fraction of MFN2 is also present in the membrane of the ER, where it mediates the contact between ER and mitochondria by homotypic (MFN2-MFN2) and/or heterotypic (MFN2-MFN1) binding [29][30][31]. The GTPase OPA1 is incorporated into the inner mitochondrial membrane, where it mediates fusion of the inner membrane and christae formation [28,[32][33][34]. It was shown that YME1L1 and m-AAA protease, members of the family of AAA+-ATPases regulate OPA1 processing and mitochondrial fusion [35,36]. An interaction of ATAD3A with the apoptosis inducing factor AIF was proposed [6]. During apoptosis AIF is released from the mitochondrial intermembrane space and locates to the nucleus where it interacts with histone H2AX and promotes chromatinolysis [37,38]. Additionally, an interaction between ATAD3A and calcium-binding protein S100B was shown [39]. Finally, ATAD3A bindings to the D-loop of the mitochondrial DNA (mtDNA) molecule and to ribosomes, and its involvements in the regulation of mtDNA replication, transcription of mtDNA encoded genes and mitochondrial protein synthesis were discussed [40][41][42][43][44].
An essential role of ATAD3 for development was demonstrated in Caenorhabditis elegans, because RNAi of ATAD-3 in the worm system causes severe defects, characterized by early larval arrest, gonadal dysfunction and embryonic lethality [19].
To date analyses of the localization and the function of ATAD3 family members were performed in human cell lines or in Caenorhabditis elegans. Until now there is no genetic evidence for the relevance of ATAD3 function in mammalian development or disease. In this article the early post-implantation phenotype of an Atad3 loss-of-function mutation in the mouse is described. Furthermore we analyzed the contribution of ATAD3 to the formation of native mitochondrial protein complexes.

Results
In the Mouse Two Atad3 Protein Isoforms are Generated from a Single Gene by Alternative Splicing In murine mRNA and protein databases (NCBI, Ensembl), two Atad3 isoforms are annotated, which are derived from a single gene by alternative splicing. Isoform 1 is referred as the full length Atad3 cDNA of 2412 bp with an open reading frame of 1776 bp encoding for a protein composed of 591 amino acids and with a molecular weight of 66.742 kDa. Alignment of mouse Atad3 isoform 1 cDNAs (accession numbers NM_179203 and BC058373) and the mouse genomic sequence (accession number NT039268) by BLAST (Basic Local Alignment Search Tool) indicates that the murine Atad3 gene is located on chromosome 4 and is composed of 16 exons, extending over a genomic locus of around 20.5 kb (Fig. 1A). Additionally a highly (96%) homologous sequence of similar length (2316 bp) is located on chromosome 15. This sequence likely is thought to be a pseudo-gene as it is typically composed of only a single exon-like section without any intronic interruptions. Atad3 isoform 2 encodes a shorter protein of 512 amino acids and a molecular weight of 57 kDa. Isoform 2 is generated by alternative splicing of exons 13 and 14, which leads to a subsequent translational frame shift. The murine Atad3 protein isoform 1 shows an identity of 92.1% in its amino acid sequence to the human orthologue ATAD3A (NP_001164007) which has a molecular weight of 66 kDa. Both murine isoforms contain two N-terminal coiled-coil domains, central trans-membrane segments, and Walker A and Walker B motifs, respectively. Interestingly, the C-terminal portion of the AAA+-ATPase domain, directly positioned after the Walker B motif in isoform 1, is missing in isoform 2.
Gene Trap Disruption of the Murine Atad3 Gene Leads to a Loss-of-function Mutation The E14TG2a.4 (129SV2) ES cell clone E118D03 (offered by the German Gene Trap Consortium) carrying a gene trap mutation in one Atad3 allele (Atad3 GT ) was used to establish a stable mouse line that had transmitted the mutation into the germ line. In this ES cell clone, the gene trap vector rFlipROSAbetageo(Cre)0 is integrated after nt 402 into the first intron of the Atad3 gene, generating a fusion transcript by splicing Atad3 exon 1 at its splice donor site (SD) to the splice acceptor site (SA) of a transgenic cassette (bgeo), which encodes for the bacterial LacZ reporter gene and a neomycin phosphotransferase selection marker. Termination of transcription is mediated by a SV40 polyadenylation signal (Fig. 1A). A RT-PCR approach for amplification of a sequence containing exons 11 to 16 failed to detect a cDNA transcript, which proves that the described gene trap event in the Atad3 locus leads to a complete loss of the 3?encoded region in Atad3 GT/GT tissues (Fig. 1B) and therefore represents a loss-of-function mutation. The resulting fusion protein contains only the first 67 amino acids of the wildtype Atad3 protein, i.e. the N-terminal part of the first coiled-coil domain. As the trans-membrane and the AAA+-ATPase domain are completely missing, the mutant protein is rendered dysfunctional.
Genotyping of mice and embryos was performed by PCR, employing three primers. The wildtype allele is represented by an 813 bp long fragment, whereas the mutant allele (Atad3 GT ) is characterized by a 273 bp fragment of endogenous and transgenic origin (Fig. 1A, C).

Atad3 GT/GT Embryos Exhibit Retarded Post-implantation Development and Die Around E7.5
Genotyping showed that heterozygous Atad3 (Atad3 GT/+ ) mice are viable and fertile. Atad3 GT/+ mice exhibit no obvious phenotype. When offspring from heterozygous parents was genotyped, no homozygous mutants (Atad3 GT/GT ) were obtained, suggesting that the mutation results in recessive lethality during embryonic development. Indeed, the genotype distribution of embryos obtained from heterozygous intercrosses which were isolated between the stages E6.5 to E8.5 reveals that Atad3 GT/GT embryos die before E8.5. Between E6.5 and E8.5 the ratio of vital Atad3 GT/GT individuals decreases from 20.6% to 0.0%, whereas the ratio of detectable resorptions increases markedly from 5.9% to 32.9% ( Table 1). Because of the complete degradation of the respective embryonic tissues, resorptions were not genotyped. Detectable numbers of Atad3 GT/GT embryos and resorptions at the analyzed embryonic stages are found to be close to the expected Mendelian ratio of 25%. All Atad3 GT/GT embryos are developmentally retarded and show the same abnormal morphology. The phenotype is characterized by a low variability in size and morphology of the mutant embryos at E6.5 (n .14) and E7.5 (n .12) and a constant time point of lethality between E7.5 and E8.5. Compared to wildtype embryos at the egg cylinder stage E6.5 ( Fig. 2A), Atad3 GT/GT embryos show a total growth reduction, have an oval to conic shape, and specifically the proximo-distal axis is not extended (Fig. 2B). Furthermore, the ectoplacental cone, marked by its red colour is not visible in Atad3 GT/GT embryos, indicating that the differentiation of extra-embryonic tissue is disturbed and reduced (Fig. 2B). As the overall growth of murine embryos is minimal between E5.5 and E7.5, only an embryo of the final vital stage E7.5 is depicted in Figure 2B. Histological analysis gives a more precise view on the developmental retardation of Atad3 GT/GT embryos. Along their proximo-distal axis, wildtype egg cylinder stage embryos have developed three tissues, which are the embryonic ectoderm, the extra-embryonic ectoderm and the ectoplacental cone (Fig. 2C). Embryonic ectoderm and extraembryonic ectoderm are surrounded by the endoderm. In contrast, Atad3 GT/GT embryos (n = 3) at the gastrula stage (E7.5) resemble wildtype embryos of the stage E5.5, because internal cavitation is completely missing. The ectoplacental cone and also the extra-embryonic ectoderm are at least strongly reduced, maybe even completely absent. Additionally, the embryonic ectoderm and endoderm appear less differentiated (Fig. 2D). Absence of a proamniotic canal clearly indicates that the Recognition sites (rec) for Flp and Cre recombination enzymes allow first the reconstitution of the wildtype mRNA and secondly a subsequent conditional mutagenesis. In the lower scheme, arrows indicate the positions of the PCR primers EF (endogenous intron 1 forward primer), ER (endogenous intron 1 reverse primer) and VR (gene trap vector reverse primer) used for genotyping. Constitution of wildtype (813 bp) and mutant (273 bp) PCR fragments are shown below. B RT-PCR analysis of E6.5 embryonic samples shows that the complete 3? region (exons 11 to 16) of the Atad3 cDNA is missing in Atad3 GT/GT embryos, instead in wildtype and Atad3 G/+ tissues a 805 bp fragment represents the Atad3 cDNA. Amplification of ribosomal protein S6 (RPS6) cDNA serves as quantity control. C For genotyping of the Atad3 locus, wildtype and mutant genomic PCR amplificates are separated in a 1.5% agarose gel and identify samples from wildtype (+/+), heterozygous (GT/+) and homozygous mutant (GT/GT) individuals. doi:10.1371/journal.pone.0054799.g001 Atad3 Function in the Mouse PLOS ONE | www.plosone.org development of the embryonic ectoderm is also affected by the mutation. But since firstly, the effect of the mutation appears to be more dramatic on the formation and differentiation of extraembryonic tissues, and since secondly, the extra-embryonic tissue is known to have a strong influence on the proximo-distal growth and survival of the complete embryo during early gastrulation, further analyses were focused on the importance of Atad3 function on trophoblast development.

In vitro Cultured Atad3 GT/GT Embryos Only Form a Minimal Trophoblast Outgrowth
To investigate the effect of Atad3 deficiency on the formation and function of extra-embryonic tissue on the subcellular level, mitochondrial morphology and trophoblast differentiation was analyzed in outgrowths of E6.5 embryos, in?vitro. Cultivated postimplantation embryos continue with the imminent steps of the developmental program. In wildtype embryos of this stage (n = 26), trophoblast derived tissue grows out radially from an already existing small ectoplacental cone, and forms more differentiated  (Fig. 3A). Although attachment to the gelatine covered dish is mainly mediated by trophoblast tissue, most Atad3 GT/GT embryos (n = 9) are able to adhere indicating the existence of minimal tissue emerging from the polar trophectoderm. Compared to the wildtype tissues, the size of the epiblast remains clearly diminished in Atad3 GT/GT embryos even after three days of culture (Fig. 3B). During three to four days of culture, Atad3 GT/GT embryos form only minimal outgrowths (Fig. 3B). The variability in sizes of the outgrowths is up to 50%. In wildtype embryos, immuno-staining reveals a moderate amount of Atad3 protein in both the epiblast and the trophoblast (Fig. 3C). In contrast, Atad3 protein is only weakly expressed in the epiblasts of Atad3 GT/GT outgrowths and is almost undetectable in the trophoblast (Fig. 3D). Existence of Atad3 protein in the mutant epiblast might be due to persisting protein contribution of the oocyte throughout pre-implantation development until the early post-implantation stages.

Mitochondrial Morphology is Modified during Trophoblast Development
The modification of the mitochondrial morphology and the mitochondrial network during trophoblast differentiation was studied by co-immunostaining for Atad3 and Mash2 (mouse achaete-scute homolog 2) in wildtype outgrowths (n = 13). Atad3 and its orthologues in other species (Homo sapiens, Drosophila melanogaster) are excellent trackers for mitochondrial populations in cells, as they are abundantly expressed in this organelle and span the inner membrane, the inter-membrane space and probably also the outer membrane [2]. Differentiation of the trophoblast lineage from the proximity of the center to the distal area of the outgrowth is characterized by the expression and nuclear import of the bHLH transcription factor Mash2, which indicates differentiated trophoblasts and trophoblast giant cells. In cells of the proximal trophoblast, where Mash2 protein is localized to the cytoplasm (Fig. 4B), the mitochondria are small and diffusely distributed (Fig. 4A, C). In the distal zone of the outgrowths the cells contain enlarged, swollen mitochondria (Fig. 4D, F, G), and additionally small mitochondria arranged in arrays from the center to the periphery of the cell (Fig. 4D, F). In these cells, Mash2 protein has translocated into the nucleus as expected (Fig. 4E-G), but surprisingly is also detected in the matrix of the swollen mitochondria (Fig. 4G).

Atad3 GT/GT Embryos are Defective in Trophoblast Stem Cell Maintenance and Trophoblast Differentiation
Next we wanted to elucidate whether Atad3 deficiency had an effect on mitochondrial morphogenesis, intrinsic apoptosis and differentiation of the trophoblast. If cell death were the reason for the reduction of trophoblast size, intrinsic apoptosis might be expected, which is characterized by the release of cytochrome c from mitochondria to the cytoplasm. In wildtype cells (n = 8) the punctate cytochrome c expression as well as the explicit colocalization with the mitochondrial tracker Atad3 indicates that intrinsic apoptosis does normally not occur in the trophoblast (Fig. 5A'). In Atad3 GT/GT cells (n = 3), a robust punctate cytochrome c pattern is also apparent, indicating that intrinsic apoptosis in the mutant trophoblast can be excluded (Fig. 5B'). Additionally, the cytochrome c expression pattern itself highlights regularly formed mitochondria in the few cells, of which the Atad3 GT/GT trophoblast consists. Fragmentation of the nuclei is a general hallmark for apoptosis. But neither in wildtype (n = 26) nor in Atad3 GT/GT embryos (n = 9), DAPI staining reveals any fragmented nuclei. Since accelerated apoptosis could be ruled out, the competence of Atad3-defective trophoblast stem cells to differentiate into mature trophoblast cells was monitored by immuno-staining for the differentiation marker Mash2. And indeed, Mash2 is rarely detectable in cells of the complete Atad3 GT/GT trophoblast outgrowth (n = 13) (Fig. 5D') as compared to wildtype cells (n = 3) (Fig. 5C'), proving the disability of trophoblast stem cells or their very early descendants to differentiate into later trophoblast cell types or trophoblast giant cells. Additionally, the expression of several marker genes for trophoblast stem cells (Cdx2), extra-embryonic ectoderm (Bmp4) and ectoplacental cone (Mash2, Hand1) in wildtype (n = 3 pools of minimum 4 embryos) and Atad3 GT/GT embryos (n = 3 pools of minimum 4 embryos) of the stage E6.5 was monitored by RT-PCR (Fig. 5E). Interestingly, Cdx2 and Bmp4 mRNAs are not verifiable at all. Likewise, Mash2 and Hand1 transcripts are rarely traceable in Atad3 GT/GT embryos. This indicates that not only the differentiation within the early trophoblast is impaired, but already the maintenance of the trophoblast stem cell pool is affected in Atad3 GT/GT embryos.

ATAD3 Contributes to Five Mitochondrial Protein Complexes of Different Molecular Weight
Apart from investigating its function during mouse development, we were interested in the mitochondrial topology of ATAD3 protein complexes. It was already proposed that ATAD3 forms oligomers which might span both the inner and outer mitochondrial membranes [2]. Therefore, native mitochondria from diverse human cell lines (HeLa, Jurkat E6, HEK293) and murine ES cells were isolated and subjected to Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) and immuno-blotting. In all analyzed cells of human and murine origin, a highly similar pattern of five protein complexes containing ATAD3A/Atad3 was found (Fig. 6A). The largest complex of about 800 to 900 kDa is the most abundant one. The smaller sub-complexes have estimated molecular weights of about 720, 600, 480 and 240 kDa (Fig. 6B). Since the proteins maintain in their native conformation during BN-PAGE, the size estimation may have an expected size error of up to 15%. Coincidentally, some of the sub-complexes co-migrate with bands of the used protein standard (Apoferritin, B-phycoerythrin). To exclude any effects from contamination of the samples, the pattern was verified in several experiments and was also seen in lanes distant from the one which contained the protein standard. Also, the variation of the detergent concentrations ranging from a detergent -protein ratio from 2:1 to 20:1 had no influence on the complex pattern (data not shown). Since several proteins, especially mediators of mitochondrial fission and fusion, had been proposed to interact with ATAD3A, the association of MFN1, MFN2 and DRP1 to mitochondrial protein complexes was likewise tested by BN-PAGE. Intriguingly, MFN1 is found in two complexes, which are a little bit bigger than the ATAD3A  Figure 6B). We thus conclude that ATAD3A likely is a component of multiple mitochondrial protein complexes, which might explain previous findings pointing to a variety of ATAD3 localizations and functions in mitochondria.

Discussion
Previous studies in human cell lines and in Caenorhabditis elegans showed that ATAD3 AAA+-ATPases are localized to mitochondria, where they probably are arranged in oligomers that span both mitochondrial membranes with the enzymatic domain positioned in the matrix [2,7,19]. It is proposed that ATAD3 is implicated in the regulation of mitochondrial and ER dynamics, as interactions with mitochondrial fission (DRP1) and fusion (mitofusins, OPA1) proteins could be proofed [2,6,7,19]. The ability of ATAD3 to bind mtDNA is discussed controversially [40][41][42][43][44].
In Caenorhabditis elegans it was demonstrated that proper ATAD-3 function is necessary for larval development [19]. Several studies with human cancer cells pointed out a role of ATAD3A and ATAD3B in tumor progression [1,6,[45][46][47]. In order to elucidate the relevance of ATAD3 for mammalian development and disease, we analyzed an Atad3 deficient mouse line. In these mice a loss-of-function mutation in the Atad3 gene was established by gene trapping. The resulting mutant protein is neither able to enter the mitochondrion nor to hydrolyze ATP. This article describes the essential function of Atad3 for the progression of the first steps of post-implantation development. Atad3 deficient embryos die around E7.5 due to retardation in growth and a defective development of the trophoblast lineage. The polar trophectoderm of the blastocyst gives rise to trophoblast stem cells, which are the source of the trophoblast lineage towards the establishment of secondary trophoblast giant cells and the embryonic component of the placenta. Since a minimal trophoblast outgrowth is detectable in Atad3 deficient embryos in?vitro, we assume that TS cells can be established in principle, but that their maintenance as well as their subsequent differentiation is disturbed. Intrinsic and extrinsic apoptotic events are unlikely to During pre-implantation development from the fertilized egg to the blastocyst stage the embryo generates energy mainly by glycolysis because of the hypoxic atmosphere in the oviduct and a strongly reduced biogenesis of new mitochondria [48][49][50]. Nearly all mitochondria in the pre-implantation embryo are derived from the ooplasm [51,52]. The first embryo-own mitochondria are generated around implantation of the blastocyst into the uterus, mainly in the trophectoderm, where 80% of the embryos total ATP is synthesized and 90% of the amino acid turnover takes place [53,54]. This rapid generation of mitochondria is due to the rapid energy requirement and oxygen consumption of the embryo, which, at this stage, resides in an oxygenized atmosphere for the first time. Therefore, ATP production switches from glycolysis in the cytoplasm to oxidative phosphorylation (OXPHOS) in the mitochondria [55]. Increased mitochondrial activity (energy metabolism and biosynthesis) is necessary for cellular differentiation processes and is therefore mediated by the new formation and elongation of mitochondria leading to the expansion of the mitochondrial network [55][56][57].
Atad3 function is essential for early post-implantation development at a time-point when mitochondrial biogenesis, i.e. the formation of new mitochondria, and ATP production through OXPHOS are required [58,59]. Therefore, Atad3 might play a role in the biogenesis of mitochondria in trophoblast stem cells and in differentiating trophoblasts by controlling one of the following processes: growth (swelling) of mitochondria, replication of mtDNA, transcription or translation of mtDNA encoded proteins, mitochondrial protein synthesis, folding of mtDNA encoded proteins, assembly of mitochondrial protein complexes (DRP1 oligomers, OPA1 oligomers, prohibitin oligomers, mtDNA replication machinery etc.), uptake of iron into mitochondria and incorporation of iron into complexes of the respiratory chain or cytochrome c. A loss of Atad3 function might influence mitochondrial morphology and disturb mitochondrial dynamics and also alter mitochondrial activity (inner membrane potential, oxygen consumption, ATP production).
The phenotype of a loss-of-function mutation of the Atad3 gene in the mouse is similar to the phenotype of ATAD-3 deficiency in Caenorhabditis elegans, in which reduced growth of embryos and a failure of early embryonic development is observed [19]. The phenotype of the prohibitin knockout mouse is very similar to the phenotype of the Atad3 gene trap mouse, described in this article. Prohibitin knockout mice are embryonically lethal between E3.5 and E8.5 due to a rapid retardation in growth and development [60]. In mammals as well as in Drosophila melanogaster and in yeast two prohibitin genes PHB1 and PHB2 exist. Both prohibitins are located in the inner mitochondrial membrane where they form a ring complex and mediate cristae formation [61][62][63][64][65]. Prohibitins are associated to the m-AAA protease [65,66].
In addition to the elucidation of Atad3 function in the mouse embryo, we started analyzing the contribution of ATAD3 to native mitochondrial protein complexes in order to obtain novel clues regarding its cellular function. In mitochondria of human and murine cells, ATAD3 isoforms are organized in five different protein complexes of distinct sizes, one main complex of about 800-900 kDa and four smaller sub-complexes. Since oligomerization of this protein was proposed before [2], these five complexes might reflect definite stages of ATAD3 assembly. DRP1 and MFN2 are contained in complexes of similar sizes as compared to ATAD3 sub-complex III. The precise compositions of these native complexes, as well as their exact relations to the ATAD3A complexes need further investigation.
In the future, it will be important to analyze ATAD3 function and protein interaction network in cell types, in which a switch from glycolysis to OXPHOS occurs, i.e. in which de novo biogenesis of mitochondria is of tremendous importance. Since ATP production by glycolysis is postulated to be characteristic for various types of stem cells [55,[67][68][69][70][71] and because cell differentiation requires OXPHOS and expansion of the mitochondrial network [56,57,70,72,73], it will be interesting to study the role of ATAD3 in stem cells which are differentiating into their first progeny. With respect to the increasing assessment of mitochondrial function for tumorigenesis [74] and the efforts of investigating the relevance of ATAD3 up-regulation in cancer [1,6,45], studies in tumor stem cells might be of value, too.

Ethics Statement
All animal experiments were conducted in a licensed animal facility in accordance with the German law on the protection of experimental animals and were approved by local authorities of the state of Nordrhein-Westfalen (Landesamt für Natur, Umwelt und Verbraucherschutz NRW). The approval number is 8.87-50.10.31.08.158.

Generation of an Atad3 Gene Trap Mouse Line and Genotyping
An Atad3 gene trap mouse line (Atad3 GT/+ ) was generated by injection of E14TG2a.4 ES cells of the annotated clone E118D03 (obtained from the German Gene Trap Consortium, Munich, Germany) into C57BL/6N blastocysts. Chimeric offspring showing germ line transmission were backcrossed into the C57BL/6J genetic background.