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Dynamic interactions between components of the outer (OM) and inner (IM) membranes control a number of critical mitochondrial functions such as channeling of metabolites and coordinated fission and fusion. We identify here the mitochondrial AAA+ ATPase protein ATAD3A specific to multicellular eukaryotes as a participant in these interactions. The N-terminal domain interacts with the OM. A central transmembrane segment (TMS) anchors the protein in the IM and positions the C-terminal AAA+ ATPase domain in the matrix. Invalidation studies in Drosophila and in a human steroidogenic cell line showed that ATAD3A is required for normal cell growth and cholesterol channeling at contact sites. Using dominant-negative mutants, including a defective ATP-binding mutant and a truncated 50-amino-acid N-terminus mutant, we showed that ATAD3A regulates dynamic interactions between the mitochondrial OM and IM sensed by the cell fission machinery. The capacity of ATAD3A to impact essential mitochondrial functions and organization suggests that it possesses unique properties in regulating mitochondrial dynamics and cellular functions in multicellular organisms.
Mitochondria not only supply cells with the bulk of their ATP but also contribute to the fine regulation of metabolism, calcium homeostasis, and apoptosis (27). Coordination of these functions is dependent on the dynamic nature of mitochondria (5). These organelles constantly fuse and divide to form small spheres, short rods, or long tubules and are actively transported to specific subcellular locations. These processes are essential for mammalian development, and defects can lead to degenerative diseases and cancers (9, 17). In eukaryotes, these organellar gymnastics are controlled by numerous pathways that preserve proper mitochondrial morphology and function (30, 45). The best-understood mitochondrial process is the fusion and fission pathways, which rely on conserved GTPases, and their binding partners to regulate organelle connectivity (10, 18, 45). There are also evidences that dynamic interactions between the outer membrane (OM) and inner membrane (IM) exist for coordinated fusion and fission, channeling of metabolites, and protein transport, but proteins playing a role in these interactions have yet to be identified (34). In the present study, we provide a detailed biochemical and functional characterization of the mitochondrial AAA+ ATPase ATAD3A protein that is present exclusively in multicellular eukaryotes and which participates in the control of mitochondrial dynamics at the interface between the IMs and OMs. Proteins related to the Atad3A genes have been previously identified in proteomic surveys of mouse brain mitochondria (28) and liver mitochondrial inner membrane (8), as mitochondrial DNA-binding proteins (4, 21, 44) and as nuclear mRNA-associated proteins (6). The Atad3A protein has also been identified as a cell surface antigen in some human tumors (16). Functional genomics identified the Drosophila Atad3A ortholog (bor) as a major gene positively regulated by the TOR (for target of rapamycin) signaling pathway involved in cell growth and division (19). In our laboratory, we identified ATAD3A as a specific target for the Ca2+/Zn2+-binding S100B protein (B. Gilquin et al., unpublished data). We here show that ATAD3A is anchored into the mitochondrial IM at contact sites with the OM. The N-terminal domain of ATAD3A interacts with the inner surface of the OM and its C-terminal AAA ATPase domain localizes in a specific matrix compartment. Thanks to its simultaneous interaction with two membranes, ATAD3A regulates mitochondrial dynamics at the interface between the IMs and OMs and controls diverse cell responses ranging from cell growth, channeling of cholesterol, and mitochondrial fission.
w; PBc05441/TM6b (42) and UAS-MitoGFP (32) strains were from the Bloomington Stock Center. The y, w, hsFLP; Act5C>CD2>Gal4 strain was kindly provided by Renald Delanoue (ISBDC-UMR CNRS 6543, Université de Nice). The UAS-ATAD3AIR transgenic line was obtained from the Vienna Drosophila RNAi Center (11).
U373 cells were purchased from the American Type Culture Collection and maintained in Dulbecco modified Eagle medium (DMEM-Gutamax), supplemented with 10% fetal calf serum, 100 U of penicillin/ml, and 100 μg of streptomycin/ml.
Affinity-purified polyclonal antibodies to ATAD3A were raised in rabbit. Pan-specific N-terminal antibodies were obtained against the RPAPKDKWSNFDPTGC peptide, and human-specific C-terminal antibodies against the CLKAEGPGRGDEPSPS peptide (23). Monoclonal anti-Myc IgG was home-made hybridoma supernatant. Rabbit polyclonal anti-ATP synthase was a generous gift of G. Brandolin. The following commercial primary antibodies were used: mouse monoclonal anti-ATP synthase beta, rabbit anti-CytC, rabbit anti-OPA1, and rabbit antiprohibitin (Abcam); mouse monoclonal anti-GM130 and anti-DRP1 (BD Biosciences); and mouse anti-TOM20 (Santa Cruz). Secondary antibodies conjugated to cyanin 3 were from Jackson Immunoresearch Laboratories. Secondary antibodies conjugated to Alexa Fluor 488 were from Molecular Probes, Inc.
The yeast two-hybrid assay was performed by using the Matchmaker two-hybrid system 3 (Clontech) according to the manufacturer's instructions. For assays of protein interaction, the AH109 yeast strains carrying pGBKT7-ATAD3A1-245 and the empty pGBKT7 DNA-BD vector were transformed with the prey vectors, the pGADT7-ATAD3A1-245 or the empty pGADT7 AD vector, plated on the SD/−Leu/−Trp medium, and then incubated at 30°C. After 3 days, the transformants were replica plated to the SD/−Ade/−His/−Leu/−Trp medium containing X-α-Gal and incubated at 30°C for 6 days. In this yeast two-hybrid system, the transformants that were able to grow and form blue colonies on the SD/−Ade/−His/−Leu/−Trp/X-α-Gal plates contained the cDNA clones encoding interacting proteins. The growth phenotype of each transformant was reevaluated by spot test.
Cells grown in 100-mm plates were resuspended in 1 ml of buffer containing 0.28 M sucrose, 1 mM EDTA, and 10 mM Tris-HCl (pH 7.4). Cell suspension was homogenized with 20 passages through a G25 needle. The homogenate was centrifuged at 1,200 × g for 10 min. The supernatant was subsequently centrifuged at 11,000 × g for 15 min. The pellet contained mitochondria and mitochondrion-associated membranes.
Submitochondrial fractionation was performed according to the method of Pon et al. (33). Crude mitochondria were resuspended in 1 ml of swelling buffer (10 mM HEPES-KOH [pH 7.4], 0.5 mM EDTA). After a 10-min incubation on ice, 0.38 ml of sucrose 55% (wt/vol) was added, and mitochondria were further incubated for 15 min on ice. After sonication (those pulses of 1 min each), remaining intact mitochondria and large fragments were removed by centrifugation (15,000 × g for 10 min). The supernatant was centrifuged at 200,000 × g for 60 min at 4°C in a Beckman Optima MAX-XP centrifuge equipped with a TLS-55 rotor. The pellet was resuspended in 100 μl of buffer containing 10 mM HEPES-KOH (pH 7.4), 10 mM KCl, 15% sucrose, and 0.5 mM EDTA and loaded onto a discontinuous sucrose gradient (made with 280 μl of 55% [wt/vol] sucrose, 1,000 μl of 46% [wt/vol] sucrose, 460 μl of 38% [wt/vol] sucrose, and 280 μl of 29% [wt/vol] sucrose in 10 mM HEPES-KOH [pH 7.4] and 10 mM KCl). After centrifugation (100,000 × g for 6 h, 4°C), 150-μl fractions were collected from bottom to top. Then, 15 μl was loaded on SDS-PAGE gels for Western blot analyses.
A standardized method was used. Mitochondrial pellets were resuspended in either isotonic buffer containing 0.28 M sucrose in 20 mM HEPES (pH 7.5) or in hypotonic buffer containing 20 mM HEPES (pH 7.5), in the absence or presence of 120 mM NaCl. Mitochondrial preparations (150 μg/ml) were incubated with various amounts of trypsin for various amounts of time at 30°C. For combined trypsin digestion with detergents, Triton X-100 (10% solution) or digitonin (5-mg/ml solution) were added and incubated for 5 min with the mitochondrial preparation prior to trypsin. Digestion was stopped by the addition of a trypsin inhibitor (Pefabloc) and SDS loading buffer containing 4 mM EGTA and 20 mM dithiothreitol (DTT), after which the samples were kept at 95°C for 5 min.
The mitochondrial preparation was solubilized in 40 mM Tris (pH 7.4)-150 mM NaCl buffer (control), 1 M NaCl-0.1 M Na2CO3 (pH 11.5) (NaCO3)-2 M urea, or 0.5% Triton X-100 (Triton) as indicated on the figure panels. After centrifugation at 11,000 × g for 15 min, the soluble protein supernatants (S) and the membranous pellets (P) were analyzed by immunoblot.
Total cell lysates in 1 ml of immunoprecipitation buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.3% Triton X-100, 4 mM EGTA, and protease inhibitor cocktail) were incubated with anti-Myc monoclonal antibody, together with protein G-Sepharose (Pharmacia) for 1 h of rotation at 4°C. The immunoprecipitates were washed three times in incubation buffer and transferred to a new Eppendorf tube, and the beads were boiled in 1× Laemmli with 20 mM DTT. Proteins were separated by SDS-PAGE and analyzed by Western blotting.
For cross-linking experiments, mitochondria were prepared in buffer containing 0.28 M sucrose, 1 mM EDTA, and 20 mM HEPES (pH 7.4). Crude mitochondria were incubated at 30°C in the presence of 0.5 mM DSP or DTSSP. Cross-linking was stopped by the addition of SDS loading buffer without reducing agents. The proteins were resolved on by SDS-PAGE on 10.5 or 6% polyacrylamide gels.
For immunocytochemical analysis, cells were plated onto poly-l-lysine (Sigma)-coated glass coverslips. U373 cells were fixed either with methanol (5 min) or 4% paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized with 0.2% Triton X-100 for 10 min, washed in Tris-buffered saline (TBS), and blocked in TBS containing 5% normal goat serum (NGS) for 30 min. After incubation with primary antibodies in NGS-TBS overnight at 4°C, the cells were washed in TBS and stained with secondary antibodies. Images were obtained with a Zeiss Axiovert 200M microscope or with a Leica TCS SP2 confocal microscope. Hoechst, Alexa 488, and cyanine 3 (Cy3) fluorescences were excited and determined sequentially (400 Hz line by line) by using wavelengths of 405 nm for Hoechst, 488 nm for Alexa 488, and 543 nm for Cy3 excitation. Fluorescence emissions were collected from 415 to 465 nm for Hoechst, from 500 to 542 nm for Alexa 488, and from 560 to 655 nm for Cy3. Quantifications were performed by using the Leica analysis software.
cDNA and siRNA transfections of U373 cells were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols in Opti-MEM culture medium. For siRNA, cells growing on 10-cm dishes at 50% confluence were transfected with an equimolar mix (100 pM) of three different or individual siRNAs directed against human ATAD3A/B (23), with a single Drp1 siRNA (15), or with scrambled nucleotide. The cells were transfected a second time at 24 h. At 24 h after the last transfection, cells were treated with trypsin and seeded onto plastic dishes or poly-l-lysine (Sigma)-coated glass coverslips.
NCI H295R cells were cultured as previously described (3). Cells were harvested by trypsinization and pelleted by centrifugation at 200 × g for 5 min, resuspended in growth medium, counted, and then resuspended again at 2 × 106 cells/ml in 100 μl of Amaxa cell line optimization Nucleofector solution R. Then, 0.5 μg of ATAD3A siRNA was added, and the mixture were transferred into the Amaxa electroporation cuvette. Electroporation was performed using program T-16. A total of 500 μl of DMEM/F-12 medium containing 2% Ultroser (Ciphergen, Cergy-Saint-Christophe, France) and 1% ITS+ (insulin, transferrin, and selenium premix; BD Biosciences Bedford, MA) was added immediately to the electroporation mixture, and the cells were transferred to six-well plates containing DMEM/F-12 medium supplemented with 2% Ultroser and 1% ITS+. At 48 h posttransfection, the cells were incubated in fresh DMEM/F-12 containing forskolin (FSK; 10 μM) or angiotensin II (10 nM) for 3 h at 37 C. After the incubation period, aliquots of the culture medium were assayed for cortisol or aldosterone content by radioimmunoassay (RIA).
Atad3 genes are conserved in multicellular organisms, but absent in unicellular organisms, including yeast. In the human genome, two genes encoding ATAD3 proteins, Atad3A and Atad3B, are located on chromosome 1 at locus p36.33. The ExPASy Molecular Biology Server revealed two alternatively spliced human transcripts derived from the Atad3A gene: Q9NVI7-2/ATAD3A (length, 586 amino acids) and variant Q9NVI7/ATAD3A-2 (length, 634 amino acids).
Characterization of ATAD3 transcripts in human U373 and HeLa cells by reverse transcription-PCR (RT-PCR) revealed that only ATAD3A (Q9NVI7-2) mRNA is expressed in these two cell lines (data not shown). The ATAD3A isoform is conserved in all mammals and Drosophila. The Atad3B gene is human specific and ATAD3B protein (648 amino acids; 72 kDa) is only expressed at very low level in specific human cell lines (23). Computer-assisted structural predictions identified two coiled-coil domains (CC1, amino acids 85 to 115; CC2, amino acids 180 to 220) with high oligomerization probability within the N terminus of ATAD3A. The coiled-coil domains are followed by a predicted transmembrane segment (TMS; amino acids 248 to 264) positioned in the central part of the molecule (Fig. (Fig.1a).1a). The C terminus harbors a conserved ATPase domain characteristic of the AAA+-ATPase subfamily with ATP-binding and ATPase domains (Walker A and Walker B), including an SRH motif, sensor I and sensor II residues, and an Arg finger (Fig. (Fig.1b)1b) (13).
Differential solubility assays showed that ATAD3A behaves as an integral mitochondrial membrane protein. ATAD3A is resistant to carbonate and high salt extraction and is soluble only in buffers containing Triton X-100 (see below). Submitochondrial membrane vesicle fractionation by density gradient centrifugation showed that ATAD3A partitions in heavy-density fractions but is also present in intermediate and light fractions (Fig. (Fig.2a).2a). These results suggest that ATAD3A is a protein anchored to the IM that can also interact with the OM (33).
To examine the submitochondrial localization of ATAD3A domains in more details, we analyzed the accessibility of the protein to trypsin digestion under various conditions. In isotonic buffer containing 0.28 M sucrose, ATAD3A was protected from proteolysis (Fig. (Fig.2b).2b). In hypotonic swelling conditions, and in the absence of salt, ATAD3A became highly sensitive to trypsin (Fig. 2b and c). At a low trypsin/protein ratio, ATAD3A proteolysis generated two fragments sequentially, ATAD3A* and ΔN-ATAD3A (37 kDa). The ATAD3A* fragment that is recognized by both N-ter and C-ter antibodies was produced with kinetics that paralleled proteolysis of the OM protein TOM20 (Fig. (Fig.2c).2c). This indicates that the first 40 amino acids are positioned close to the mitochondrial surface. At higher trypsin/protein ratios, the ΔN-ATAD3A fragment that is specifically recognized by ATAD3A C-ter antibody remained protected from digestion, whereas OPA1, an inner membrane space (IMS) protein that peripherally associated with the outer surface of the IM (18), is fully degraded (Fig. (Fig.2b).2b). Under the same conditions, prohibitin, an oligomeric transmembrane protein of the IM with a domain exposed to the IMS, is digested at much lower rate (Fig. (Fig.2b).2b). To investigate whether resistance of the C-terminal ΔN-ATAD3 fragment to trypsin is dependent on its specific submitochondrial localization, we combined trypsin digestion with Triton X-100 treatment in hypotonic buffer. Under these conditions, full proteolysis of the ATAD3A protein was observed (Fig. (Fig.2d).2d). Similarly, if mitochondria were preincubated with a low trypsin concentration in hypotonic swelling conditions to promote formation of the ΔN-ATAD3A fragment, subsequent addition of Triton X-100 induced full proteolysis of this fragment at very low Triton X-100 concentrations (Fig. (Fig.2e).2e). At low trypsin concentrations, Triton X-100 does not increase OPA1 proteolysis as OPA1 is an IMS protein whose accessibility to protease does not require IM permeabilization (Fig. (Fig.2e).2e). These results indicated that the ATAD3A C-terminal domain is localized in a matrix compartment.
When protease treatment of mitochondria was carried out in hypotonic buffer that contained 120 mM NaCl, different proteolysis patterns were observed (Fig. (Fig.2f).2f). In the absence of Triton X-100, ATAD3A, and OPA1 were partially protected from proteolysis. The protective effect of NaCl is assumed to be the result of mitochondrial condensation by the salt which, in turn, promoted OM resealing and shrinkage of the IMS. In the presence of Triton X-100, ATAD3A and OPA1 proteolyses were restored. However, low-molecular-weight trypsin-resistant ATAD3A fragments could be observed that were not present in the absence of salt (Fig. (Fig.2f).2f). Notably, an N-terminal 25-kDa fragment (*) remained visible even at high trypsin concentrations. To investigate whether salt protects ATAD3A from proteolysis via strengthened interactions between mitochondrial membranes, we combined trypsin digestion with digitonin extraction in an isotonic buffer in the absence or presence of 120 mM NaCl. A relatively low concentration of digitonin (0.65 to 0.75%) generate holes in and removes most of the OM, making IMS proteins accessible to cleavage by exogenous proteases (20). However, contact sites between the OM and the IM can withstand this relatively harsh treatment (20, 39). It is thus expected that proteins localized at contact sites will be protected from proteolysis in condensed mitochondria. In the absence and in the presence of 120 mM NaCl, solubilization of the OM was detectable by full digestion of OPA1 at low trypsin concentrations (Fig. (Fig.2g).2g). OPA1 was even more efficiently proteolysed in the presence of NaCl. Considering ATAD3A, in the absence of NaCl, the digestion profile was similar to that observed for mitoplasts under hypotonic conditions. However, in the presence of 120 mM NaCl, ATAD3A was protected from proteolysis (Fig. (Fig.2g).2g). Only the first 40 amino acids on ATAD3A were accessible to trypsin, resulting in the production of a single ATAD3A* fragment recognized by both C-ter and N-ter antibodies. As digitonin also punctures the IM, thereby exposing matrix proteins to protease digestion (18), full ATAD3A proteolysis occurred at high trypsin concentrations with no intermediate ΔN-ATAD3A fragment (Fig. (Fig.22 h).
These data led us to conclude that ATAD3A is anchored into the mitochondrial IM and enriched in sites with the potential to form contacts with the OM. The N-terminal domain interacts with the inner surface of the OM and the C-terminal AAA ATPase domain localizes in a specific matrix compartment. Uncertainty remains regarding the exact localization of the 40 first amino acids, but the high sensitivity of this region to protease attack suggests that they may be located close to the mitochondrial surface (see also the discussion section and Fig. Fig.1010).
The complex topology of ATAD3A and its interactions with mitochondrial membranes, in addition to the absence of a predicted mitochondrial targeting signal, raises questions regarding the mitochondrial targeting and anchoring mechanisms. To identify the import signal for ATAD3A, we constructed several Myc-tagged truncated versions of the protein and examined their cellular localization (Fig. (Fig.1c).1c). We first analyzed the 245-586 ATAD3A-Myc protein that carries the predicted TMS and the AAA domain. When expressed in U373 cells, this mutant protein localized exclusively to mitochondria (Fig. (Fig.3a).3a). Solubility experiments demonstrated that the 245-586 ATAD3A-Myc protein behaved, like endogenous wild-type protein, as an integral mitochondrial membrane protein (Fig. (Fig.3b).3b). Trypsin sensitivity experiments in hypotonic conditions demonstrated that the membrane-bound 245-586 ATAD3A-Myc protein was protected from protease attack but was digested in the presence of Triton X-100 (Fig. (Fig.3c).3c). Hence, the C-terminal ATAD3A domain contains all of the information necessary for mitochondrial targeting, membrane insertion, and the positioning of the C-terminal AAA ATPase domain in a matrix compartment.
To investigate the contribution of the TMS to mitochondrial targeting, we next analyzed the effect of TMS (residues 246 to 264) on the localization of different N-terminal half mutants. We used truncated Δ50 N-terminal mutants because we found that the first 50 amino acids may participate in interactions with the mitochondrial outer membrane (MOM) (see below). When expressed in U373 cells, the Δ50-250 ATAD3A-Myc protein lacking the TMS localized diffusely in the cytoplasm and accumulated on perinuclear structures with no mitochondrial labeling (Fig. (Fig.3d).3d). The Δ50-280 ATAD3A-Myc protein with the TMS showed the same immunostaining pattern (Fig. (Fig.3e).3e). Further addition of 10 amino acids was sufficient to target the Δ50-290 ATAD3A-Myc protein to mitochondria, where the protein also promoted mitochondrial chain fragmentation (Fig. (Fig.3f).3f). We concluded that both the TMS and an adjacent auxiliary import region cooperate for mitochondrial import.
To analyze the mechanism of interaction between ATAD3A N terminus and the MOM, we used the 1-250 ATAD3A-Myc mutant lacking the TMS. When expressed in U373 cells, a large fraction of the 1-250 ATAD3A-Myc-tagged protein colocalized and cosedimented with the mitochondrial fraction (Fig. 4a and b). Indirect immunofluorescence analysis of transfected cells showed that a population of the 1-250 ATAD3A-Myc protein localizes along the mitochondrial tubules (Fig. (Fig.4a).4a). Solubility experiments revealed that the 1-250 ATAD3A-Myc protein is peripherally associated with the mitochondrial OM. It was resistant to NaCl extraction but was solubilized by carbonate buffer and 2 M urea (Fig. (Fig.4b).4b). Trypsin digestion experiments demonstrated that a 25-kDa fragment harboring the N-ter epitope was selectively resistant to proteolysis (*), whereas the C-terminus Myc tag epitope was digested (Fig. (Fig.4c).4c). The same trypsin-resistant 25-kDa fragment was also observed for ATAD3A digestion in the presence of Triton X-100 and NaCl (Fig. (Fig.2f).2f). This confirms that, upon interaction with mitochondrial OM, the ATAD3A N terminus can be protected from trypsin digestion. To further localize the OM interaction domain, we deleted the first 50 amino acids and found that this was sufficient to abrogate interactions of the Δ50-250 ATAD3A-Myc mutant protein with the mitochondrial OM (Fig. (Fig.3d).3d). The 50 first N-terminal amino acids were nevertheless insufficient for reconstituting a functional mitochondrial OM interaction domain. Indeed, the 1-220 ATAD3A-Myc mutant did not interact or colocalize with mitochondria (Fig. (Fig.1c1c).
We could then demonstrate that oligomerization of the N-terminal domain of ATAD3A is probably required for MOM interaction. As expected from the presence of two coiled-coil domains, yeast two-hybrid experiments confirmed that the ATAD3A N terminus (amino acids 1 to 245) forms oligomers (Fig. (Fig.4d).4d). The ATAD3A N terminus can also mediate formation of hetero-oligomers with the full-length ATAD3A cotranslated in rabbit reticulocytes (Fig. (Fig.4e).4e). Dimerization requires the full-length coiled-coil domains, as revealed by the lost of heterodimerization of the 1-220 ATAD3A mutant (Fig. (Fig.4e,4e, lanes 1). Finally, cross-linking experiments with the hydrophilic cross-linker dithiobis(sulfosuccinimidyl propionate) (DTSSP) confirmed that the 1-250 ATAD3A-Myc protein does associate as a dimer when bound to the mitochondrial OM (Fig. (Fig.4f).4f). Together, these results indicate that the N terminus of ATAD3A can physically interact with the mitochondrial OM and that this interaction requires the sequence comprising the first 50 N-terminal amino acids plus specific functional features provided by transoligomerization.
The high degree of similarity between the Drosophila ATAD3A (dATAD3A) ortholog and human ATAD3A (70%), suggested that the cellular functions of ATAD3A might be conserved between humans and Drosophila. As an initial approach to identifying the cellular function of ATAD3A, we disrupted dAtad3A gene expression in Drosophila melanogaster. To begin a mutational analysis of dATAD3A, we searched different Drosophila databases and identified a piggyBac transposon inserted in the dATAD3A gene designated PBc05441 (42). We confirmed by sequencing the flanking region that this insertion was located in the 5′-untranslated region of the dATAD3A transcription unit, and it will thus be referred to as dATAD3Ac05441. Homozygous mutants showed growth arrest during larval development, indicating that dATAD3A is required for organism growth (Fig. (Fig.5a).5a). These results complement a recent study showing that Caenorhabditis elegans ATAD3A is essential for mitochondrial activity and development (22).
We then used the “Flp-out” recombination technique (31, 38) to generate clones of fat body cells expressing a mitochondrial green fluorescent protein (GFP) (32) alone or in combination with an RNAi construct targeting dATAD3A (Fig. 5b and c). Fat body cells are determined during embryogenesis and show an enormous increase in cell size during larval stages without cell division. Recombined fat body cells in which dATAD3A expression was knocked down displayed strong size reduction, demonstrating that dATAD3A is required for cell growth in a cell-autonomous manner. Our genetic analysis is consistent with genomic analysis that identified dATAD3A as a major gene positively regulated by the TOR signaling pathway involved in cell growth (19). The mitochondrial GFP also revealed a more fragmented mitochondrial network in cells expressing dATAD3A RNAi (Fig. 5b and c, lower panels).
To investigate whether the growth phenotype associated with ATAD3A downregulation is linked with changes at contact sites and altered mitochondrial metabolism, we focused on mitochondrial cholesterol metabolism. In steroidogenic tissues, tropic hormones stimulate steroid biosynthesis from cholesterol through the activation of the cyclic AMP or the calcium messenger systems. The rate-limiting step in all steroidogenic pathways is the transfer of cholesterol from the mitochondrial OM to the IM, where the first step of the steroidogenic cascade, namely, the conversion of cholesterol to pregnenolone by the cytochrome P450 side chain cleavage (P450scc), occurs (7, 24, 36, 43). Cholesterol is imported into the mitochondrial IM with the assistance of a transduceosome complex that organizes at the contact sites (reviewed in reference 36). To confirm that ATAD3A functions at contact sites, we investigated its contribution to steroid biosynthesis in the human steroidogenic cell line NCI-H295R, which retains several differentiated functions and is therefore useful for defining the cellular mechanisms regulating steroid production. In NCI-H295R cells only ATAD3A but not ATAD3B is expressed (Fig. (Fig.6a).6a). A marked difference in the expression levels of ATAD3A in NCI-H295R and U373 cells also suggests that ATAD3 proteins are differentially regulated between these two human cell lines (Fig. (Fig.6a).6a). Stimulation with angiotensin II (AII), a Ca2+-mobilizing steroidogenic hormone, specifically enhanced ATAD3A level in NCI-H295R cells (Fig. 6a and b). In contrast, FSK, which directly activates the transduceosome complex, has no such effect (Fig. (Fig.6b).6b). Similar results were obtained with primary bovine adrenocortical cell cultures stimulated with AII (not shown). These results suggest that ATAD3A could participate in the formation of contact sites modulated by hormonal stimulation for cholesterol import into the IM. A direct contribution of ATAD3A to cholesterol import was next confirmed by analyses of the effects of short-term ATAD3A downregulation on cortisol and aldosterone production in NCI-H295R cells stimulated with AII or FSK. Western blot analyses showed marked downregulation of ATAD3A protein in cells transfected with hATAD3A siRNA (Fig. (Fig.6c).6c). Short-term ATAD3A downregulation does not induce major changes in mitochondrial network organization (Fig. 6d and e) and did not significantly impact the basal production levels of cortisol and aldosterone in resting cells (Fig. (Fig.6f).6f). In contrast, the stimulatory effects of AII and FSK on cortisol and aldosterone syntheses were markedly inhibited (Fig. (Fig.6f).6f). Taken together, these data suggest that ATAD3A is required for enhanced channeling of cholesterol for hormone-dependent steroidogenesis. They also strongly support the hypothesis that altered cell growth associated with ATAD3A downregulation could be linked with altered mitochondrial metabolism resulting from changes at contact sites.
Dynamic interactions between the OM and IM also play important roles in regulating mitochondrial fusion and fission processes (5, 34, 45). To further support a role of ATAD3A at contact sites, we thought to analyze the short-term effect of dominant-negative ATAD3A mutants on mitochondrial network organization in U373 cells. To this end, we considered two remarkable features of the AAA+ ATPase subfamily of proteins, namely, their assembly into functional oligomers and the dependence of the active oligomers on the nucleotide-binding state and the ATPase activity of the AAA domain subunits (1, 12, 35, 37). It was expected that defective ATP-binding ATAD3A mutants or defective ATP hydrolysis mutants could function as dominant-negative proteins by interfering with normal oligomer functions. To test this possibility, we prepared Myc-tagged wild-type ATAD3A and mutant proteins with well-described AAA mutations in the Walker A and Walker B domains that, respectively, do not bind ATP (358K/E WA ATAD3A) or bind ATP but are defective in ATP hydrolysis (413E/Q WB-ATAD3A) (1) (Fig. 1b and c). Coimmunoprecipitation studies confirmed that Myc-tagged ATAD3A constructs are competent to oligomerize with endogenous wild-type ATAD3A (Fig. (Fig.7b).7b). Overexpression of these proteins in U373 cells revealed that WA ATAD3A produced immediate fragmentation of the mitochondrial network, whereas overexpression of wild-type ATAD3A (Fig. (Fig.7a)7a) or WB mutant (Fig. (Fig.7c)7c) produced no such phenotype. Quantitative indirect immunofluorescence analysis with C-ter ATAD3A antibodies demonstrated that a 4-fold expression of WA ATAD3A-Myc over endogenous wild-type protein was sufficient to fragment mitochondrial chains (Fig. (Fig.7d).7d). These findings were reproduced with mouse NIH 3T3 cells (data not shown).
To confirm that the rapid mitochondrion fragmentation phenotypes induced by WA ATAD3A mutants were not a secondary consequence of mitochondrial dysfunction, we first verified that in early transfected cells fragmented mitochondria could still be labeled with the membrane potential-dependent dye MitoTracker Red CMXRos (Fig. (Fig.8a).8a). We also ensured that mitochondrial fragmentation did not correlate with leakage of cytochrome c (not shown) and did not affect mtDNA nucleoid core organization (Fig. (Fig.8b).8b). We finally showed that the fragmentation phenotype associated with WA ATAD3A mutant expression is linked to an activation of the cell fission machinery (Fig. 8c and d). In mammalian cells, mitochondrial fission is regulated by Drp1, a cytosolic GTPase (40). Transfection of U373 cells with Drp1 siRNA induced a profound decrease in Drp1 protein (Fig. (Fig.8c)8c) and inhibited mitochondrial fragmentation induced by WA-ATAD3A overexpression (Fig. (Fig.8d).8d). Counting the number of WA-ATAD3A-expressing cells revealed that 60% had fragmented mitochondria, whereas this dropped to less than 10% in cells transfected with Drp1 siRNA. Together, these results suggest that the loss of function of ATAD3A at contact sites between the OM and IM is sensed by the cell fission machinery.
To investigate the molecular mechanisms associated with loss of function of the WA mutant, we first compared the partitioning of WA and WB mutants with submitochondrial membrane vesicles fractionated by density gradient centrifugation (Fig. (Fig.9a).9a). The WB ATAD3A mutant was present in all fractions, whereas the WA mutant concentrated in intermediate and heavy fractions. This suggested that the WA ATAD3A mutant was less prone to interact with the OM, but the protein still localized in the vicinity of contact sites. Because interaction with the OM requires transoligomerization of ATAD3A N terminus (Fig. (Fig.4),4), we compared the susceptibility of the wild-type ATAD3A and WA mutant to the hydrophilic cross-linker DTSSP (Fig. (Fig.9b).9b). With control mitochondria, cross-linked ATAD3A oligomers migrated as a single band on SDS-PAGE, with the apparent molecular mass varying between 150 and 300 kDa as a function of the acrylamide concentration. In 6% PAGE cross-linked ATAD3A oligomers migrated in the position of the 150-kDa marker protein (Fig. (Fig.9a,9a, left panel). Mass spectrometry analysis of trypsin-digested peptides derived from cross-linked ATAD3A oligomers revealed that they were exclusively composed of ATAD3A peptides, indicating that they are exclusively constituted with ATAD3A molecules (data not shown). Under the same conditions, prohibitin, an oligomeric transmembrane protein of the IM, was not cross-linked with DTSSP (not shown). If purified mitochondria expressing wild-type ATAD3A-Myc are incubated in the presence of DTSSP, the Myc-tagged molecules become cross-linked as homo-oligomers and hetero-oligomers with the endogenous ATAD3A molecules (Fig. (Fig.9b,9b, right panel, lanes 1 to 3). Hetero-oligomers (*) could be identified because they migrated below the homo-oligomers and reacted with both anti-Myc and anti-ATAD3A C-ter antibodies (not shown). With mitochondria expressing the WA ATAD3A-myc mutant, hetero-oligomers cross-linked with endogenous ATAD3A were not observed at all (Fig. (Fig.9b,9b, right panel, lanes 4 to 6). Moreover, the WA mutant protein was less prone to cross-linking by DTSSP, although it spontaneously formed covalent dimers that can be reduced by DTT (results not shown). These results indicate that loss of function by the WA-ATAD3A mutant could be due to decreased transoligomerization of the N-terminal domains.
The interaction of ATAD3A with the MOM requires both transoligomerization of the N terminus and the first 50 amino acids (Fig. (Fig.4).4). To confirm that decreased interaction of the WA mutant with MOM could contribute to the mitochondrial chain fragmentation phenotype, we analyzed the effect of ectopic expression of the truncated Δ50 ATAD3A-Myc mutant on mitochondrial tubular network in U373 cells (Fig. 9c and d). Coimmunoprecipitation studies revealed that the Δ50 ATAD3A-Myc mutant supports the formation of a heterocomplex with endogenous wild-type protein (Fig. (Fig.9c).9c). Indirect immunofluorescence demonstrated that a 4- to 5-fold expression of Δ50 ATAD3A-Myc over endogenous wild-type protein induced fragmentation of the mitochondrial chains, similar to that observed with the WA mutant (Fig. (Fig.9d).9d). We concluded that the interaction of ATAD3A N termini with MOM is required for correct mitochondria homeostasis and is regulated by ATP.
ATAD3A belongs to a newly recognized family of mitochondrial inner membrane AAA+ ATPase proteins specific to multicellular eukaryotes. Several other mitochondrial AAA+ ATPases of the inner membrane have already been identified (25, 26). These are ATP-dependent proteases that combine both chaperonelike and proteolytic activities. AAA proteases are characterized by a conserved proteolytic domain that follows the AAA+ module. This domain is not present in ATAD3A. Thus, ATAD3A is unlikely to function as a mitochondrial AAA+ protease. Detail analyses of mitochondrial ATAD3A topology showed that the protein is enriched in regions that may form contact between the IM and the OM and shows a tripartite organization (Fig. (Fig.10).10). The N-terminal domain (residues 1 to 245) is positioned in the IMS and interacts with the OM. A central TMS (residues 246 to 264) spans the IM and position the C-terminal AAA domain within a matrix compartment. Mitochondrial IM targeting information and sorting signals are encoded by the TMS with an adjacent auxiliary import region (comprising residues 280 to 290) (Fig. (Fig.3).3). The mitochondrial import information of ATAD3A show similarities with the mitochondrial IM AAA-ATPase BCS1 protein (14, 41). Although BCS1 and ATAD3A show similarities in their IM anchorage and the matrix localization of their C-terminal AAA ATPase domain, only ATAD3A has an additional N terminus interacting with the inner surface of the OM making this protein unique among the mitochondrial AAA ATPase family members thus far identified. Confocal microscope analyses revealed punctuate ATAD3A C-ter immunostaining pattern and no overlapping with ATP synthase F1 subunit (Fig. (Fig.6d)6d) or with mitochondrial DNA (Fig. (Fig.8b).8b). This suggested that ATAD3A C terminus domain may be positioned in a specific matrix subcompartment. The exact submitochondrial localization of the 40 first N-terminal amino acids is not yet clearly resolved, but several observations suggest that these residues could penetrate the OM lipid bilayer. First, only these residues are readily accessible to protease attack in digitonin-treated lysates, suggesting that they are positioned close to the mitochondrial surface. Second, the 50 first amino acids were required to anchor the truncated 1-250 ATAD3A mutant to the OM. Finally, proteolysis studies on mitochondria expressing an EGFP-Nter-ATAD3A fusion protein revealed that the N-terminal EGFP moiety is exposed to the cytosol and highly sensitive to protease attack. Trypsin digestion of the EGFP-ATAD3A fusion protein in isotonic buffer generated a proteolytic product with the mobility and antigenic characteristics of the ATAD3A* fragment (data not shown). Because deletion of the 50 first amino acids does not interfere with mitochondrial localization of the truncated Δ50-ATAD3A mutants, these residues do not function as a mitochondrial targeting sequence but rather regulate the interaction of ATAD3A with the mitochondrial OM. Further analysis of the Δ50-ATAD3A mutant properties revealed that this truncated mutant behaved as a dominant-negative mutant and induced mitochondrial chain fragmentation, as also observed with the ATP-binding deficient WA mutant. Together, these results suggest that ATAD3A mediates dynamic interactions between the IM and the OM that are regulated by the N terminus and the C terminus AAA ATPase activity. The finding that ATP binding-deficient but not ATP hydrolysis-deficient ATAD3A mutants behave as dominant-negative proteins with respect to mitochondrial function is consistent with a previous study showing that ATP binding is more relevant than ATP hydrolysis for some biological activities of AAA ATPase proteins (37). Given the consensus ring-shaped oligomer conformation conferred by the AAA+ module (29), we propose that the ATAD3A AAA+ domains (amino acids 320 to 586) also associate as ring-shaped oligomers. Two helices (amino acids 268 to 280 and 290 to 310) connect the oligomeric AAA module to the TMS that span the IM (amino acids 244 to 264) and which could potentially organize a porelike structure. The formation of a porelike structure has been proposed as a general mechanism for membrane hexameric AAA+ ATPase (2, 26). The N termini (amino acids 1 to 244) of the ATAD3A complexes are projected into the IMS, where they can transoligomerize in an ATP-bound dependent manner and interact with the OM (Fig. (Fig.10).10). With such a topology, ATAD3A could function both in regulating interactions between the mitochondrial IM an OM and also in mitochondrial import processes. This would explain the high sensitivity of ATAD3A to the hydrophilic cross-linker DTSSP (Fig. (Fig.9b).9b). Interactions between the mitochondrial IM and OM control a number of central mitochondrial functions, such as channeling of metabolites, protein transport, coordinated fusion and fission, and mitochondrial DNA inheritance (34). It is thus not surprising that either ATAD3A downregulation or overexpression of ATAD3A dominant-negative mutants promoted diversified cell responses ranging from altered cell growth at the organism and cellular levels (Fig. (Fig.5),5), altered mitochondrial channeling processes (Fig. (Fig.6),6), and mitochondrial fission (Fig. (Fig.77 and and8).8). Two previous studies also reported the interaction of ATAD3A with mitochondrial DNA and possible regulation of mtDNA maintenance (21, 44). Subsequently Bogenhagen group's found that, although both ATAD3A and ATAD3B were present in HeLa cell nucleoid preparation, neither ATAD3 protein was cross-linked with mtDNA in isolated formaldehyde-cross-linked HeLa cell nucleoids, indicating that they are unlikely to bind mtDNA. These researchers concluded that ATAD3A is an example of a protein found in association with nucleoids but not in direct contact with DNA (4). This conclusion is in agreement with our observation that neither the ATAD3A N terminus nor the C terminus localizes with mtDNA (Fig. (Fig.8b).8b). It remains nevertheless possible that the ATAD3A C terminus, through interactions with proteins of the IM, contributes to support mtDNA maintenance within nucleoids.
In conclusion, our study has identified for the first time a mitochondrial protein specific to multicellular eukaryotes that connects the mitochondrial inner membrane to the OM. The capacity of ATAD3A to impact on essential mitochondrial functions and organization suggests that this family of proteins possesses unique properties in regulating cellular functions in multicellular organisms.
We thank Jonhatan LaMarre (University of Guelph) for critical reading of the manuscript, Didier Grunwald for help with the confocal microscopy, and Nathalie Bertacchi for expert technical assistance. We are grateful to Renald Delanoue, the Bloomington Stock Center, and the Vienna Drosophila RNAi Center for the fly strains.
This study was supported by a grant from the Association pour la Recherche sur le Cancer (ARC 4829) (J.B.), the Institut National du Cancer (PL114) (J.B.), and CREST, JST (O.K.).
We have no competing financial interests.
Published ahead of print on 12 February 2010.