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The translocator maintenance protein Tam41 is required for mitochondrial cardiolipin biosynthesis
Correspondence to Nikolaus Pfanner: nikolaus.pfanner{at}biochemie.uni-freiburg.de
The mitochondrial inner membrane contains different translocator systems for the import of presequence-carrying proteins and carrier proteins. The translocator assembly and maintenance protein 41 (Tam41/mitochondrial matrix protein 37) was identified as a new member of the mitochondrial protein translocator systems by its role in maintaining the integrity and activity of the presequence translocase of the inner membrane (TIM23 complex). Here we demonstrate that the assembly of proteins imported by the carrier translocase, TIM22 complex, is even more strongly affected by the lack of Tam41. Moreover, respiratory chain supercomplexes and the inner membrane potential are impaired by lack of Tam41. The phenotype of Tam41-deficient mitochondria thus resembles that of mitochondria lacking cardiolipin. Indeed, we found that Tam41 is required for the biosynthesis of the dimeric phospholipid cardiolipin. The pleiotropic effects of the translocator maintenance protein on preprotein import and respiratory chain can be attributed to its role in biosynthesis of mitochondrial cardiolipin.
Abbreviations used in this paper: AAC, ADP/ATP carrier; Crd1, cardiolipin synthase; DIC, dicarboxylate carrier; LCMS, liquid chromatography/mass spectrometry; Mmp37, mitochondrial matrix protein 37; PA, phosphatidic acid; PAM, presequence translocase-associated motor; PG, phosphatidylglycerol; PGP, phosphatidylglycerophosphate; Pgs1, PGP synthase; Tam41, translocator assembly and maintenance protein 41; TIM, translocase of the inner membrane.
© 2008 Kutik et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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as driving force.
The presequence translocase (TIM23 complex) recognizes the preproteins with cleavable N-terminal presequences and inserts them into the Tim23 import channel in a -dependent manner. Preproteins carrying a hydrophobic sorting signal are laterally released into the inner membrane, whereas the majority of cleavable preproteins are completely translocated into the matrix. Reconstitution experiments with proteoliposomes revealed that the TIM23 complex and an energized cardiolipin-rich membrane represented a minimal system for membrane integration of cleavable preproteins (van der Laan et al., 2007). In organello, however, the TIM23 complex dynamically interacts with several further protein machineries: the translocase of the outer membrane for preprotein transfer from the outer to the inner membrane (Chacinska et al., 2005; Mokranjac et al., 2005), complexes III and IV of the respiratory chain to stimulate the -driven membrane insertion of preproteins with sorting signal (van der Laan et al., 2006; Wiedemann et al., 2007; Saddar et al., 2008), and the presequence translocase-associated motor (PAM) with the heat shock protein 70 that drives protein transport into the matrix at the expense of ATP (Kutik et al., 2007; Neupert and Herrmann, 2007; D'Silva et al., 2008).
The TIM23 complex is a multistep machine and its assembly and mode of cooperation with the motor PAM are only partially understood. Two recent studies identified a mitochondrial protein, which is peripherally attached to the inner membrane from the matrix side and involved in the assembly and maintenance of the activity of the TIM23 complex (Gallas et al., 2006; Tamura et al., 2006). The protein was termed translocator assembly and maintenance protein 41 (Tam41) or mitochondrial matrix protein 37 (Mmp37). Yeast cells lacking Tam41/Mmp37 show a temperature-sensitive growth defect, and the import of presequence-carrying mitochondrial preproteins is impaired at elevated temperature in vivo and in organello. Tam41 does not stably bind to the TIM23 complex, yet its absence influences the integrity of the TIM23 complex and the cooperation with the motor PAM. It was thus concluded that Tam41 is not a structural subunit of the TIM23 complex but a new member of the mitochondrial translocator systems required to maintain the proper assembly state and activity of the TIM23 complex (Gallas et al., 2006; Tamura et al., 2006). Although both studies agreed well on the effects of Tam41/Mmp37 on the presequence pathway, different findings were reported on the second protein import pathway to the inner membrane, the carrier pathway. Gallas et al. (2006) reported that tam41 mitochondria were impaired in the import of a noncleavable carrier precursor, whereas Tamura et al. (2006) did not observe a defect in the translocation of carrier precursors to a protease-protected location. The molecular function of Tam41 remained open.
We used a native assembly assay to characterize the biogenesis of noncleavable carrier proteins in mitochondria lacking Tam41. Surprisingly, we observed a strong defect in carrier assembly even at low temperature, suggesting that the defect of tam41 mitochondria may be more pronounced in the biogenesis of carrier proteins than in the presequence pathway. The subsequent analysis revealed pleiotropic effects of Tam41 on the mitochondrial membrane potential and the assembly state of respiratory chain supercomplexes. We report that these seemingly nonrelated effects are caused by the involvement of Tam41 in the biosynthesis of cardiolipin.
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cells are temperature sensitive for growth (Gallas et al., 2006; Tamura et al., 2006), the cells were grown at low temperature to minimize indirect effects. Mitochondria were isolated and subjected to a short heat shock. The steady-state levels of proteins from the four mitochondrial compartments were comparable for tam41 and wild-type mitochondria, whereas the import of presequence-carrying preproteins was impaired in the mutant mitochondria (unpublished data), in agreement with the reported defect of tam41 mitochondria in the presequence pathway (Gallas et al., 2006; Tamura et al., 2006). These two studies reported different observations for the import of noncleavable carrier proteins as analyzed by protease protection assays. Carrier proteins are imported in a multistep process (Fig. 1 A) and thus precursors accumulated at stage III on the intermembrane space side can be protected against externally added protease when low to moderate concentrations of protease are used (Pfanner and Neupert, 1987; Ryan et al., 1999).
Importantly, mitochondria with a defect of or the TIM22 complex can efficiently accumulate carrier proteins at stage III and, thus, depending on the strength of the protease treatment and the stability of the outer membrane, protease protection of carrier precursors can be observed despite an inner membrane import defect. To directly monitor the assembly stages of mitochondrial carrier proteins, we used a blue native gel assay, which separates stage III (intermediate in intermembrane space) from stage V (mature assembled carrier) upon lysis of mitochondria with a mild detergent such as digitonin (Ryan et al., 1999; Dyall et al., 2003; Rehling et al., 2003). Surprisingly, the -dependent formation of stage V carrier was blocked in tam41 mitochondria, as analyzed by importing the 35S-labeled precursors of the ADP/ATP carrier (AAC) and dicarboxylate carrier (DIC; Fig. 1 B). Instead a smaller form (AAC* and DIC*) was observed, migrating above the stage III intermediate. The formation of AAC* and DIC* was inhibited by dissipation of in contrast to the stage III intermediate (Fig. 1 B), demonstrating that the generation of AAC* and DIC* depended on the activity of the inner membrane. Even without heat shock treatment, carrier assembly to the oligomeric form (stage V) was completely blocked into tam41 mitochondria (Fig. 1 C). AAC imported into tam41 mitochondria was resistant to extraction at pH 11.5 (Fig. 1 D). Together with the dependence of import, this indicates that the carrier was inserted into the lipid phase of the inner membrane. Thus tam41 mitochondria are defective in carrier assembly, suggesting that the function of Tam41 extends beyond the maintenance of the TIM23 complex.
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mitochondria, shown here with the 35S-labeled cleavable preprotein of the Rieske Fe/S-protein. Although the import into the mutant mitochondria was impaired after the heat shock, it was only moderately affected at low temperature (Fig. 1, E and F). We assessed the of tam41 mitochondria by fluorescence quenching. When the yeast cells were grown at low temperature, the fluorescence decrease of tam41 mitochondria was close to that of wild-type mitochondria (Fig. 2 A), indicating no major alteration of .
When the cells were grown at elevated temperature, however, a strong decrease of the of tam41 mitochondria was observed (Fig. 2 A). For in vitro protein import experiments, the mitochondria were isolated from cells grown at low temperature and subjected to a short heat shock. This in vitro heat shock led to a reduction of the fluorescence quenching, i.e., a reduction of (Fig. 2 B). Thus, both protein import via the presequence pathway and the inner membrane potential are impaired in tam41 mitochondria in a temperature-sensitive manner. In contrast, the assembly of carrier proteins is also blocked at low temperature. Because under these conditions the of the mutant mitochondria is close to that of wild-type mitochondria, cannot be the critical determinant for the lack of carrier assembly in tam41 mitochondria.
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mitochondria than the biogenesis of presequence-containing proteins. Neither maintenance of TIM23 function nor generation seems to be a primary target of the functional defect of tam41 mitochondria.
Alteration of inner membrane protein complexes in mitochondria lacking Tam41 or cardiolipin biosynthetic enzymes
AAC binds cardiolipin, a unique phospholipid with dimeric structure, and it was reported that the lack of cardiolipin synthase (Crd1) alters the oligomeric state of AAC (Hoffmann et al., 1994; Jiang et al., 2000; Nury et al., 2005; Claypool et al., 2008). A direct comparison of tam41 and crd1 mitochondria by Western blot analysis of blue native gels revealed a remarkable similarity of the pattern of AAC in both mutant mitochondria (Fig. 3 A).
Oligomeric forms, including the dominant AACV, were virtually absent in both mutants, and the carrier molecules migrated in the low molecular mass range (Fig. 3 A). Mitochondria lacking phosphatidylglycerophosphate (PGP) synthase (Pgs1), which functions upstream of Crd1 in cardiolipin biosynthesis (Chang et al., 1998; Mileykovskaya et al., 2005; Li et al., 2007), showed a similar alteration of the AAC pattern (Fig. 3 A). We also studied mitochondria lacking Taz1, the orthologue of human tafazzin, which is affected in the Barth syndrome (Vreken et al., 2000; Brandner et al., 2005; Claypool et al., 2006; McKenzie et al., 2006). The putative acyltransferase or transacylase Taz1 is involved in the last step of cardiolipin maturation, the remodeling of fatty acid side chains (Gu et al., 2004; Valianpour et al., 2005; Xu et al., 2006; Li et al., 2007). In taz1 mitochondria, the higher oligomers of AAC were absent, whereas AACV was still present and only slightly altered in its mobility (Fig. 3 A; Brandner et al., 2005). Thus, the blue native pattern of AAC in tam41 mitochondria was different from that of taz1 mitochondria but similar to the pattern of crd1 and pgs1 mitochondria.
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mitochondria. Indeed, tam41, crd1, and pgs1 mitochondria showed a similar alteration of the mobility of TIM23 complexes (Fig. 3 B). A slight mobility shift was observed for the TIM22 complex in the mutant mitochondria (Fig. 3 C), whereas the soluble Tim8–Tim13 complex of the intermembrane space was not altered (Fig. 3 D). Cells lacking Tam41 were shown to accumulate mitochondrial precursor proteins in vivo (Gallas et al., 2006; Tamura et al., 2006). We observed that not only tam41 cells but also crd1 cells accumulated the precursor of the cleavable protein Mdj1 (Fig. 3 E). Thus the lack of cardiolipin leads to similar effects on TIM complexes and preprotein accumulation as the lack of Tam41. To exclude that the lack of Tam41 led to a loss of Crd1 or Pgs1, we determined the steady-state levels of the proteins and did not observe a difference between wild-type and tam41 mitochondria (Fig. 3 F).
Cardiolipin stabilizes supercomplexes of the mitochondrial respiratory chain, in particular complexes up to 1 Md formed by complexes III and IV (Schägger, 2002; Zhang et al., 2002, 2005; Pfeiffer et al., 2003; Zhong et al., 2004). Assuming that Tam41 plays a role in the biosynthesis of cardiolipin, the III and IV supercomplexes should be altered in the deletion mutant. We analyzed digitonin-lysed mitochondria by blue native electrophoresis and immunodecoration for cytochrome c1 (complex III) and Cox4 (complex IV). Indeed, the supercomplexes were strongly altered in both tam41 and crd1 mitochondria (Fig. 3 G).
Collectively, the characterization of tam41 mitochondria reveals pleiotropic effects on complexes of the mitochondrial inner membrane and a remarkable resemblance of the defects observed in mitochondria lacking cardiolipin.
tam41 mitochondria are deficient in cardiolipin
To directly address if the lack of Tam41 affected the levels of mitochondrial cardiolipin, we extracted lipids from isolated wild-type and tam41 mitochondria and subjected them to mass spectrometry. Fig. 4 A shows the liquid chromatography/mass spectrometry (LCMS) profiles of wild-type and tam41 mitochondrial lipid extracts.
The major ions in the spectra correspond to phosphatidylethanolamine and phosphatidylinositol, whereas cardiolipins appear as minor ions in the lipid profiles (Fig. 4 A, top). Using an unbiased profiling approach (Guan et al., 2006; Shui et al., 2007), we compared the lipid profile of tam41 mitochondria relative to the wild-type profile and expressed this as a differential profile. The main difference observed was a down-regulation of several ions (mass-to-charge ratio [m/z] of 685.5, 699.5, and 713.5 and 1371.9, 1399.9, and 1427.9) in tam41, corresponding to the doubly and singly charged species of cardiolipin molecules found in Saccharomyces cerevisiae (Fig. 4 A, middle; ions 1117.8 and 1145.8 were identified as fragments of cardiolipin). For comparison, we also analyzed lipid extracts of crd1 and taz1 mitochondria. As expected, monolysocardiolipin accumulated in taz1 mitochondria (m/z 1055.8, 1137.8, and 1165.8) and phosphatidylglycerol (PG) accumulated in crd1 mitochondria (m/z 719.5, 747.5, and 775.5; Fig. 4 A, bottom; Tuller et al., 1998; Jiang et al., 2000; Zhang et al., 2003; Gu et al., 2004; Valianpour et al., 2005; Claypool et al., 2006).
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mitochondria (Fig. 4 B). To directly determine if the lack of Tam41 was responsible for the observed defects, Tam41 was reexpressed from a plasmid in tam41 cells. Thereby, the growth of the cells at elevated temperature was restored (Fig. 4 C) and cardiolipin was synthesized (Fig. 4 D). Thus, the expression of Tam41 in tam41 cells is sufficient for cardiolipin synthesis and cell viability at elevated temperature.
Upon in vivo labeling with [32PO4], the lipids of enriched mitochondria were analyzed by one- and two-dimensional separation (Fig. 5, A and B).
Cardiolipin and PG were not detected in tam41 mitochondria. Tandem mass spectrometry–based quantification of PG levels demonstrated that the levels of PG were significantly reduced in tam41 mitochondria in contrast to the strong increase in crd1 mitochondria (Fig. 5 C; Tuller et al., 1998; Jiang et al., 2000; Zhang et al., 2003). These results demonstrate that Tam41 does not function at the stage of Crd1, but the reduced levels of PG in tam41 indicate that Tam41 acts upstream of Crd1. There was no evidence that tam41 mitochondria accumulated large amounts of PGP (the immediate precursor of PG), which would run very close to the origin in the two-dimensional system (Fig. 5 B). Collectively, the results obtained so far raise the possibility that Tam41 functions at an early step of cardiolipin biosynthesis. To address this, we determined the levels of phosphatidic acid (PA), the first metabolite in the biosynthesis of cardiolipin, and indeed found a considerable increase in tam41 mitochondria but not in crd1 mitochondria (Fig. 5 D). The increased levels of PA were also observed by one- and two-dimensional separation of [32PO4] lipids (Fig. 5, A and B). Reexpression of Tam41 in tam41 cells decreased the levels of PA to wild-type levels (Fig. 5 E), demonstrating that the increase of PA was caused by the lack of Tam41.
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mitochondria showed a reduced and a defect in the assembly of carrier proteins, which are imported via the TIM22 complex. Importantly, at low temperature, tam41 mitochondria were still strongly impaired in carrier assembly, whereas and presequence pathway were not or only moderately affected, suggesting that the presequence pathway was not the primary site of action of Tam41.
Third, various inner membrane protein complexes, including supercomplexes of the respiratory chain, carrier complexes, and TIM translocases, were altered in tam41 mitochondria, resembling the broad spectrum of defects observed in mitochondria lacking cardiolipin (Jiang et al., 2000; Schägger, 2002; Zhang et al., 2002, 2005; Pfeiffer et al., 2003; Zhong et al., 2004; Nury et al., 2005; van der Laan et al., 2007; Claypool et al., 2008). Finally, a detailed analysis by mass spectrometry and in vivo radiolabeling of lipids proved that the levels of cardiolipin, as well as of PG, were significantly reduced in tam41 mitochondria. The sequence of cardiolipin synthesis in yeast is PA to CDP-diacylglycerol to PGP to PG to cardiolipin (Mileykovskaya et al., 2005; Li et al., 2007). We did not observe an accumulation of PGP, but a significant accumulation of PA in tam41 mitochondria. In contrast, mitochondria lacking Crd1 accumulate large amounts of PG but not PA (Tuller et al., 1998; Jiang et al., 2000; Zhang et al., 2003; this study), demonstrating that Tam41 acts upstream of Crd1. Moreover, tam41 mitochondria contain wild-type levels of Crd1 and Pgs1. The accumulation of the early metabolite PA thus suggests that Tam41 is required at an early stage of cardiolipin biosynthesis.
In summary, the available results indicate that Tam41 is required for the biosynthetic pathway of cardiolipin or the regulation of the pathway. This dimeric mitochondrial phospholipid exerts a broad influence on numerous functions of the inner membrane, including the assembly states of the respiratory chain, metabolite carriers, and the TIM23 complex. The apparently pleiotropic effects of Tam41 can thus be explained by its involvement in lipid biosynthesis.
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(fomp7-A; YBG-0800-A) was generated by plasmid shuffling (Chacinska et al., 2005). pgs1, taz1, and crd1 strains have been described previously (Chang et al., 1998; Brandner et al., 2005; Euroscarf). The region encoding Tam41, plus 500 bp upstream/downstream, was cloned into pFL39 using SacI and BamHI restriction sites, yielding pFL39-TAM41.
Growth conditions and mitochondrial protein import
Cells were grown in liquid YP medium (1% [wt/vol] yeast extract; 2% [wt/vol] Bacto Peptone; and either 3% [vol/vol] glycerol (YPG), 2% [wt/vol] sucrose [YPS], or 2.2% [vol/vol] lactic acid; 0.05% [wt/vol] glucose; 0.04% [wt/vol] CaCl2; 0.03% [wt/vol] KH2PO4; 0.1% [wt/vol] NH4Cl; 0.05% [wt/vol] NaCl, pH 4.8 [YPLac]) or on 2% (wt/vol) glucose (YPD) agar plates. Mitochondria were isolated by differential centrifugation and stored at –80°C in SEM (250 mM sucrose, 1 mM EDTA, and 10 mM MOPS/KOH, pH 7.2). Proteins were synthesized in rabbit reticulocyte lysate in the presence of [35S]methionine and incubated with isolated mitochondria in import buffer (3% [wt/vol] BSA, 250 mM sucrose, 80 mM KCl, 5 mM methionine, 5 mM MgCl2, 2 mM KH2PO4, 10 mM MOPS-KOH, pH 7.2, 2 mM NADH, 4 mM ATP, 20 mM creatine phosphate, and 0.1 mg/ml creatine kinase; Ryan et al., 1999). Where indicated, mitochondria were subjected to a 15-min heat shock (37°C). was dissipated by addition of 8 µM antimycin A, 20 µM oligomycin, and 1 µM valinomycin before the import experiment. After import, mitochondria were treated with 50 µg/ml of proteinase K for 15 min on ice. Analysis was performed by digital autoradiography (Storm imaging system; GE Healthcare) and ImageQuant software (GE Healthcare). For alkaline extraction, mitochondria were resuspended in 0.1 M Na2CO3 and incubated for 15 min on ice. Pellet and supernatant fractions of mitochondrial proteins were separated by 60-min centrifugation at 100,000 g. Fractions were precipitated by trichloroacetic acid precipitation (12% vol/vol).
Lipid analysis
Mitochondrial lipids were extracted using chloroform/methanol/water (10:10:3 vol/vol) and desalted using butanol extraction. Tetramyristoyl cardiolipin (Avanti Polar Lipids, Inc.) was added as internal standard. Profiles of polar lipids were obtained using a micromass spectrometer (Q-Tof micro; Waters) in the negative electrospray ionization ion mode with upfront LCMS separation as described previously (Shui et al., 2007). Mass spectra were recorded from m/z of 600 to 1500. Lipid chromatograms combined to generate averaged spectra and profiles of wild-type and mutant conditions were compared in a nontargeted fashion based on a chemometric method (Guan et al., 2006). Ions corresponding to cardiolipin were selected and their chromatograms were extracted using MassLynx 4.0 software (Waters). Integrated peak areas and their normalized values (using tetramyristoyl cardiolipin as internal standards) were used for semiquantitative analysis (Shui et al., 2007). Quantification of individual molecular species of PA and PG was performed using multiple reaction monitoring with a 4000 Q-Trap mass spectrometer (Applied Biosystems) as described by Fei et al. (2008).
For in vivo labeling, cells were grown at least six generations to late log phase at 25°C in YPG medium supplemented with 10 µCi/ml of [32PO4]. A mitochondrial enriched fraction was prepared as previously described (Chang et al., 1998). In brief, cells were disrupted using silica/zirconium beads in a Mini-Bead-Beater 8 (BioSpec Products) and unbroken cells were removed by centrifugation. Enriched mitochondria were isolated by a final centrifugation at 27,000 g. Enriched mitochondria were suspended in 0.5 M NaCl and 0.1 N HCl/methanol/chloroform (1:2:1 vol/vol) and, after vortexing, a two-phase system was generated by adding an additional part of the NaCl solution. The chloroform phase was applied to either 10 x 10- or 10 x 20-cm HPTLC Silica 60 plates (EMD) for two- or one-dimensional separation of lipids, respectively. For two-dimensional separation, chloroform/methanol/25% NH3 (65:35:5 vol/vol) was used in the first dimension and chloroform/methanol/acetone/acetic acid/water (50:20:10:10:5 vol/vol) was used in the second dimension. For one-dimensional separation, chloroform/methanol/acetic acid (65:25:8) was used. Radiolabel was imaged using a Molecular Imager FX (Bio-Rad Laboratories). Lipids were identified based on mobilities of known standards.
Miscellaneous
of isolated mitochondria was assessed by fluorescence quenching using the potential-sensitive fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide in membrane potential buffer (0.6 M sorbitol, 0.1% [wt/vol] BSA, 10 mM MgCl2, 0.5 mM EDTA, and 20 mM KPi, pH 7.2) as described previously (Brandner et al., 2005). In some figures, nonrelevant gel lanes were removed. For Western blot analysis, mitochondrial proteins or protein complexes were separated by SDS-PAGE or blue native electrophoresis (Ryan et al., 1999; Schägger, 2002) and blotted on polyvinylidene fluoride membranes.
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft (P. Rehling and N. Pfanner), Sonderforschungsbereich 746, Excellence Initiative of the German Federal and State Governments (EXC 294), Gottfried Wilhelm Leibniz Program, Fonds der Chemischen Industrie (N. Pfanner), Singapore National Research Foundation, National University of Singapore via the Life Science Institute and the Academic Research Fund, Biomedical Research Council of Singapore (M.R. Wenk), National Institutes of Health (grant GM56389 to W. Dowhan), and Boehringer Ingelheim Fonds (predoctoral fellowship to S. Kutik).
Submitted: 6 June 2008
Accepted: 25 November 2008
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