Role of the Deafness Dystonia Peptide 1 (DDP1) in Import of Human Tim23 into the Inner Membrane of Mitochondria*

Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder. We show that DDP1 acts with human Tim13 in a complex in the intermembrane space. The DDP1 (cid:1) hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria. The human DDP1 (cid:1) hTim13 complex complements the function of the TIM8 (cid:1) 13 complex in yeast and facilitates import of yeast and human Tim23. Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction. hTim13 to immunoprecipitation using either affinity-purified anti- DDP1 or anti-hTim13 IgG. Prior to immunoprecipitation, Triton X-100 was diluted to a final concentration of 0.2%. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting using antibodies against DDP1 and hTim13. Immunoprecipitation— Isolated mitochondria were resuspended at a concentration of 0.2 mg/ml in 1 ml of column buffer (1 (cid:3) phosphate- buffered saline, 10% glycerol, m M phenylmethylsulfonyl fluoride) with 0.5% Triton X-100 and lysed on ice for 30 min. After a clarifying spin (30 min, 100,000 (cid:3) g ), the extracts were split into two aliquots, diluted to a final concentration of Triton X-100 of 0.2%, and immunoprecipitated using antibodies against either DDP1 or hTim13 prebound to protein A-Sepharose beads. The immunocomplexes were dissociated in SDS- containing sample buffer and analyzed by SDS-PAGE and immuno-staining with anti-DDP1 and anti-hTim13 antibodies. MBP-DDP1, MBP-hTim13, and MBP-lac cells lysed clarifying

Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder. We show that DDP1 acts with human Tim13 in a complex in the intermembrane space. The DDP1⅐hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria. The human DDP1⅐hTim13 complex complements the function of the TIM8⅐13 complex in yeast and facilitates import of yeast and human Tim23. Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction.
The vast majority of mitochondrial proteins are encoded as precursors in the nuclear genome. Mitochondrial biogenesis is, therefore, dependent on the import and sorting of the nuclear encoded precursor proteins into mitochondrial subcompartments. In eukaryotes three distinct preprotein import systems located in the mitochondrial outer and inner membrane have been described (1)(2)(3)(4)(5). The outer membrane contains a general preprotein translocase, the TOM 1 complex, which mediates the recognition and binding of preproteins and their transfer across the outer membrane. This complex is most likely used by all nuclear encoded precursors. Import into and across the inner membrane is mediated by two distinct inner membrane translocases, the TIM22 and the TIM23 complexes. Both TIM complexes cooperate with the TOM complex but differ in their substrate specificity (6 -11). The TIM23 complex mediates import of preproteins with a positively charged matrix targeting signal into the mitochondrial matrix space and into the inner membrane (6,12,13). The translocation of such precursors into the matrix requires the membrane potential ⌬ across the inner membrane and ATP in the matrix. The ⌬ drives the translocation of the presequences through the protein-conducting channel of the TIM23 complex which is formed by the membrane-integrated proteins Tim23 and Tim17 (6,12). A molecular motor that is attached to the inner side of this channel then promotes further translocation of the mature portion of the preproteins into the matrix. This motor consists of the peripheral membrane protein Tim44, the mitochondrial Hsp70, and the nucleotide exchange factor Mge1. Together, these components in repeated ATP-dependent reaction cycles facilitate the vectorial translocation into the matrix in a stepwise manner (14).
The TIM22 complex mediates the insertion of a class of hydrophobic proteins with internal targeting signals into the inner membrane (7)(8)(9)(10)(11)(15)(16)(17). Typical substrates are members of the mitochondrial carrier family and other integral inner membrane proteins that are synthesized without a matrix-targeting signal. Insertion of these precursors into the inner membrane is strictly dependent on ⌬ but does not require ATP in the matrix.
The transfer of carrier proteins from the TOM complex to the TIM22 complex involves the assistance of three small, structurally related proteins of the intermembrane space, Tim9, Tim10, and Tim12 (8,10,11,15,17). These proteins belong to an evolutionary conserved family of zinc finger proteins characterized by a Cys 4 motif (5,18). Tim9 and Tim10 form a hetero-oligomeric complex of 70 kDa which interacts with translocation intermediates of the precursors which are partially translocated across the TOM complex (10,15). From the TIM9⅐10 complex the carrier proteins are handed over to the TIM9⅐10⅐12 complex tightly associated with the TIM22 complex in the inner membrane. The insertion of the translocation intermediate into the inner membrane by the TIM22 complex is strictly dependent on the membrane potential ⌬.
The family of small zinc finger proteins comprises two further components, Tim8 and Tim13 (18 -20). Their precise function was not known until recently. In the yeast Saccharomyces cerevisiae, Tim8 and Tim13 are also organized in a heterooligomeric complex in the intermembrane space. They are, in contrast to Tim9, Tim10, and Tim12, not essential for the viability of yeast cells (19). Recently it was shown that the TIM8⅐13 complex of yeast interacts with translocation intermediates of Tim23, the major component of the translocase for matrix-targeted preproteins (17,21,22). The TIM8⅐13 complex assists the import of Tim23 by binding to the partially trans-located precursor in the intermembrane space still associated with the TOM complex. It is only strictly required when the membrane potential is low and the interaction with TIM22 complex is inefficient (21). Under these conditions the TIM8⅐13 complex was proposed to function in accumulating the Tim23 translocation intermediate thereby shifting the equilibrium toward direct interaction with the membrane-integrated portion of the TIM22 complex (21).
The human homolog of Tim8 is encoded by the DDP1 (deafness dystonia peptide 1) gene. Mutations in the DDP1 cause the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder characterized by sensorineural hearing loss, dystonia, mental retardation, and blindness (19,23,24). Most of the DDP1 mutations are loss-of-function mutations predicted to lead to an absent or a truncated gene product. So far, only one missense mutation was found causing a cysteine to tryptophan exchange (C66W) within the Cys 4 motif (25).
In the present study, we analyzed the structural organization of the human zinc finger proteins DDP1 and hTim13 and their functional role in mitochondrial preprotein import. We show that DDP1 interacts with hTim13 in the mitochondrial intermembrane space thereby forming a hetero-oligomeric complex of 70 kDa. When expressed in yeast, the DDP1⅐hTim13 complex facilitates import of hTim23. In human mitochondria the DDP1⅐hTim13 complex is in direct contact with translocation intermediates of hTim23. This suggests that an impaired mitochondrial preprotein import is the pathogenetic basis of the Mohr-Tranebjaerg syndrome.
Preparation of Mitochondria and Subcellular Fractions-Mitochondria from yeast were isolated as described (26). Mitochondria from frozen HeLa cells (Computer Cell Culture Center, Mons, Belgium) or mouse liver were prepared in a medium containing 0.25 M sucrose, 5 mM EDTA, 20 mM Tris/HCl, pH 7.4, by differential centrifugation and subsequent sucrose step gradient ultracentrifugation as described previously (27). To analyze the subcellular localization of human small Tim proteins, the postmitochondrial supernatant after a 10,000 ϫ g centrifugation step was centrifuged in a TL100 ultracentrifuge at 100,000 ϫ g and 4°C for 30 min to yield the cytoplasmic fraction (supernatant) and the microsomal fraction (pellet). To release soluble components of the intermembrane space into the supernatant, mitochondria prepared from HeLa cells were solubilized using increasing concentrations of digitonin (0.05-0.3% w/v or 0.75-4.5 g of digitonin/g of mitochondrial protein) and centrifuged at 100,000 ϫ g for 30 min.
Fractions in which DDP1 and hTim13 coeluted were subsequently subjected to immunoprecipitation using either affinity-purified anti-DDP1 or anti-hTim13 IgG. Prior to immunoprecipitation, Triton X-100 was diluted to a final concentration of 0.2%. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting using antibodies against DDP1 and hTim13.
Immunoprecipitation-Isolated mitochondria were resuspended at a concentration of 0.2 mg/ml in 1 ml of column buffer (1 ϫ phosphatebuffered saline, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride) with 0.5% Triton X-100 and lysed on ice for 30 min. After a clarifying spin (30 min, 100,000 ϫ g), the extracts were split into two aliquots, diluted to a final concentration of Triton X-100 of 0.2%, and immunoprecipitated using antibodies against either DDP1 or hTim13 prebound to protein A-Sepharose beads. The immunocomplexes were dissociated in SDScontaining sample buffer and analyzed by SDS-PAGE and immunostaining with anti-DDP1 and anti-hTim13 antibodies.
Determination of Zinc-MBP-DDP1, MBP-hTim13, and MBP-lac␣ fusion proteins were expressed in XL1-blue Escherichia coli cells at 30°C in the presence of 1 mM zinc acetate for 16 h. Cells were lysed by sonication in a buffer containing 1 mM zinc acetate, 20 mM HEPES/ KOH, pH 7.4, 100 mM NaCl, 20 mM ␤-mercaptoethanol. A clarifying spin was performed, and the supernatant was either incubated with 40 mM N-ethylmaleimide (NEM) for 1 h or applied directly to an amylose column for affinity purification. After binding the column was washed with 50 ml of lysis buffer lacking the zinc acetate. Elution from the resin was obtained using 10 mM maltose. Concentrations of the eluted proteins were adjusted to 300 g/ml, and the Zn 2ϩ content was analyzed using inductively coupled plasma atomic emission spectroscopy (ICP) in a Varian-Vista Simultan spectrometer.
In Vitro Synthesis of Precursor Proteins and Import into Mitochondria-Precursor proteins were synthesized by coupled transcription/ translation in rabbit reticulocyte lysate (Promega) in the presence of [ 35 S]methionine (30). Import reactions into isolated yeast mitochondria or mitochondria prepared from mouse liver were carried out for 10 -20 min at 25°C in 180 l of import buffer (0.6 M sorbitol, 0.1 mg/ml bovine serum albumin, 80 mM KCl, 10 mM Mg(OAc) 2 , 2.5 mM EDTA, 2 mM KH 2 PO 4 , 50 mM HEPES/KOH, pH 7.2) in the presence of 2.5 mM ATP and 5 mM NADH. To energize isolated mitochondria from mouse liver 2.5 mM succinate was added additionally. The reactions contained 180 g of mitochondrial protein and 4% reticulocyte lysate with the radiolabeled precursor protein. Membrane potential was dissipated by omission of NADH and preincubation for 5 min at 25°C with 1 M valinomycin and 25 M FCCP. Aliquots (50 l) each corresponding to 50 g of mitochondrial protein were either incubated with protease (50 g/ml trypsin or 30 g/ml proteinase K) for 20 min on ice followed by incubation with protease inhibitor (5 min on ice) or left untreated. Mitochondria were washed twice in HS buffer (20 mM HEPES/KOH, pH 7.4, 0.6 M sorbitol), subjected to SDS-PAGE, and blotted onto nitrocellulose membranes.
For cross-link experiments, import reactions were performed in import buffer using 7.5% reticulocyte lysate with the radiolabeled precursor protein. A 100-g aliquot of freshly isolated mouse liver mitochondria was used for total cross-links and 30 g for cross-linking and subsequent immunoprecipitation. After import, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was added to a final concentration of 100 M and incubated 20 min on ice. The cross-linking reaction was quenched with 80 mM Tris, pH 8.0. Mitochondria were either analyzed directly by SDS-PAGE (total cross-links) or lysed in 0.5% Triton X-100 (30 min on ice) and subjected to immunoprecipitation.

RESULTS
DDP1 and hTim13 are Soluble Components of the Mitochondrial Intermembrane Space-The identification of DDP1, hTim13, hTim9, hTim10a, and hTim10b as components of mitochondrial import systems is based on their sequence similarities to yeast Tim8, Tim13, Tim9, Tim10, and Tim12, respectively, structurally related zinc finger proteins identified in the mitochondrial intermembrane space of yeast (18). To determine the intracellular location of the human homologs immunoblot analyses of subcellular fractions prepared from HeLa cells were performed. Western blotting using affinity-purified antibodies indicated that DDP1, hTim13, hTim9, Tim10a, and hTim10b colocalized with human Tim23 in the mitochondrial fraction but were absent in the cytoplasmic or the microsomal fraction (Fig. 1A). When isolated HeLa cell mitochondria were treated with proteinase K, neither protein was accessible to proteases (not shown), whereas all of them were released from the mitochondria when lysed with 0.1% digitonin (Fig. 1B). To analyze the submitochondrial location of DDP1 and hTim13 in more detail, mitochondria were treated in a stepwise manner with increasing concentrations of digitonin and fractionated by centrifugation. At a concentration of 0.1% digitonin the outer membrane was selectively opened, but the inner membrane remained intact because hTim23, a component of the inner membrane (Fig. 1B), and hTim44, a peripheral mitochondrial matrix protein, were recovered in the pellet fraction (Fig. 1, B and C). Under these conditions DDP1 and hTim13 were completely released into the supernatant, demonstrating that both proteins are located in the intermembrane space.
DDP1 and hTim13 Are Zinc-binding Proteins-DDP1 and hTim13 belong to an evolutionary conserved protein family whose members share four conserved cysteine residues (Cys 4 motif) (5,18). The Cys 4 motif is considered to constitute a metal binding site. The cysteine residues of yeast Tim10 and Tim12 have previously been shown to bind zinc, and zinc binding is required for the interaction of Tim10 and Tim12 with ADP/ATP carrier during import (8).
We constructed chimeric proteins consisting of DDP1 or of hTim13 fused to the MBP. The recombinant proteins were expressed in E. coli in the presence of 1 mM Zn 2ϩ ions and affinity purified on amylose resin ( Fig. 2A). As a control, MBP protein fused to the ␣-fragment of the ␤-galactosidase (MBP-␣) was purified ( Fig. 2A). MBP-DDP1, MBP-hTim13, and MBP-␣ were analyzed using ICP. The atomic emission was determined at the zinc-specific wavelengths of 206.200 and 213.857 nm. The Zn 2ϩ content was calculated based on the counts obtained at the major wavelength at 213.857 (Fig. 2B). The preparations of MBP-DDP1 and MBP-hTim13 contained Zn 2ϩ in ϳ1:1 stoichiometry (Fig. 2C). In contrast, the control preparation of MBP-␣ contained only background levels of zinc. This demonstrates that DDP1 and hTim13 are zinc-binding proteins.
To test whether cysteine residues are involved in Zn 2ϩ binding the sulfhydryl groups of MBP-DDP1 were modified with NEM prior to affinity purification. The Zn 2ϩ content of NEMtreated MBP-DDP1 was reduced to almost background levels (Fig. 2, B and C). Thus, the Cys 4 motif appears to mediate the binding of Zn 2ϩ .
DDP1 and hTim13 Are Organized in Hetero-oligomeric Complexes-Using recombinant MBP-DDP1 and MBP-hTim13 for calibration, we estimated that mitochondria from HeLa cells contain approximately equal amounts of DDP1 and hTim13, corresponding to about 10 -50 pg/g of mitochondrial protein (Fig. 3A). To assess the organization of DDP1 and hTim13, mitochondria were lysed with 0.5% Triton X-100 and subjected to gel filtration analysis on a Superose-12 column. DDP1 and hTim13 coeluted in fractions corresponding to an apparent molecular mass of ϳ70 kDa (Fig. 3B). DDP1 and hTim13 were absent in fractions corresponding to higher molecular masses. In addition, mitochondria were analyzed by blue native gel electrophoresis (28,29). DDP1 and hTim13 were monitored by immunoblotting with affinity-purified antibodies. Each antibody detected a major band with an electrophoretic mobility corresponding to a native molecular mass of ϳ70 kDa (Fig. 3C). A minor fraction of hTim13 was found in a complex of approximately 110 kDa. Thus, DDP1 and hTim13 appear to be organized in complexes of 70 kDa.
According to the predicted masses of DDP1 and hTim13 these complexes may be composed of six or seven subunits. To investigate whether DDP1 and hTim13 form homo-or heterooligomeric complexes, coimmunoprecipitations were performed using either total mitochondria or the 70-kDa fraction from the gel filtration column (Fig. 3D). When total mitochondria were lysed with Triton X-100, antibodies against DDP1 precipitated both DDP1 and hTim13. Likewise, antibodies against hTim13 FIG. 1. Localization of the small human Tim proteins to the mitochondrial intermembrane space. A, subcellular localization of Tim proteins. Human HeLa cell homogenates were fractionated into cytosol, microsomal fraction, and mitochondrial fraction. Equal amounts were subjected to SDS-PAGE and analyzed by immunoblotting, using affinity-purified antibodies against hTim9, hTim10a, hTim10b, hTim13, DDP1, Grp78, and antisera against hTim23 and argininosuccinate lyase (ASL). B, submitochondrial localization of the small Tim proteins. Isolated mitochondria from HeLa cells were treated with 0.1% digitonin, and membranes were reisolated by centrifugation. The membrane pellet (P) and supernatant (S) were analyzed by SDS-PAGE and Western blot using affinity-purified antibodies against the small Tim proteins and antisera against hTim23 and hTim44. C, controlled opening of the outer membrane of mitochondria from HeLa cells. Mitochondria were incubated with digitonin (Dig.) at increasing concentrations. The membrane pellets were separated from the soluble content by centrifugation. Proteins from the supernatants subsequently were precipitated using trichloroacetic acid. Trichloroacetic acid-precipitated supernatants were analyzed by SDS-PAGE and Western blotting using antibodies against hTim44, hTim13, and DDP1. The amounts of protein released into the supernatant were quantified. precipitated hTim13 and DDP1 (Fig. 3D, left panel). Antibodies against DDP1 and hTim13 did not precipitate hTim9, hTim10a, or hTim10b (not shown). Both antibodies depleted their cognate antigen and the corresponding partner protein from mitochondrial extracts. Yet, Western blot analysis detected significantly lower amounts of the partner proteins in the immunoprecipitates. When DDP1 and hTim13 were immunoprecipitated directly from the 70-kDa fraction of the gel filtration column similar relative amounts of the respective partner proteins were determined (Fig. 3D, right panel). This suggests that DDP1 and hTim13 are organized in heterohexameric complexes containing approximately equal amounts of each component. The lower efficiency of coprecipitation of the respective partner proteins is likely to result from dissociation of the complex during immunoprecipitation or the subsequent washing procedures.
DDP1 and hTim13 Are Orthologs of Yeast Tim8/Tim13-Tim8 and Tim13, the yeast homologs of DDP1 and hTim13, are not essential for cell viability. The fungal TIM8⅐13 complex interacts with the translocation intermediate of Tim23 and facilitates its import by trapping the incoming precursor in the intermembrane space thereby preventing retrograde translocation (21). Yeast cells carrying disruptions of TIM8 and TIM13 (⌬8/⌬13) grew normally at 30°C and 15°C in media containing the nonfermentable carbon source glycerol. In media containing glucose as a carbon source ⌬8/⌬13 grew significantly slower at 15°C (cold-sensitive). When yeast cells are grown in the presence of glucose, respiration is down-regulated, and ⌬ is lowered because it is mainly generated by the ADP/ATP carrier, which shuttles ATP from the cytosol into the matrix space in exchange for ADP from the matrix. It was suggested that the TIM8⅐13 complex is only required for import of yeast Tim23 (yTim23) when the membrane potential ⌬ is low (21).
To analyze whether DDP1 and hTim13 are able to substitute for the function of Tim8 and Tim13, we expressed the human components in the ⌬8/⌬13 yeast mutant. Both proteins were detected in mitochondria and released from the intermembrane space upon disruption of the outer membrane using digitonin at a concentration of 0.05% (Fig. 4A).
Wild-type (WT), ⌬8/⌬13, and ⌬8/⌬13 yeast cells harboring DDP1 and hTim13 (⌬8/⌬13ϩDDP1/hTim13) were grown at 15°C and 30°C in the presence of glucose. The cells grew normally at 30°C (Fig. 4, B and C, left panels). At 15°C the growth rate of ⌬8/⌬13 cells was reduced drastically compared with WT cells, whereas the ⌬8/⌬13ϩDDP1/hTim13 cells grew nearly like WT (Fig. 4, B and C, right panels). Thus, DDP1 and hTim13 rescue the cold-sensitive deletion phenotype in yeast. This indicates that the human homologs are able to complement the function of the yeast TIM8-13 complex. The DDP1⅐hTim13 Complex Facilitates Import of Yeast and Human Tim23-We have investigated the role of DDP1 and hTim13 in the import of mitochondrial precursor proteins into yeast mitochondria. Mitochondria isolated from WT, ⌬8/⌬13, and ⌬8/⌬13ϩDDP1/hTim13 yeast cells were either energized with NADH or preincubated with valinomycin and FCCP to dissipate the membrane potential. Radiolabeled precursor of yeast Tim23 and of human Tim23 was added, and import was measured. In the absence of ⌬, yTim23 accumulated at stage III, a translocation intermediate that is partially resistant to trypsin treatment (Fig. 5, upper left panel). In ⌬8/⌬13 mitochondria the amount of the stage III intermediate was reduced to ϳ50% of the WT level, indicating that the TIM8⅐13 complex is required to trap the translocation intermediate in the intermembrane space. In mitochondria from ⌬8/⌬13ϩDDP1/ hTim13 cells the formation of the stage III intermediate was restored to WT levels. This indicates that the DDP1⅐hTim13 complex interacts with the Tim23 precursor of yeast. When human Tim23 was incubated with the mitochondria in the absence of ⌬ no significant amounts of trypsin-protected species were detected (Fig. 5, upper right panel). Similarly, no resistant intermediates were detected when proteinase K was used (not shown). Although stage III intermediates of hTim23 were detected by cross-linking (see below), it is apparently not possible to characterize them by protease protection assays.
In the presence of ⌬, yTim23 was equally well imported into yeast WT and ⌬8/⌬13 mitochondria (Fig. 5, lower left panel), supporting the notion that the TIM8⅐13 complex is not strictly required for the biogenesis of yTim23 when the membrane potential is sufficiently strong (21). Yet, the import efficiency of yTim23 was increased significantly in mitochondria harboring the DDP1⅐hT13 complex, demonstrating that the human or- thologs contribute to import of the yeast precursor (Fig. 5, lower  left panel).
At high levels of ⌬, hTim23 was imported efficiently into WT mitochondria (Fig. 5, lower right panel). The import of hTim23 was reduced significantly in mitochondria from ⌬8/⌬13 cells, suggesting that it is dependent on the TIM8⅐13 complex even at high ⌬. When the human DDP1⅐hTim13 complex was present the import efficiency of hTim23 was restored. This indicates that the DDP1⅐hTim13 complex facilitates translocation and insertion of hTim23 into the mitochondrial inner membrane.
In conclusion, the DDP1⅐hTim13 complex acts in the import of Tim23 and assists the translocation of the precursor across the outer membrane in a manner similar to the yeast TIM8⅐13 complex. In contrast to yTim23, the import of hTim23 is gen-erally dependent on the assistance of the DDP1⅐hTim13 complex even when the membrane potential is high.
The DDP1⅐hTim13 Complex Interacts with the Stage III Translocation Intermediate of Human Tim23 in Mammalian Mitochondria-Import of hTim23 into isolated mouse liver mitochondria was dependent on ⌬ (data not shown). To address whether the DDP1⅐hTim13 complex is in contact with translocation intermediates (stage III) in mammalian mitochondria, we performed cross-linking. Mouse liver mitochondria were preincubated to dissipate the ⌬. Subsequently, radiolabeled hTim23 was added to accumulate stage III intermediates, and cross-linking with MBS was performed. Two major hTim23 cross-linking adducts were detected which correspond to apparent molecular masses of ϳ33 and 35 kDa (Fig. 6A). To characterize these cross-linking products immunoprecipitations with FIG. 3. DDP1 and hTim13 are organized in hetero-oligomeric complexes of 70 kDa. A, levels of DDP1 and hTim13 in mitochondria from of HeLa cells. Anti-peptide antibodies directed against the C termini of DDP1 and hTim13 were affinity purified, and recombinant MBP-DDP1 and MBP-hTim13 were used for calibration of their reactivity on Western blots (left panel). 30 g of HeLa cell mitochondria was subjected to SDS-PAGE and analyzed by Western blotting and immunodecoration with the anti-DDP1 and anti-hTim13 (right panel). B, gel filtration analysis of human DDP1 and hTim13. Mitochondria from HeLa cells were solubilized with 0.5% Triton X-100 and analyzed by gel filtration on a Superose-12 column. Fractions were analyzed by SDS-PAGE and Western blotting (upper panels) and quantified by densitometry (lower panels). DDP1 and hTim13 eluted from the column in peak fractions corresponding to an apparent molecular mass of ϳ70 kDa. The elution of calibration standards (see "Experimental Procedures") is indicated by arrows. C, DDP1 and hTim13 are part of a 70-kDa complex. Isolated mitochondria were solubilized in 0.5% Triton X-100 and separated by blue native electrophoresis (6 -16.5% polyacrylamide). Immunoblots were decorated with affinity-purified antibodies against DDP1 or hTim13. The protein complexes at 70 kDa (dark arrowhead) and 110 kDa (asterisk) are indicated. The molecular masses of standards run in adjacent lanes are indicated. D, DDP1 and hTim13 form hetero-oligomeric complexes. Isolated mitochondria were solubilized with Triton X-100 and subjected to immunoprecipitation with affinity-purified antibodies (␣-DDP1 and ␣-hTim13); preimmune IgG of DDP1 (left panel) and Tim13 (not shown) was used for control. The solubilized mitochondrial extract was fractionated by gel filtration, and the 70-kDa fraction was subjected to immunoprecipitation (right panel). Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with antibodies against DDP1 and hTim13.
antibodies against DDP1 and hTim13 were performed (Fig.  6B). Under native conditions each of the antibodies precipitated both cross-linking products. This indicates that the hTim23 precursor interacts with the intact DDP1⅐hTim13 complex in the intermembrane space of mammalian mitochondria.

DISCUSSION
The Mohr-Tranebjaerg syndrome is considered to be the first mitochondrial disorder caused by a defect in the mitochondrial import machinery. The suggestion of the underlying pathomechanism is based on the homology of the dysfunctional DDP1 to a family of small yeast proteins that act along the TIM22-dependent import pathway. In yeast, this protein family comprises the essential proteins Tim9, Tim10, and Tim12, and the nonessential components Tim8 and Tim13 (8,10,11,15,19). They form three distinct hetero-oligomeric complexes in the intermembrane space which facilitate the transfer of hydrophobic precursors from the TOM complex to the TIM22 complex in the inner membrane. These "small" TIM complexes bind to the translocation intermediates during import thereby maintaining the precursors in an import-competent conformation (17,21). The essential components differ from the nonessential components by their specificity for substrate preproteins. The TIM9⅐10 and the TIM9⅐10⅐12 complexes mediate the import of mitochondrial carrier proteins, whereas the TIM8⅐13 complex interacts specifically with translocation intermediates of Tim23, the major component of the inner membrane translocase for matrix targeted preproteins.
By analogy to the function of Tim8 and Tim13 in yeast it was suggested that the Mohr-Tranebjaerg syndrome is caused by a defect in the biogenesis of the human TIM23 complex (17,21). Human cDNAs of DDP1 and hTim13 were expressed in yeast cells carrying a double deletion of yTim8 and yTim13 (⌬8/⌬13). Mitochondria from WT, ⌬8/⌬13, and ⌬8/⌬13 cells harboring the human components (⌬8/⌬13ϩDDP1/hTim13) were isolated. Equal amounts of mitochondrial protein were subjected to SDS-PAGE and analyzed by Western blotting using antibodies against the mitochondrial yeast proteins yTim23, yTim8, yTim13, and against DDP1 and hTim13. B and C, complementation of the cold-sensitive phenotype of ⌬8/⌬13 in the presence of glucose. WT, ⌬8/⌬13, and ⌬8/⌬13ϩDDP1/hTim13 strains were grown at 30°C to an A 578 of 1.0. B, the cultures were subjected to serial 10-fold dilutions, and 2-l aliquots were spotted on YPD plates and incubated at 30°C and 15°C. C, the cultures were diluted to A 578 ϭ 0.05 in fresh YPD medium, and the liquid cultures were incubated at 15°C and 30°C. Once a day the cultures were rediluted to A 578 ϭ 0.05 in YPD medium prewarmed to the respective temperature. Aliquots were withdrawn after the indicated time periods, and the cell number was determined. The cell number at t ϭ 0 h was set equal to 1.
FIG. 5. The human DDP1⅐hTim13 complex facilitates the import of yeast and human Tim23 into yeast mitochondria. Radiolabeled hTim23 and yTim23 were synthesized in reticulocyte lysate and incubated for 15 min with mitochondria isolated from WT, ⌬8/⌬13, and ⌬8/⌬13ϩDDP1/hTim13 strain. The membrane potential was either dissipated with FCCP/valinomycin (upper panels; Ϫ⌬) or strengthened with NADH (lower panels; ϩ⌬). Samples were treated with 50 g/ml trypsin, analyzed by SDS-PAGE and Western blotting, and quantified using a PhosphorImaging system.
FIG. 6. The mammalian DDP1⅐hTim13 complex interacts with translocation intermediates of hTim23. Freshly isolated mouse liver mitochondria were pretreated with FCCP/valinomycin to dissipate the membrane potential. Radiolabeled human Tim23 precursor was added, and cross-linking with 100 M MBS was performed where indicated (A). Mitochondria were solubilized with 0.5% Triton X-100, and immunoprecipitation with affinity-purified antibodies against DDP1 and hTim13 and preimmune IgG to DDP1 was carried out under nondenaturing conditions. Samples were subjected to SDS-PAGE, Western blotting, and autoradiography (B). hTim23-specific cross-link adducts (X-links) are indicated.
However, the TIM8⅐13 complex in yeast is not strictly required for the import of Tim23. A requirement of the TIM8⅐13 complex was only observed when membrane insertion of Tim23 was compromised (21). It was not clear whether the human DDP1 and hTim13 are true orthologs of Tim8 and Tim13 and fulfill a corresponding function in the import of Tim23.
In the present study, we analyzed the structural organization and function of DDP1 and hTim13. We show that both proteins are located in the intermembrane space. They are present in roughly equimolar amounts and form hetero-oligomeric assemblies with an apparent molecular mass of ϳ70 kDa. The 70-kDa complex appears to be composed of three molecules of DDP1 and three molecules of hTim13. However, it cannot be decided whether this stoichiometry is fixed or determined by the relative expression levels of the components. DDP1 and hTim13 belong to a family of evolutionary conserved proteins of the intermembrane space. In humans, six members have been identified all of which share four conserved cysteine residues (Cys 4 motif) considered to constitute a metal binding site. We determined that DDP1 and hTim13 bind Zn 2ϩ ions in a 1:1 stoichiometry and thus appear to form zinc fingers. As in yeast these zinc fingers might be crucial for the recognition and binding of translocation intermediates by DDP1 and hTim13.
We demonstrated that the human DDP1⅐hTim13 complex is functional in yeast. It rescues the growth defect observed at low temperature in the yeast ⌬tim8/⌬tim13 mutant, indicating that it is a true ortholog. Moreover, mitochondria from the complemented strain import yTim23 with a higher efficiency than WT yeast mitochondria. Thus, DDP1 and hTim13 so far are the only components of the mitochondrial inner membrane translocase that are functional across species. Considering the rather low sequence similarity between the human and yeast orthologs and its restriction to regions around the conserved cysteine pairs (18), the putative zinc finger might be the functionally important element of the DDP1⅐hTim13 complex.
In all but one patient with Mohr-Tranebjaerg syndrome this putative zinc finger is not expressed because of nonsense or frameshift mutations located upstream the Cys 4 motif. In one patient a missense mutation, C66W, leads to a nonfunctional zinc finger. Studies on the mutant DDP1 C66W revealed that it does not accumulate in the intermembrane space of mitochondria from patient cell lines. 2 This suggests that the mutant DDP1 protein is not able to fold properly and is degraded rapidly; this also explains the full-blown clinical phenotype observed in a patient harboring the mutant C66W allele on the X chromosome.
Has the DDP1⅐hTim13 complex a role in the biogenesis of the human TIM23 complex? Import of hTim23 into yeast mitochondria appears to be strongly dependent on the assistance of DDP1 and hTim13, whereas import of yTim23 does not require the yeast TIM8⅐13 complex under normal conditions. In particular, import of hTim23 into yeast mitochondria required the assistance of the DDP1⅐hTim13 complex even when the ⌬ was high. Under these conditions biogenesis of yTim23 is independent of the yeast TIM8⅐13 complex. Furthermore, hTim23 was found to be in contact with the DDP1⅐hTim13 complex during import in isolated mammalian mitochondria. Together, these observations provide direct evidence that the DDP1⅐hTim13 complex fulfills an important role in import of Tim23 in mammals by trapping translocation intermediates in the intermembrane space (Fig. 7A). Mutations in DDP1 are likely to cause a defect in the biogenesis of the Tim23 in humans (Fig. 7B).
In summary, the DDP1⅐hTim13 complex acts in the inter-membrane space and specifically assists the import of the Tim23 precursors into the mitochondrial inner membrane. The DDP1⅐hTim13 complex functions in a manner comparable to that of the yeast TIM8⅐13 complex. Whereas import of yeast Tim23 does not require the TIM8⅐13 complex under normal conditions, import of hTim23, by contrast, appears to rely on the assistance of the DDP1⅐hTim13 complex under all conditions. Accordingly, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may therefore be based on a defective TIM23 translocase leading to severe pleitropic mitochondrial dysfunction. FIG. 7. Role of the DDP1⅐hTim13 complex in the import of hTim23 and proposed pathomechanism of the Mohr-Tranebjaerg syndrome. A, intact mitochondria. hTim23 precursor bound to receptors on the surface of the outer membrane is translocated across the TOM complex where it is trapped by the DDP1⅐hTim13 complex in the intermembrane space. The TIM22 complex interacts with the accumulated precursor and mediates insertion into the inner membrane in a ⌬-dependent manner. B, loss of DDP1 function. In the absence of the DDP1⅐hTim13 complex (Mohr-Tranebjaerg syndrome) hTim23 precursor cannot be trapped in the intermembrane space and accumulates bound to the receptors on the surface of the mitochondria. Because of the reduced concentration of translocation intermediates in the intermembrane space, insertion of hTim23 into the inner membrane by the TIM22 complex is compromised. IM, inner membrane; OM, outer membrane.