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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Mol Biol. Author manuscript; available in PMC 2012 August 19.
Published in final edited form as:
PMCID: PMC3146634

Structural Basis for the Function of Tim50 in the Mitochondrial Presequence Translocase


Many mitochondrial proteins are synthesized as preproteins carrying amino-terminal presequences in the cytosol. The preproteins are imported by the translocase of the outer mitochondrial membrane (TOM) and the presequence translocase of the inner membrane (TIM). Tim50 and Tim23 transfer preproteins through the intermembrane space to the inner membrane. We report the crystal structure of the intermembrane space domain of yeast Tim50 to 1.83 Å resolution. A protruding β-hairpin of Tim50 is crucial for interaction with Tim23, providing a molecular basis for the cooperation of Tim50 and Tim23 in preprotein translocation to the protein-conducting channel of the mitochondrial inner membrane.

Keywords: mitochondrial inner membrane, preprotein, protein sorting, Saccharomyces cerevisiae, Tim23

Mitochondria play central functions in cellular bioenergetics, metabolism, ion homeostasis and apoptosis.1-5 99% of the ~1,000 different mitochondrial proteins are synthesized in the cytosol and imported into mitochondria.1,2,6-8 More than half of the mitochondrial preproteins are synthesized with N-terminal presequences that form positively charged amphipathic α-helices and target the preproteins to mitochondria. The preproteins are imported by the general translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex).2,9-14 After passing through the TOM complex, the preproteins are received by Tim50, an essential component of the TIM23 complex exposed to the intermembrane space (IMS).15-17 Tim50 interacts with the N-terminal IMS-domain of Tim23 to transport preproteins to the transmembrane channel formed by the C-terminal domain of Tim23.13,18-22 No structural information on Tim50 has been reported and thus the molecular mechanism of Tim50 and its mode of interaction with Tim23 are unknown.

In this study, we report the crystal structure of the IMS-domain of Tim50 that contains a large groove as putative binding site for presequences and an exposed β-hairpin. We show that the β-hairpin is crucial for the interaction of Tim50 with Tim23, suggesting a cooperative function of these two essential TIM proteins in preprotein import.

Structure of the IMS-domain of Tim50

Tim50 consists of an N-terminal presequence, a transmembrane anchor and a large C-terminal IMS-domain (residues 133-476 in Saccharomyces cerevisiae).15-17 We expressed the C-terminal domain in E. coli and generated a trypsin-resistant fragment (residues 164-361). The crystal structure of the trypsin-resistant fragment was determined to 1.83 Å resolution using the molecular replacement method (Table 1). The final model of Tim50IMS contains residues 176-361, representing the conserved core of Tim50 (Fig. S1). Tim50IMS forms a monomer in the crystal structure and consists of five α-helices (A1-A5) and nine β-strands (B1-B9) (Fig. 1a). The core of the structure is constituted by a parallel β-sheet formed by B1, B4, B5, B8 and B9 with B1 in the middle. A proline-rich region forms an extended loop region at the N-terminus of the structure. Three α-helices A3, A4 and A5 constitute an anti-parallel bundle at the C-terminus of the structure. A β-hairpin protruding out of the Tim50 molecular surface by ~15 Å is formed by B2 and B3 and the short loop between B2 and B3 (Fig. 1a). The protruding β-hairpin represents the largest conserved area on the Tim50 molecular surface (Fig. 1b). Close to the protruding β-hairpin, Tim50IMS contains a large groove with a dimension of 15×10×5 Å (L×W×D) (Fig. 1b and c). The bottom of the groove is formed by the loops between B1/B2, B4/A2, B5/B6 and B8/B9.

Fig. 1
The Tim50 IMS-domain structure. (a) Ribbons drawing of the Tim50IMS monomer structure in side-by-side stereo mode. α-helices (A1 to A5) and β-strands (B1 to B9) are shown in light blue and green, respectively. Turns are shown in dark blue. ...
Table 1
Data collection and refinement statistics for Tim50IMS structure

Interaction of Tim50 with Tim23 in organello

To map the region of Tim50 that interacts with Tim23, we established an in organello assay by importing modified forms of Tim50 into mitochondria that carried a protein A-tag at Tim23. It was shown that the N-terminal presequence and transmembrane domain of Tim50 are not essential for its function.21 However, the crystallized Tim50IMS also lacks 115 C-terminal residues. To test if this C-terminal region of Tim50 was required for its biogenesis or binding to Tim23, we synthesized and radiolabeled a C-terminally truncated Tim50. Tim501-361 was efficiently imported into yeast mitochondria (Fig. S2a) and co-precipitated by protein A-tagged Tim23 after lysis of the mitochondria with a non-ionic detergent (Fig. S2b). Control proteins like Atp19 of the mitochondrial ATP synthase and Tim10 of the carrier translocase were not co-precipitated. Thus, the C-terminal region of Tim50 (residues 362-476) is neither essential for biogenesis of Tim50 nor binding to Tim23. The exact function of the C-terminal region of Tim50 is unknown; it may potentially contribute to the interaction with preproteins or the cooperation of Tim50 with the TOM complex or further TIM subunits.

Using a chemical crosslinking reagent, the only cysteine of yeast Tim50 (C268) was reported to be in proximity to Tim23.23 C268 is located at the bottom of the large groove and near the protruding β-hairpin (Fig. 1c). We performed a structure-based mutagenesis of Tim50 residues located at the protruding β-hairpin or the groove. The resulting mutant forms of Tim501-361 were efficiently imported into mitochondria (Fig. 2a, lanes 3-8), but differed significantly in their interaction with Tim23, as analyzed by pull-down with tagged Tim23 (Fig. 2a, lanes 11-16). Replacement of R214 and K217 of the β-hairpin strongly reduced the ability of Tim50 to bind to Tim23. In contrast, replacement of the residues E199 and D200 did not impair the Tim50-Tim23 interaction, whereas replacement of D222, Y223 and E310 slightly enhanced the interaction (Fig. 2a and b). In the crystal structure, residues R214 and K217 are located at the lateral surface of the protruding β-hairpin; E199, D200 and E310 are located in the large groove; and residues D222 and Y223 are positioned at the side of the Tim50 molecule below the β-hairpin (Fig. 1b and c; Fig. S3). In addition to the in organello interaction studies, we also analyzed the interaction of recombinant Tim50164-361 with Tim23IMS (residues 1-96; pI 4.12) by crosslinking. Replacement of R214 or K217 inhibited the Tim50-Tim23 interaction in vitro (Fig. 2c) comparable to the findings in organello. Taken together, these results indicate that the protruding β-hairpin of Tim50IMS, including the positively charged residues 214 and 217, is critical for binding to the acidic IMS-domain of Tim23.

Fig. 2
Interaction of mutant forms of Tim50 with Tim23. (a) Radiolabeled Tim501-361 and mutant forms were imported into yeast mitochondria carrying protein A-tagged Tim23 or into wild-type (WT) mitochondria. After lysis with digitonin and affinity-purification, ...

Model for cooperation of Tim50 and Tim23 in protein import

The large groove in Tim50IMS contains several exposed negatively charged residues (Fig. 1c). We speculate that this groove is ideally suited as binding site for positively charged presequences/preproteins (additionally, hydrophobic residues located around the rim of the groove may contribute to an interaction with the hydrophobic surfaces of the amphipathic presequences), though an involvement of the groove in the cooperation of Tim50 with the TOM complex or further TIM subunits cannot be excluded. The crystal structure of Tim50IMS was determined by molecular replacement using the phosphatase Scp1 that is specific for the C-terminal domain of RNA polymerase II.24 Scp1 is the top structural homologue of Tim50IMS (Z-score of 22.8), exhibiting similar protein folding with rms derivation of 2.2 Å for the main chain atoms (Fig. 3) (the sequence identity between human Scp1 and yeast Tim50IMS is 33.5% [calculated by ClustalW]). Interestingly, the crystal structure of Scp1 was solved in complex with the C-terminal phosphorylated peptide of RNA Polymerase II.24 The peptide forms a U-turn in a groove that corresponds to the putative presequence-binding groove in Tim50IMS (Fig. 3). In Tim50IMS, the protruding β-hairpin swings away from the groove, providing more space for the presequence-binding groove to likely accommodate an α-helix formed by the presequence. The bottom of the peptide-binding groove of Scp1 contains a DXDX(T/V) phosphatase motif24 that is not present in Tim50 (and Tim50 does not show phosphatase activity).15 The bottom of the putative presequence-binding groove of Tim50IMS is only weakly conserved (Fig. 1c), which may reflect that Tim50 has to interact with a large variety of mitochondrial presequences.

Fig. 3
Superimposition of Tim50IMS (green) and Scp1 structures (blue). The orientation of Tim50 is similar to that in Fig. 1a. The bound peptide substrate of Scp1 is shown in red.

The close proximity between the putative preprotein-binding groove and the Tim23-interacting β-hairpin of Tim50 can provide a molecular explanation for the observation that binding of preproteins to Tim50 depends on the interaction of Tim50 with Tim23.21 Though NMR spectroscopy analysis indicated that Tim23IMS is intrinsically disordered,25 recent studies provided evidence that the Tim23 sites for preprotein binding and interaction with Tim50 are in close proximity. Crosslinking studies mapped a region of Tim23 including residues 70 and 71 interacting with Tim50.20,22 NMR spectroscopy suggested that a Tim23 region from residues 71–84 is involved in presequence binding.25 We propose that for Tim50 as well as Tim23, the site of preprotein recognition is in close proximity to the Tim50-Tim23 interaction site, leading to a working model that the receptor module of the presequence translocase is formed by a composite presequence binding pocket that involves both Tim50 and Tim23.

In summary, our structural and functional analysis of Tim50 revealed that the protruding β-hairpin is important for recruiting Tim23, supporting the hypothesis that Tim50 and Tim23 function cooperatively to direct preproteins to the transmembrane channel formed by the C-terminal domain of Tim23.

Supplementary Material



We are grateful to the staff scientists in APS beamline SER-CAT for their help in data collection. We thank I. Perschil, C. Prinz and A. Schulze-Specking for expert technical assistance. This work was supported by grants from NIH (R01 GM65959) and Army Research Office (51894LS) (to B.D.S), Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 746, Excellence Initiative of the German Federal & State Governments (EXC 294 BIOSS; GSC-4 Spemann Graduate School), Bundesministerium für Bildung und Forschung, Landesforschungspreis Baden-Württemberg, Gottfried Wilhelm Leibniz Program and the Fonds der Chemischen Industrie.

Abbreviations used

intermembrane space
TIM23 complex
presequence translocase of the inner mitochondrial membrane
presequence translocase of inner membrane subunit of ~50 kDa
TOM complex
translocase of the outer mitochondrial membrane


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Accession code Atomic coordinates have been deposited in the Protein Data Bank under accession code 3QLE.

Supplementary material Supplementary material associated with this article can be found online at doi: xxx.


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