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Mitochondrial Biogenesis, Switching the Sorting Pathway of the Intermembrane Space Receptor Mia40*

  • Agnieszka Chacinska
    Footnotes
    Affiliations
    Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany

    Centre for Biological Signalling Studies (bioss), Universität Freiburg, 79104 Freiburg, Germany
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  • Bernard Guiard
    Footnotes
    Affiliations
    Centre de Génétique Moléculaire, CNRS, 91190 Gif-sur-Yvette, France
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  • Judith M. Müller
    Affiliations
    Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany
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  • Agnes Schulze-Specking
    Affiliations
    Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany
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  • Kipros Gabriel
    Footnotes
    Affiliations
    Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany
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  • Stephan Kutik
    Affiliations
    Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany

    Centre for Biological Signalling Studies (bioss), Universität Freiburg, 79104 Freiburg, Germany
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  • Nikolaus Pfanner
    Correspondence
    To whom correspondence should be addressed: Inst. für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, Stefan-Meier-Straße 17, D-79104 Freiburg, Germany. Tel.: 49-761-203-5224; Fax: 49-761-203-5261
    Affiliations
    Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany

    Centre for Biological Signalling Studies (bioss), Universität Freiburg, 79104 Freiburg, Germany
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  • Author Footnotes
    * This work was supported in part by Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 746), the Excellence Initiative of the German Federal and State Governments (EXC 294), the Gottfried Wilhelm Leibniz Program, and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.
    1 Both authors contributed equally to this work.
    2 Supported by a postdoctoral fellowship from the Alexander von Humboldt Foundation.
    3 Present address: Dept. of Genetics, University of Melbourne, Parkville 3010, Australia.
Open AccessPublished:September 08, 2008DOI:https://doi.org/10.1074/jbc.M805356200
      Mitochondrial precursor proteins are directed into the intermembrane space via two different routes, the presequence pathway and the redox-dependent MIA pathway. The pathways were assumed to be independent and transport different proteins. We report that the intermembrane space receptor Mia40 can switch between both pathways. In fungi, Mia40 is synthesized as large protein with an N-terminal presequence, whereas in metazoans and plants, Mia40 consists only of the conserved C-terminal domain. Human MIA40 and the C-terminal domain of yeast Mia40 (termed Mia40core) rescued the viability of Mia40-deficient yeast independently of the presence of a presequence. Purified Mia40core was imported into mitochondria via the MIA pathway. With cells expressing both full-length Mia40 and Mia40core, we demonstrate that yeast Mia40 contains dual targeting information, directing the large precursor onto the presequence pathway and the smaller Mia40core onto the MIA pathway, raising interesting implications for the evolution of mitochondrial protein sorting.
      Mitochondria contain two aqueous compartments, the intermembrane space (IMS)
      The abbreviations used are: IMS, intermembrane space; hMIA40, human MIA40; MOPS, 4-morpholinepropanesulfonic acid; Ni-NTA, nickel-nitrilotriacetic acid.
      5The abbreviations used are: IMS, intermembrane space; hMIA40, human MIA40; MOPS, 4-morpholinepropanesulfonic acid; Ni-NTA, nickel-nitrilotriacetic acid.
      and the matrix. Most mitochondrial proteins are synthesized as precursors in the cytosol and recognized by the TOM (translocase of outer membrane) complex. After translocation through TOM, precursor proteins destined for the matrix employ the presequence TIM23 complex (translocase of inner membrane) and the associated motor. The precursors carry N-terminal presequences that specifically interact with TOM and TIM23 subunits and are proteolytically removed upon arrival in the matrix (
      • Dolezal P.
      • Likic V.
      • Tachezy J.
      • Lithgow T.
      ,
      • Oka T.
      • Mihara K.A.
      ,
      • Jensen R.E.
      • Johnson A.E.
      ,
      • Neupert W.
      • Herrmann J.M.
      ,
      • Endo T.
      • Yamamoto H.
      • Esaki M.
      ,
      • Truscott K.N.
      • Brandner K.
      • Pfanner N.
      ). Translocation of the presequences across the inner membrane requires the membrane potential (Δψ).
      Although the presequence pathway is used by virtually all proteins imported into the matrix, two different pathways have been characterized for protein transport into the IMS. (i) Precursors such as cytochrome b2 contain bipartite targeting information in their N-terminal region, a presequence-type matrix targeting signal followed by a hydrophobic sorting signal, which arrests translocation in the TIM23 complex. The preprotein is laterally transferred into the inner membrane, and the IMS domain either can be released by proteolytic cleavage or remains attached to the inner membrane via the hydrophobic segment (
      • Neupert W.
      • Herrmann J.M.
      ,
      • Endo T.
      • Yamamoto H.
      • Esaki M.
      ,
      • Glick B.S.
      • Brandt A.
      • Cunningham K.
      • Müller S.
      • Hallberg R.L.
      • Schatz G.
      ,
      • Chacinska A.
      • Lind M.
      • Frazier A.E.
      • Dudek J.
      • Meisinger C.
      • Geissler A.
      • Sickmann A.
      • Meyer H.E.
      • Truscott K.N.
      • Guiard B.
      • Pfanner N.
      • Rehling P.
      ,
      • van der Laan M.
      • Meinecke M.
      • Dudek J.
      • Hutu D.P.
      • Lind M.
      • Perschil I.
      • Guiard B.
      • Wagner R.
      • Pfanner N.
      • Rehling P.
      ). (ii) Many IMS proteins are small (<15–20 kDa) and contain conserved cysteine residues that form disulfide bonds and/or bind metal ions (
      • Neupert W.
      • Herrmann J.M.
      ,
      • Koehler C.M.
      ,
      • Stojanovski D.
      • Müller J.M.
      • Milenkovic D.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ). These proteins are synthesized without a presequence and are imported by the MIA (mitochondrial IMS import and assembly) machinery (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Tokatlidis K.
      ). Although the presequence-carrying preproteins depend on Δψ for import, the MIA machinery does not require Δψ yet functions in a redox-regulated manner by forming transient disulfide bonds with precursor proteins. Two essential members of the MIA machinery have been identified, Mia40 (Tim40) and Erv1. Mia40 acts as a receptor in the IMS. After translocation across the outer membrane, the precursor proteins form mixed disulfides with Mia40 (
      • Milenkovic D.
      • Gabriel K.
      • Guiard B.
      • Schulze-Specking A.
      • Pfanner N.
      • Chacinska A.
      ,
      • Sideris D.P.
      • Tokatlidis K.
      ). Erv1 is a sulfhydryl oxidase that functions in a disulfide relay to oxidize Mia40 and promote substrate release from Mia40 (
      • Mesecke N.
      • Terziyska N.
      • Kozany C.
      • Baumann F.
      • Neupert W.
      • Hell K.
      • Herrmann J.M.
      ,
      • Hofhaus G.
      • Lee J.E.
      • Tews I.
      • Rosenberg B.
      • Lisowsky T.
      ,
      • Allen S.
      • Balabanidou V.
      • Sideris D.P.
      • Lisowsky T.
      • Tokatlidis K.
      ,
      • Rissler M.
      • Wiedemann N.
      • Pfannschmidt S.
      • Gabriel K.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ,
      • Grumbt B.
      • Stroobant V.
      • Terziyska N.
      • Israel L.
      • Hell K.
      ,
      • Müller J.M.
      • Milenkovic D.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ).
      Although most studies analyzing the MIA pathway used precursor proteins smaller than 12 kDa, a recent study revealed that the MIA machinery can handle larger substrates such as the IMS proteins Mic14 (14 kDa) and Mic17 (17 kDa) (
      • Gabriel K.
      • Milenkovic D.
      • Chacinska A.
      • Müller J.
      • Guiard B.
      • Pfanner N.
      • Meisinger C.
      ). Moreover, the components of the MIA machinery are encoded by nuclear genes, synthesized in the cytosol, and imported into mitochondria. Remarkably, the precursor of Erv1 with a molecular mass of 22 kDa is imported by the MIA machinery, representing the largest MIA substrate known (
      • Gabriel K.
      • Milenkovic D.
      • Chacinska A.
      • Müller J.
      • Guiard B.
      • Pfanner N.
      • Meisinger C.
      ,
      • Terziyska N.
      • Grumbt B.
      • Bien M.
      • Neupert W.
      • Herrmann J.M.
      • Hell K.
      ). In contrast, yeast Mia40 is synthesized with an N-terminal presequence. This preprotein of 45 kDa is imported via the presequence pathway in a Δψ-dependent manner (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Naoé M.
      • Ohwa Y.
      • Ishikawa D.
      • Ohshima C.
      • Nishikawa S.
      • Yamamoto H.
      • Endo T.
      ). A hydrophobic segment following the presequence arrests translocation of Mia40 in the inner membrane (
      • Naoé M.
      • Ohwa Y.
      • Ishikawa D.
      • Ohshima C.
      • Nishikawa S.
      • Yamamoto H.
      • Endo T.
      ,
      • Terziyska N.
      • Lutz T.
      • Kozany C.
      • Mokranjac D.
      • Mesecke N.
      • Neupert W.
      • Herrmann J.M.
      • Hell K.
      ). Mature Mia40 is bound to the inner membrane, although Naoé et al. (
      • Naoé M.
      • Ohwa Y.
      • Ishikawa D.
      • Ohshima C.
      • Nishikawa S.
      • Yamamoto H.
      • Endo T.
      ) showed that Mia40 is also functional as soluble protein when it is targeted to the IMS by the bipartite signal of cytochrome b2.
      Hofmann et al. (
      • Hofmann S.
      • Rothbauer U.
      • Mühlenbein N.
      • Baiker K.
      • Hell K.
      • Bauer M.F.
      ) reported that human MIA40 (hMIA40) is considerably smaller than its yeast counterpart and not attached to the inner membrane. hMIA40 lacks an N-terminal presequence but is homologous to the C-terminal portion of yeast Mia40, including the conserved six cysteine residues. In a heterologous assay, hMIA40 was imported into yeast mitochondria, and several of its cysteine residues were required for import and/or stability of the protein (
      • Hofmann S.
      • Rothbauer U.
      • Mühlenbein N.
      • Baiker K.
      • Hell K.
      • Bauer M.F.
      ). hMIA40 has a molecular mass of 16 kDa and thus would fall in the size range of substrates that can be handled by the MIA pathway (
      • Gabriel K.
      • Milenkovic D.
      • Chacinska A.
      • Müller J.
      • Guiard B.
      • Pfanner N.
      • Meisinger C.
      ,
      • Terziyska N.
      • Grumbt B.
      • Bien M.
      • Neupert W.
      • Herrmann J.M.
      • Hell K.
      ), yet experimental evidence that hMIA40 is imported by the MIA system has been lacking so far. Grumbt et al. (
      • Grumbt B.
      • Stroobant V.
      • Terziyska N.
      • Israel L.
      • Hell K.
      ) reported that a recombinant C-terminal fragment of yeast Mia40 is competent in binding to Erv1 in vitro, raising the possibility that the C-terminal domain represents a functional domain. However, it has not been shown if the C-terminal domain alone can bind precursor proteins and is functional in vivo.
      We report that not only hMIA40 but also the C-terminal domain of yeast Mia40 (termed Mia40core) can replace full-length Mia40 of yeast. Remarkably, hMIA40 and Mia40core were functional in vivo, demonstrating that the C-terminal half of yeast Mia40 is sufficient for function and import into mitochondria. We show that Mia40core is imported into mitochondria by the MIA machinery, and thus, yeast Mia40 contains dual targeting information for both the presequence and MIA pathways.

      EXPERIMENTAL PROCEDURES

      Yeast Strains—The Saccharomyces cerevisiae strains used in this study are derivatives of YPH499 (MATa, ade2-101, his3-Δ200, leu2-Δ1, ura3-52, trp1-Δ63, lys2-801). The conditional mia40-3, erv1-2, and erv1-5 mutants were characterized previously (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Rissler M.
      • Wiedemann N.
      • Pfannschmidt S.
      • Gabriel K.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ,
      • Müller J.M.
      • Milenkovic D.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ). hMIA40 was derived from the cDNA clone IRATp970F0452D (RZPD). pFL39-derived plasmids with mia40 versions under the control of an endogenous promoter and terminator, pGB8220 (MIA40), pGB9308 (b2-MIA40Δ1-70/mia40Δ1), pGB9310 (b2-MIA40core/mia40Δ3), pGB9334 (MIA40core), and pGB9348 (hMIA40), were introduced into YPH499 mia40::ADE2 containing pMIA40 (pGB8210; URA3-containing Yep352 with MIA40). pMIA40 was removed by plasmid shuffling upon 5-fluoroorotic acid treatment at 24 °C on plates with rich synthetic medium. Mitochondria were isolated by differential centrifugation from cells grown on YPG medium (1% (w/v) yeast extract, 2% (w/v) Bacto-peptone, and 3% (w/v) glycerol) at 19 or 24 °C. The temperature-sensitive allele of erv1-2 (
      • Rissler M.
      • Wiedemann N.
      • Pfannschmidt S.
      • Gabriel K.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ) was integrated into the genome, giving rise to erv1-2int. Wild-type YPH499 and erv1-2int were transformed with pGB9334 (MIA40core) and grown on selective medium supplemented with 0.2% (w/v) glucose and 2% (w/v) glycerol at 28 °C for Western blot analysis of mitochondria.
      Phylogenetic Analysis—Mia40 homologs were identified by PSI-BLAST searches and confirmed by reciprocal PSI-BLAST analysis. 24 sequences from model organisms were selected and aligned with ClustalW2. From the resulting multiple sequence alignment, a subalignment was selected comprising residues 282–350 of S. cerevisiae Mia40 and used for the calculation of a PHYLIP tree.
      Import of Recombinant Mia40—Recombinant Mia40core (pN-His10-Mia40Δ1–225) with a His10 tag at the N terminus was expressed in Escherichia coli and purified as described for full-length soluble Mia40 (renamed due to the change in amino acid numbering by the Saccharomyces Genome Database from pN-His10-Mia40Δ1–94 (
      • Milenkovic D.
      • Gabriel K.
      • Guiard B.
      • Schulze-Specking A.
      • Pfanner N.
      • Chacinska A.
      ) to pN-His10-Mia40Δ1–70). For import into mitochondria, Mia40core was reduced by 50 mm 1,4-dithiothreitol and denatured in 8 m urea, 30 mm MOPS/KOH, and 10 mm 1,4-dithiothreitol (pH 7.2). Mitochondria (50–75 μg of protein) were incubated with 1.25–2.5 μg of Mia40core in a 100-μl reaction at 30 °C. Where indicated, 50 mm iodoacetamide was added, or Δψ was dissipated by addition of 1 μm valinomycin, 8 μm antimycin, and 20 μm oligomycin. Non-imported material was removed by treatment with 50 μg/ml proteinase K. Import was assayed by immunodecoration with antibodies raised against intact Mia40. Nickel-nitrilotriacetic acid (Ni-NTA) affinity purification of imported Mia40core was performed according to the procedure described for endogenous Mia40-His10 (
      • Milenkovic D.
      • Gabriel K.
      • Guiard B.
      • Schulze-Specking A.
      • Pfanner N.
      • Chacinska A.
      ).
      Miscellaneous—To generate mitoplasts, mitochondria were subjected to hypo-osmotic swelling in 1 mm EDTA and 10 mm MOPS/KOH (pH 7.2), followed by 250 mm NaCl where indicated. Mitochondria and mitoplasts were subjected to Western blot analysis with different antibodies. The 35S-labeled precursors of Tim9 and Tim8 were synthesized and subjected to binding assays with purified Mia40 or imported into isolated mitochondria. After import of 35S-labeled Tim9, the samples were treated with 50 μg/ml proteinase K where indicated and analyzed by reducing or nonreducing SDS-PAGE to detect disulfide-bonded conjugates (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Milenkovic D.
      • Gabriel K.
      • Guiard B.
      • Schulze-Specking A.
      • Pfanner N.
      • Chacinska A.
      ,
      • Rissler M.
      • Wiedemann N.
      • Pfannschmidt S.
      • Gabriel K.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ). Formation of the oxidized Tim8 monomer was analyzed as described previously (
      • Müller J.M.
      • Milenkovic D.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ). Briefly, following the import of 35S-labeled Tim8, mitochondria were solubilized in digitonin solubilization buffer (1% (w/v) digitonin, 20 mm Tris-HCl, 0.5 mm EDTA, 10% (v/v) glycerol, 50 mm NaCl, and 1 mm phenylmethylsulfonyl fluoride (pH 7.4)) supplemented with 50 mm iodoacetamide. The samples were analyzed by blue native electrophoresis and digital autoradiography.

      RESULTS AND DISCUSSION

      The Essential Function of Mia40 Is Located in Its C-terminal Domain—To determine whether the C-terminal domain of yeast Mia40 (termed Mia40core) is competent in the binding of precursor proteins, we expressed the domain (amino acid residues 226–403 of S. cerevisiae Mia40) in E. coli cells. For comparison, we also expressed the large form of Mia40 lacking the N-terminal targeting segments (
      • Milenkovic D.
      • Gabriel K.
      • Guiard B.
      • Schulze-Specking A.
      • Pfanner N.
      • Chacinska A.
      ). Because disulfide-linked Mia40-precursor conjugates remain stable under nonreducing conditions (
      • Milenkovic D.
      • Gabriel K.
      • Guiard B.
      • Schulze-Specking A.
      • Pfanner N.
      • Chacinska A.
      ,
      • Sideris D.P.
      • Tokatlidis K.
      ), we used nonreducing SDS-PAGE to directly monitor the binding of precursor proteins to the purified Mia40 constructs. Upon incubation with the 35S-labeled precursor of Tim9, both recombinant proteins formed Mia40-Tim9 conjugates with comparable efficiency (Fig. 1), indicating that Mia40core efficiently binds substrate proteins in vitro.
      Figure thumbnail gr1
      FIGURE 1The C-terminal half of recombinant Mia40 is sufficient for the binding of precursor proteins. Recombinant forms of S. cerevisiae Mia40 and Mia40core were purified and incubated with 35S-labeled Tim9 at 15 °C for the indicated time periods. The samples were analyzed by nonreducing SDS-PAGE and digital autoradiography.
      To address whether Mia40core is functional in vivo, we attached the bipartite presequence of cytochrome b2 to its N terminus (b2-Mia40core) (Fig. 2A) and expressed it in yeast cells that lacked the chromosomal copy of MIA40 but expressed full-length Mia40 from a URA3-containing plasmid. Upon loss of the URA3-containing plasmid, the cells containing only b2-Mia40core were viable (Fig. 2B, right panel). The presequence of cytochrome b2 was also attached to the large form of Mia40 (b2-Mia40) (Fig. 2A) (
      • Naoé M.
      • Ohwa Y.
      • Ishikawa D.
      • Ohshima C.
      • Nishikawa S.
      • Yamamoto H.
      • Endo T.
      ). The growth behavior of yeast cells expressing b2-Mia40core, b2-Mia40, or wild-type Mia40 was indistinguishable (Fig. 2B), demonstrating that the C-terminal domain contains the essential part of Mia40.
      Figure thumbnail gr2
      FIGURE 2The C-terminal half of Mia40 is functional. A, a schematic representation of Mia40 constructs fused to the cytochrome (Cyt.) b2 presequence (plus linker amino acids) is shown. The numbering of amino acid residues is according to the current assignment in the Saccharomyces Genome Database. TM, transmembrane segment. B, the growth of yeast at 24 °C before and after removal of wild-type MIA40 by 5-fluoroorotic acid (5-FOA) treatment is shown. C and D, the immunodecoration of yeast mitochondria (Mitoch.) and mitoplasts (Mitopl.) is shown. i, intermediate; m, mature; CCHL, cytochrome c heme lyase. E, 35S-labeled Tim9 was imported into isolated yeast mitochondria at 30 °C for the indicated time periods. The mitochondria were treated with proteinase K (Prot. K) and analyzed by SDS-PAGE and digital autoradiography. F, 35S-labeled Tim9 imported into isolated mitochondria was analyzed by nonreducing SDS-PAGE and digital autoradiography. G, 35S-labeled Tim8 imported into isolated mitochondria was analyzed by blue native electrophoresis and digital autoradiography.
      The presence of the Mia40 constructs in isolated mitochondria was analyzed by immunodecoration. Cytochrome b2 is first processed by the matrix processing peptidase and then by the inner membrane peptidase, leading to the mature form released to the IMS. The efficiency of the second cleavage depends on the presence of the heme-binding domain of mature cytochrome b2 (
      • Naoé M.
      • Ohwa Y.
      • Ishikawa D.
      • Ohshima C.
      • Nishikawa S.
      • Yamamoto H.
      • Endo T.
      ). As the b2 constructs contain only the presequence part, their processing was not complete, and two forms, intermediate and mature, were observed for b2-Mia40core (Fig. 2C), similar to the behavior of b2-Mia40 (
      • Naoé M.
      • Ohwa Y.
      • Ishikawa D.
      • Ohshima C.
      • Nishikawa S.
      • Yamamoto H.
      • Endo T.
      ). Upon swelling of mitochondria, intermediate b2-Mia40core remained membrane-bound (Fig. 2C), like wild-type Mia40 and intermediate b2-Mia40 (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Naoé M.
      • Ohwa Y.
      • Ishikawa D.
      • Ohshima C.
      • Nishikawa S.
      • Yamamoto H.
      • Endo T.
      ,
      • Terziyska N.
      • Lutz T.
      • Kozany C.
      • Mokranjac D.
      • Mesecke N.
      • Neupert W.
      • Herrmann J.M.
      • Hell K.
      ). The mature forms of b2-Mia40core and b2-Mia40 were released by swelling, like the soluble IMS protein cytochrome b2 (Fig. 2C).
      We performed several assays to determine whether mitochondria containing b2-Mia40core were functional in protein import to the IMS. The steady-state levels of IMS proteins, including the MIA substrate Tim10, were similar to those of mitochondria containing the large forms of Mia40 (Fig. 2, C and D). 35S-Labeled Tim9 was incubated with isolated mitochondria, and its import was assessed by protection against added proteinase K (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Milenkovic D.
      • Gabriel K.
      • Guiard B.
      • Schulze-Specking A.
      • Pfanner N.
      • Chacinska A.
      ). b2-Mia40core mitochondria efficiently imported the precursor (Fig. 2E). The formation of Mia40-Tim9 conjugates was monitored by nonreducing SDS-PAGE (Fig. 2F). The size difference of the Tim9 conjugates with intermediate Mia40core and mature Mia40core was sufficient for separation of the conjugates on the gel. Although the smaller mature b2-Mia40core was present in lower abundance than intermediate b2-Mia40core (Fig. 2C), it efficiently formed a conjugate with Tim9 (Fig. 2F), indicating that the soluble mature b2-Mia40core is functional. To test whether b2-Mia40core mitochondria promoted oxidation of the imported substrate proteins, we used a blue native assay developed for the precursor of Tim8, which allows a direct separation of reduced and oxidized forms (
      • Müller J.M.
      • Milenkovic D.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ). b2-Mia40core mitochondria generated oxidized Tim8 in a time-dependent manner with the same efficiency as mitochondria containing large Mia40 (Fig. 2G). Thus, Mia40core targeted to mitochondria by the presequence of cytochrome b2 fully replaced the essential function of wild-type Mia40.
      Presequence-free hMIA40 and Mia40core Complement Yeast—We systematically compared the primary structures of the available Mia40 homologs and constructed a phylogenetic tree (Fig. 3A). Fungal Mia40 homologs are predicted to be larger than 25 kDa (supplemental Fig. S1), whereas metazoan and plant Mia40 homologs are typically small, in most cases below 20 kDa. Using three independent programs (Predotar (
      • Small I.
      • Peeters N.
      • Legeai F.
      • Lurin C.
      ), MitoProt (
      • Claros M.G.
      • Vincens P.
      ), and TargetP (
      • Emanuelsson O.
      • Nielsen H.
      • Brunak S.
      • von Heijne G.
      )), the Mia40 homologs from fungi but not from metazoans or plants were predicted to carry mitochondrial presequences (Fig. 3, A and B). Thus, fungal Mia40 proteins are large and possess N-terminal presequences (supplemental Fig. S1), whereas Mia40 proteins from higher eukaryotes usually correspond to the C-terminal half of fungal Mia40 and do not possess presequences. We asked whether hMIA40 (
      • Hofmann S.
      • Rothbauer U.
      • Mühlenbein N.
      • Baiker K.
      • Hell K.
      • Bauer M.F.
      ) is able to substitute for yeast Mia40. We expressed hMIA40 in yeast, and by plasmid shuffling in a yeast strain with a chromosomal deletion of MIA40, we analyzed whether hMIA40 was able to substitute for the essential function of yeast Mia40. Fig. 3C shows that hMIA40 indeed rescued cell viability. Thus, the presequence-free human protein was able to complement yeast cells lacking endogenous Mia40.
      Figure thumbnail gr3
      FIGURE 3Mia40 homologs. A, PHYLIP tree for Mia40. The tree was generated with ClustalW2 and visualized by TreeView (
      • Page R.D.
      ). B, presequence prediction for Mia40 homologs. Mitoch., mitochondrial; aa, amino acid residues. C, complementation analysis of a yeast mia40 deletion mutant by hMIA40. The growth of yeast at 24 °C before and after removal of wild-type MIA40 by 5-fluoroorotic acid (5-FOA) treatment is shown.
      As presequence-free hMIA40 is functional in yeast, we asked whether yeast Mia40core may be correctly targeted and functional in vivo without attachment of a presequence. The plasmid shuffling approach indeed revealed that presequence-free Mia40core fully rescued yeast viability (Fig. 4A). Mia40core was released upon swelling of mitochondria (Fig. 4B, lanes 2 and 3), indicating that it is a soluble IMS protein. We performed a systematic analysis to determine whether Mia40core mitochondria were functional: steady-state levels of IMS proteins (Fig. 4B, lanes 4 and 5), import of 35S-labeled Tim9 to a protease-protected location (Fig. 4C), formation of Mia40core-precursor conjugates (Fig. 4D), and oxidation of imported Tim8 (Fig. 4E). In each case, Mia40core mitochondria showed a similar activity as mitochondria with wild-type Mia40. We concluded that the C-terminal half of yeast Mia40 can be targeted to mitochondria without a presequence and is functional, like full-length Mia40.
      Figure thumbnail gr4
      FIGURE 4Mia40 without a presequence is functional. A, the growth of yeast at 24 °C before and after removal of wild-type MIA40 by 5-fluoroorotic acid (5-FOA) treatment is shown. B, the immunodecoration of mitochondria (Mitoch.) and mitoplasts (Mitopl.) is shown. CCHL, cytochrome c heme lyase; Cyt., cytochrome. C, 35S-labeled Tim9 was imported into isolated mitochondria. The mitochondria were treated with proteinase K (Prot. K) and analyzed by SDS-PAGE and digital autoradiography. D, 35S-labeled Tim9 imported into isolated mitochondria was analyzed by nonreducing SDS-PAGE and digital autoradiography. E, imported Tim8 was analyzed by blue native electrophoresis.
      Mia40core Is Imported via the MIA Pathway—To study the import of Mia40core into mitochondria, we purified the recombinant protein via an N-terminal His tag. Mia40core was transported to a protease-protected location by isolated yeast mitochondria (Fig. 5A, lanes 7–9). Its import did not require the inner Δψ (Fig. 5A, lanes 4-6), in contrast to the import of presequence-containing full-length Mia40 (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Naoé M.
      • Ohwa Y.
      • Ishikawa D.
      • Ohshima C.
      • Nishikawa S.
      • Yamamoto H.
      • Endo T.
      ). Blocking of sulfhydryl groups of Mia40core by iodoacetamide completely inhibited its import (Fig. 5A, lanes 1–3). Upon import of Mia40core, we lysed the mitochondria and performed Ni-NTA affinity chromatography. Mia40core bound Erv1 but not control proteins like cytochrome c of the IMS (Fig. 5B). Thus, Mia40core interacts with endogenous Erv1, like wild-type Mia40 (
      • Mesecke N.
      • Terziyska N.
      • Kozany C.
      • Baumann F.
      • Neupert W.
      • Hell K.
      • Herrmann J.M.
      ,
      • Rissler M.
      • Wiedemann N.
      • Pfannschmidt S.
      • Gabriel K.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ,
      • Grumbt B.
      • Stroobant V.
      • Terziyska N.
      • Israel L.
      • Hell K.
      ,
      • Terziyska N.
      • Grumbt B.
      • Bien M.
      • Neupert W.
      • Herrmann J.M.
      • Hell K.
      ).
      Figure thumbnail gr5
      FIGURE 5Purified Mia40core is imported by the MIA pathway. A, the import of recombinant Mia40core into mitochondria (Mitoch.) was performed as described under “Experimental Procedures.” The mitochondria were treated with proteinase K (Prot. K) and analyzed by SDS-PAGE and immunodecoration. B, Ni-NTA affinity purification of Mia40core was performed upon import into mitochondria and immunodecoration with different antisera. Load, 1%; Eluate, 100%. Cyt., cytochrome. C, Mia40core was imported into wild-type (WT) and mia40-3 mitochondria and analyzed as described for A. D, the import of Mia40core into wild-type and erv1 mutant mitochondria is shown.
      The Δψ-independent import of presequence-free Mia40core raised the possibility that it was a MIA substrate. To address this directly, we used conditional yeast mutants of MIA40 and ERV1 (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Rissler M.
      • Wiedemann N.
      • Pfannschmidt S.
      • Gabriel K.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ,
      • Müller J.M.
      • Milenkovic D.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ). mia40-3 mitochondria imported Mia40core with a diminished efficiency (Fig. 5C), and erv1-2 and erv1-5 mitochondria were strongly inhibited in its import (Fig. 5D). Because mia40 and erv1 mutant mitochondria import presequence-containing preproteins with wild-type efficiency (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Rissler M.
      • Wiedemann N.
      • Pfannschmidt S.
      • Gabriel K.
      • Guiard B.
      • Pfanner N.
      • Chacinska A.
      ,
      • Gabriel K.
      • Milenkovic D.
      • Chacinska A.
      • Müller J.
      • Guiard B.
      • Pfanner N.
      • Meisinger C.
      ), we conclude that Mia40core is imported via the MIA pathway.
      Taken together, these findings indicate that yeast Mia40 contains targeting information for both the presequence and MIA pathways. To directly demonstrate targeting via two pathways in vivo, we coexpressed presequence-containing full-length Mia40 as well as presequence-free Mia40core in erv1-2 yeast cells. The amount of full-length Mia40, which is imported via the TIM23 pathway, was not affected in comparison with wild-type yeast, like that of MIA-independent control proteins (Fig. 6). However, the level of Mia40core was strongly diminished in erv1-2 mitochondria, like that of Tim10 (Fig. 6, lanes 1 and 2). We concluded that yeast Mia40 contains dual targeting information.
      Figure thumbnail gr6
      FIGURE 6Mia40core depends on the MIA machinery in vivo. The steady-state protein levels of mitochondria (Mitoch.) isolated from wild-type (WT) and erv1-2 cells coexpressing Mia40core are shown. The mitochondria were analyzed by SDS-PAGE and immunodecoration. Cyt., cytochrome; CCHL, cytochrome c heme lyase. The asterisk indicates an unspecific reaction.
      Conclusions—To our knowledge, yeast Mia40 is the first protein that contains targeting information for both the presequence and MIA pathways. Mia40 proteins of fungi are large and contain N-terminal presequences that direct them onto the Δψ-dependent import route (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Naoé M.
      • Ohwa Y.
      • Ishikawa D.
      • Ohshima C.
      • Nishikawa S.
      • Yamamoto H.
      • Endo T.
      ), whereas Mia40 proteins of higher eukaryotes are small and do not contain cleavable presequences (this study and Ref.
      • Hofmann S.
      • Rothbauer U.
      • Mühlenbein N.
      • Baiker K.
      • Hell K.
      • Bauer M.F.
      ). Surprisingly, the N-terminal portion of yeast Mia40 including the presequence is dispensable for its targeting and function. Mia40 is essential for cell viability of yeast (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Naoé M.
      • Ohwa Y.
      • Ishikawa D.
      • Ohshima C.
      • Nishikawa S.
      • Yamamoto H.
      • Endo T.
      ). We have shown that this essential function is located in the C-terminal half (termed Mia40core). Import of Mia40core into mitochondria does not require a membrane potential but depends on both Mia40 and Erv1, demonstrating that Mia40core is a substrate of the MIA pathway. Thus, the targeting information of Mia40 for the MIA pathway is located in the C-terminal half of the protein.
      No prokaryotic homologs of Mia40 have been found, suggesting that the MIA machinery was developed by eukaryotic cells (
      • Chacinska A.
      • Pfannschmidt S.
      • Wiedemann N.
      • Kozjak V.
      • Sanjuán Szklarz L.K.
      • Schulze-Specking A.
      • Truscott K.N.
      • Guiard B.
      • Meisinger C.
      • Pfanner N.
      ,
      • Hofmann S.
      • Rothbauer U.
      • Mühlenbein N.
      • Baiker K.
      • Hell K.
      • Bauer M.F.
      ). Because the import of small Mia40 forms via the MIA pathway will require the presence of pre-existing Mia40, we speculate that the large, presequence-containing Mia40 forms were established early in evolution to allow import of the central MIA component via the existing presequence pathway. Once the MIA system was established, it was then possible to import Mia40 also via this pathway, and various groups of eukaryotes, including animals and plants, do so. To switch from the presequence pathway to the MIA pathway in evolution, the existence of a large Mia40 containing both a presequence and targeting information in the C-terminal half (as in present-day yeast) was advantageous because our findings show that loss of the N-terminal half does not inhibit the biogenesis and activity of Mia40, at least under the laboratory growth conditions applied. It has not been determined for what purpose (or selective advantage) all species of fungi that we analyzed have kept the N-terminal domain of Mia40 and thus likely have retained the dual targeting information.

      Acknowledgments

      We thank Drs. P. Rehling and D. Stojanovski for discussion.

      Supplementary Material

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