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

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. 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) 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 (1Dolezal P. Likic V. Tachezy J. Lithgow T. Science. 2006; 313: 314-318Crossref PubMed Scopus (432) Google Scholar, 2Oka T. Mihara K.A. Mol. Cell. 2005; 18: 145-146Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 3Jensen R.E. Johnson A.E. Nat. Struct. Biol. 2001; 8: 1008-1010Crossref PubMed Scopus (65) Google Scholar, 4Neupert W. Herrmann J.M. Annu. Rev. Biochem. 2007; 76: 723-749Crossref PubMed Scopus (1138) Google Scholar, 5Endo T. Yamamoto H. Esaki M. J. Cell Sci. 2003; 116: 3259-3267Crossref PubMed Scopus (150) Google Scholar, 6Truscott K.N. Brandner K. Pfanner N. Curr. Biol. 2003; 13: R326-R337Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). 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 (4Neupert W. Herrmann J.M. Annu. Rev. Biochem. 2007; 76: 723-749Crossref PubMed Scopus (1138) Google Scholar, 5Endo T. Yamamoto H. Esaki M. J. Cell Sci. 2003; 116: 3259-3267Crossref PubMed Scopus (150) Google Scholar, 7Glick B.S. Brandt A. Cunningham K. Müller S. Hallberg R.L. Schatz G. 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Acta. 2008; 1783: 610-617Crossref PubMed Scopus (48) Google Scholar). These proteins are synthesized without a presequence and are imported by the MIA (mitochondrial IMS import and assembly) machinery (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, 16Tokatlidis K. Cell. 2005; 121: 965-967Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). 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 (17Milenkovic D. Gabriel K. Guiard B. Schulze-Specking A. Pfanner N. Chacinska A. J. Biol. Chem. 2007; 282: 22472-22480Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Sideris D.P. Tokatlidis K. Mol. Microbiol. 2007; 65: 1360-1373Crossref PubMed Scopus (70) Google Scholar). Erv1 is a sulfhydryl oxidase that functions in a disulfide relay to oxidize Mia40 and promote substrate release from Mia40 (14Mesecke N. Terziyska N. Kozany C. Baumann F. Neupert W. Hell K. Herrmann J.M. Cell. 2005; 121: 1059-1069Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 19Hofhaus G. Lee J.E. Tews I. Rosenberg B. Lisowsky T. Eur. J. Biochem. 2003; 270: 1528-1535Crossref PubMed Scopus (71) Google Scholar, 20Allen S. Balabanidou V. Sideris D.P. Lisowsky T. Tokatlidis K. J. Mol. Biol. 2005; 353: 937-944Crossref PubMed Scopus (198) Google Scholar, 21Rissler M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (129) Google Scholar, 22Grumbt B. Stroobant V. Terziyska N. Israel L. Hell K. J. Biol. Chem. 2007; 282: 37461-37470Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 23Müller J.M. Milenkovic D. Guiard B. Pfanner N. Chacinska A. Mol. Biol. Cell. 2008; 19: 226-236Crossref PubMed Scopus (0) Google Scholar). 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) (24Gabriel K. Milenkovic D. Chacinska A. Müller J. Guiard B. Pfanner N. Meisinger C. J. Mol. Biol. 2007; 365: 612-620Crossref PubMed Scopus (130) Google Scholar). 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 (24Gabriel K. Milenkovic D. Chacinska A. Müller J. Guiard B. Pfanner N. Meisinger C. J. Mol. Biol. 2007; 365: 612-620Crossref PubMed Scopus (130) Google Scholar, 25Terziyska N. Grumbt B. Bien M. Neupert W. Herrmann J.M. Hell K. FEBS Lett. 2007; 581: 1098-1102Crossref PubMed Scopus (54) Google Scholar). 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 (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, 13Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). A hydrophobic segment following the presequence arrests translocation of Mia40 in the inner membrane (13Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 15Terziyska N. Lutz T. Kozany C. Mokranjac D. Mesecke N. Neupert W. Herrmann J.M. Hell K. FEBS Lett. 2005; 579: 179-184Crossref PubMed Scopus (145) Google Scholar). Mature Mia40 is bound to the inner membrane, although Naoé et al. (13Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) 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. (26Hofmann S. Rothbauer U. Mühlenbein N. Baiker K. Hell K. Bauer M.F. J. Mol. Biol. 2005; 353: 517-528Crossref PubMed Scopus (89) Google Scholar) 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 (26Hofmann S. Rothbauer U. Mühlenbein N. Baiker K. Hell K. Bauer M.F. J. Mol. Biol. 2005; 353: 517-528Crossref PubMed Scopus (89) Google Scholar). 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 (24Gabriel K. Milenkovic D. Chacinska A. Müller J. Guiard B. Pfanner N. Meisinger C. J. Mol. Biol. 2007; 365: 612-620Crossref PubMed Scopus (130) Google Scholar, 25Terziyska N. Grumbt B. Bien M. Neupert W. Herrmann J.M. Hell K. FEBS Lett. 2007; 581: 1098-1102Crossref PubMed Scopus (54) Google Scholar), yet experimental evidence that hMIA40 is imported by the MIA system has been lacking so far. Grumbt et al. (22Grumbt B. Stroobant V. Terziyska N. Israel L. Hell K. J. Biol. Chem. 2007; 282: 37461-37470Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) 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. 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 (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, 21Rissler M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (129) Google Scholar, 23Müller J.M. Milenkovic D. Guiard B. Pfanner N. Chacinska A. Mol. Biol. Cell. 2008; 19: 226-236Crossref PubMed Scopus (0) Google Scholar). 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 on with Mitochondria were by from cells on yeast and or The of M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (129) Google Scholar) was into the to YPH499 and were with pGB9334 and on with and for of mitochondria. were by and by from were and with the a was residues of S. cerevisiae Mia40 and used for the of a of Mia40core with a the was in and as for full-length soluble Mia40 to the in by the Saccharomyces from (17Milenkovic D. Gabriel K. Guiard B. Schulze-Specking A. Pfanner N. Chacinska A. J. Biol. Chem. 2007; 282: 22472-22480Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) to import into mitochondria, Mia40core was by and in and Mitochondria of were with of Mia40core in a was or Δψ was by of and was removed by with K. was by with of imported Mia40core was to the for endogenous (17Milenkovic D. Gabriel K. Guiard B. Schulze-Specking A. Pfanner N. Chacinska A. J. Biol. Chem. 2007; 282: 22472-22480Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). mitochondria were to in and followed by Mitochondria and were to with different The precursors of and were synthesized and to binding with Mia40 or imported into mitochondria. After import of the were with and by or to (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, D. Gabriel K. Guiard B. Schulze-Specking A. Pfanner N. Chacinska A. J. Biol. Chem. 2007; 282: 22472-22480Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 21Rissler M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (129) Google Scholar). of the was as previously J.M. Milenkovic D. Guiard B. Pfanner N. Chacinska A. Mol. Biol. Cell. 2008; 19: 226-236Crossref PubMed Scopus (0) Google Scholar). following the import of mitochondria were in and with The were by and The of Mia40 in C-terminal the C-terminal domain of yeast Mia40 (termed Mia40core) is competent in the binding of precursor we the domain residues of S. cerevisiae in we also the large form of Mia40 lacking the N-terminal targeting (17Milenkovic D. Gabriel K. Guiard B. Schulze-Specking A. Pfanner N. Chacinska A. J. Biol. Chem. 2007; 282: 22472-22480Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). under (17Milenkovic D. Gabriel K. Guiard B. Schulze-Specking A. Pfanner N. Chacinska A. J. Biol. Chem. 2007; 282: 22472-22480Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Sideris D.P. Tokatlidis K. Mol. Microbiol. 2007; 65: 1360-1373Crossref PubMed Scopus (70) Google Scholar), we used to the binding of precursor proteins to the Mia40 with the precursor of both recombinant proteins with that Mia40core substrate proteins in Mia40core is functional in vivo, we attached the bipartite presequence of cytochrome b2 to its and it in yeast cells that the of MIA40 but full-length Mia40 from a URA3-containing of the URA3-containing the cells containing only were The presequence of cytochrome b2 was also attached to the large form of Mia40 (13Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). The of yeast cells expressing or Mia40 was demonstrating that the C-terminal domain contains the essential of The presence of the Mia40 in mitochondria was by b2 is by the matrix and by the inner membrane to the form released to the IMS. The of the cleavage on the presence of the domain of cytochrome b2 (13Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). the b2 contain only the presequence their was not and two and were for to the of (13Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). of mitochondria, Mia40 and (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, 13Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 15Terziyska N. Lutz T. Kozany C. Mokranjac D. Mesecke N. Neupert W. Herrmann J.M. Hell K. FEBS Lett. 2005; 579: 179-184Crossref PubMed Scopus (145) Google Scholar). The of and were released by the soluble IMS protein cytochrome b2 We several to mitochondria containing were functional in protein import to the IMS. The of IMS including the MIA substrate were to of mitochondria containing the large of Mia40 and was with mitochondria, and its import was by (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, D. Gabriel K. Guiard B. Schulze-Specking A. Pfanner N. Chacinska A. J. Biol. Chem. 2007; 282: 22472-22480Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). mitochondria imported the precursor The of was by The size of the with Mia40core and Mia40core was sufficient for of the on the Although the smaller was in than it a with that the soluble is mitochondria of the imported substrate we used a for the precursor of which a of and J.M. Milenkovic D. Guiard B. Pfanner N. Chacinska A. Mol. Biol. Cell. 2008; 19: 226-236Crossref PubMed Scopus (0) Google Scholar). mitochondria in a manner with the as mitochondria containing large Mia40 Mia40core targeted to mitochondria by the presequence of cytochrome b2 the essential function of hMIA40 and Mia40core the of the Mia40 and a Mia40 are to be larger than kDa whereas and Mia40 are in most independent I. N. F. C. 2004; PubMed Scopus Google Scholar), P. Eur. J. Biochem. PubMed Scopus Google Scholar), and H. S. G. J. Mol. Biol. PubMed Scopus Google the Mia40 from but not from metazoans or were to carry mitochondrial presequences A and Mia40 proteins are large and N-terminal presequences whereas Mia40 proteins from to the C-terminal half of Mia40 and not We hMIA40 (26Hofmann S. Rothbauer U. Mühlenbein N. Baiker K. Hell K. Bauer M.F. J. Mol. Biol. 2005; 353: 517-528Crossref PubMed Scopus (89) Google Scholar) is to for yeast We hMIA40 in and by plasmid shuffling in a yeast with a of we hMIA40 was to for the essential function of yeast that hMIA40 rescued the human protein was to yeast cells lacking endogenous hMIA40 is functional in we yeast Mia40core be targeted and functional in without of a presequence. The plasmid shuffling revealed that Mia40core rescued yeast viability Mia40core was released upon of mitochondria and that it is a soluble IMS We a to Mia40core mitochondria were of IMS proteins and import of to a of and of imported In Mia40core mitochondria showed a as mitochondria with We that the C-terminal half of yeast Mia40 can be targeted to mitochondria without a presequence and is full-length Mia40core via the MIA study the import of Mia40core into mitochondria, we the recombinant protein via an N-terminal Mia40core was to a by yeast mitochondria import not require the inner Δψ in to the import of full-length Mia40 (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, 13Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). of sulfhydryl of Mia40core by its import import of Mia40core, we the mitochondria and Mia40core bound Erv1 but not control proteins cytochrome of the IMS Mia40core with endogenous Mia40 (14Mesecke N. Terziyska N. Kozany C. Baumann F. Neupert W. Hell K. Herrmann J.M. Cell. 2005; 121: 1059-1069Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 21Rissler M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (129) Google Scholar, 22Grumbt B. Stroobant V. Terziyska N. Israel L. Hell K. J. Biol. Chem. 2007; 282: 37461-37470Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 25Terziyska N. Grumbt B. Bien M. Neupert W. Herrmann J.M. Hell K. FEBS Lett. 2007; 581: 1098-1102Crossref PubMed Scopus (54) Google Scholar). The import of Mia40core the possibility that it was a MIA this we used conditional yeast mutants of MIA40 and (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, 21Rissler M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (129) Google Scholar, 23Müller J.M. Milenkovic D. Guiard B. Pfanner N. Chacinska A. Mol. Biol. Cell. 2008; 19: 226-236Crossref PubMed Scopus (0) Google Scholar). mitochondria imported Mia40core with a and and erv1-5 mitochondria were in its import mia40 and mitochondria import preproteins with (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, 21Rissler M. Wiedemann N. Pfannschmidt S. Gabriel K. Guiard B. Pfanner N. Chacinska A. J. Mol. Biol. 2005; 353: 485-492Crossref PubMed Scopus (129) Google Scholar, K. Milenkovic D. Chacinska A. Müller J. Guiard B. Pfanner N. Meisinger C. J. Mol. Biol. 2007; 365: 612-620Crossref PubMed Scopus (130) Google Scholar), we that Mia40core is imported via the MIA pathway. that yeast Mia40 contains targeting information for both the presequence and MIA pathways. demonstrate targeting via two pathways in vivo, we full-length Mia40 as as Mia40core in yeast The of full-length Mia40, which is imported via the TIM23 pathway, was not in with that of control proteins However, the of Mia40core was in mitochondria, that of and We that yeast Mia40 contains dual targeting yeast Mia40 is the protein that contains targeting information for both the presequence and MIA pathways. Mia40 proteins of are large and contain N-terminal presequences that onto the Δψ-dependent import (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, 13Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), whereas Mia40 proteins of are small and not contain presequences study and S. Rothbauer U. Mühlenbein N. Baiker K. Hell K. Bauer M.F. J. Mol. Biol. 2005; 353: 517-528Crossref PubMed Scopus (89) Google Scholar). the N-terminal portion of yeast Mia40 including the presequence is for its targeting and Mia40 is essential for viability of yeast (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, 13Naoé M. Ohwa Y. Ishikawa D. Ohshima C. Nishikawa S. Yamamoto H. Endo T. J. Biol. Chem. 2004; 279: 47815-47821Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). We have shown that this essential function is in the C-terminal half (termed of Mia40core into mitochondria does not require a membrane potential but on both Mia40 and demonstrating that Mia40core is a substrate of the MIA pathway. the targeting information of Mia40 for the MIA pathway is in the C-terminal half of the of Mia40 have been that the MIA machinery was by cells (12Chacinska A. Pfannschmidt S. Wiedemann N. Kozjak V. Sanjuán Szklarz L.K. Schulze-Specking A. Truscott K.N. Guiard B. Meisinger C. Pfanner N. EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (350) Google Scholar, S. Rothbauer U. Mühlenbein N. Baiker K. Hell K. Bauer M.F. J. Mol. Biol. 2005; 353: 517-528Crossref PubMed Scopus (89) Google Scholar). the import of small Mia40 via the MIA pathway require the presence of Mia40, we that the Mia40 were in evolution to import of the MIA via the presequence pathway. the MIA system was it was to import Mia40 also via this pathway, and of including and plants, switch from the presequence pathway to the MIA pathway in the of a large Mia40 containing both a presequence and targeting information in the C-terminal half in was show that of the N-terminal half does not the and of Mia40, under the has not been for all of that we have the N-terminal domain of Mia40 and thus have the dual targeting We P. Rehling and D. for with