The C66W Mutation in the Deafness Dystonia Peptide 1 (DDP1) Affects the Formation of Functional DDP1·TIM13 Complexes in the Mitochondrial Intermembrane Space
2002; Elsevier BV; Volume: 277; Issue: 26 Linguagem: Inglês
10.1074/jbc.m201154200
ISSN1083-351X
AutoresSabine Hofmann, Ulrich Rothbauer, Nicole Mühlenbein, Walter Neupert, Klaus-Dieter Gerbitz, Michael Brunner, Matthias Bauer,
Tópico(s)RNA and protein synthesis mechanisms
ResumoMohr-Tranebjaerg syndrome is a progressive, neurodegenerative disorder caused by loss-of-function mutations in theDDP1/TIMM8A gene. DDP1 belongs to a family of evolutionary conserved proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. They mediate the import and insertion of hydrophobic membrane proteins into the mitochondrial inner membrane. All of them share a conserved Cys4 metal binding site proposed to be required for the formation of zinc fingers. So far, the only missense mutation known to cause a full-blown clinical phenotype is a C66W exchange directly affecting this Cys4motif. Here, we show that the mutant human protein is efficiently imported into mitochondria and sorted into the intermembrane space. In contrast to wild-type DDP1, it does not complement the function of its yeast homologue Tim8. The C66W mutation impairs binding of Zn2+ ions via the Cys4 motif. As a consequence, the mutated DDP1 is incorrectly folded and loses its ability to assemble into a hetero-hexameric 70-kDa complex with its cognate partner protein human Tim13. Thus, an assembly defect of DDP1 is the molecular basis of Mohr-Tranebjaerg syndrome in patients carrying the C66W mutation. Mohr-Tranebjaerg syndrome is a progressive, neurodegenerative disorder caused by loss-of-function mutations in theDDP1/TIMM8A gene. DDP1 belongs to a family of evolutionary conserved proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. They mediate the import and insertion of hydrophobic membrane proteins into the mitochondrial inner membrane. All of them share a conserved Cys4 metal binding site proposed to be required for the formation of zinc fingers. So far, the only missense mutation known to cause a full-blown clinical phenotype is a C66W exchange directly affecting this Cys4motif. Here, we show that the mutant human protein is efficiently imported into mitochondria and sorted into the intermembrane space. In contrast to wild-type DDP1, it does not complement the function of its yeast homologue Tim8. The C66W mutation impairs binding of Zn2+ ions via the Cys4 motif. As a consequence, the mutated DDP1 is incorrectly folded and loses its ability to assemble into a hetero-hexameric 70-kDa complex with its cognate partner protein human Tim13. Thus, an assembly defect of DDP1 is the molecular basis of Mohr-Tranebjaerg syndrome in patients carrying the C66W mutation. translocase of the mitochondrial outer membrane translocase of the mitochondrial inner membrane membrane potential deafness dystonia peptide 1 maltose binding protein glutathioneS-transferase carbonyl cyanide-p-trifluoromethoxyphenylhydrazone inductively coupled plasma atomic emission spectroscopy phosphate-buffered saline disuccinimidyl suberate nickel-nitrilotriacetic acid reduced glutathione proteinase K o-phenanthroline With the exception of a few components of the oxidative phosphorylation machinery, all mitochondrial proteins are encoded by nuclear genes and synthesized on cytosolic ribosomes. The import of such preproteins into mitochondria and the correct sorting into mitochondrial subcompartments is mediated by a set of import systems in the outer and inner mitochondrial membrane. Three distinct preprotein import systems have been described (1Herrmann J.M. Neupert W. Curr. Opin. Microbiol. 2000; 3: 210-214Crossref PubMed Scopus (120) Google Scholar, 2Bauer M.F. Hofmann I. Neupert I. Brunner I. Trends Cell Biol. 2000; 10: 25-31Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 3Pfanner N. Geissler A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 339-349Crossref PubMed Scopus (416) Google Scholar, 4Koehler C.M. FEBS Lett. 2000; 476: 27-31Crossref PubMed Scopus (91) Google Scholar, 5Jensen R.E. Johnson A.E. Nat. Struct. Biol. 2001; 8: 1008-1010Crossref PubMed Scopus (65) Google Scholar). All preproteins most likely use the general translocase in the outer membrane, the TOM1 complex. It mediates the recognition and binding of preproteins as well as their transfer across the outer membrane. Further movement of the translocation intermediates into and across the inner membrane is mediated by two distinct translocases in the inner membrane, the TIM23 and the TIM22 complexes. The TIM23 complex mediates import of preproteins with positively charged targeting signals at their N termini into the mitochondrial matrix space and into the inner membrane (6Berthold J. Bauer M.F. Schneider H.C. Klaus C. Dietmeier K. Neupert W. Brunner M. Cell. 1995; 81: 1085-1093Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 7Bauer M.F. Sirrenberg C. Neupert W. Brunner M. Cell. 1996; 87: 33-41Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 8Donzeau M. Kaldi K. Adam A. Paschen S. Wanner G. Guiard B. Bauer M.F. Neupert W. Brunner M. Cell. 2000; 101: 401-412Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The transfer of preproteins across the inner membrane strictly requires both an electrochemical potential (Δψ) across the inner membrane and the presence of ATP in the matrix as energy sources (9Moro F. Sirrenberg C. Schneider H.-C. Neupert W. Brunner M. EMBO J. 1999; 18: 3667-36745Crossref PubMed Scopus (83) Google Scholar). The TIM22 complex is used by a class of hydrophobic inner membrane proteins with internal and so far less characterized targeting signals (10Kerscher O. Holder J. Srinivasan M. Leung R.S. Jensen R.E. J. Cell Biol. 1997; 139: 1663-1675Crossref PubMed Scopus (171) Google Scholar, 11Koehler C.M. Jarosch E. Tokatlidis K. Schmid K. Schweyen R.J. Schatz G. Science. 1998; 279: 369-373Crossref PubMed Scopus (248) Google Scholar, 12Sirrenberg C. Bauer M.F. Guiard B. Neupert W. Brunner M. Nature. 1996; 384: 582-585Crossref PubMed Scopus (252) Google Scholar, 13Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 14Koehler C.M. Merchant S. Oppliger W. Schmid K. Jarosch E. Dolfini L. Junne T. Schatz G. Tokatlidis K. EMBO J. 1998; 17: 6477-6486Crossref PubMed Scopus (161) Google Scholar, 15Endres M. Neupert W. Brunner M. EMBO J. 1999; 18: 3214-3221Crossref PubMed Scopus (149) Google Scholar). Typical substrates are the members of the mitochondrial carrier family that are synthesized without a matrix-targeting signal. In addition, the TIM22 complex appears to mediate the import of precursors of other hydrophobic membrane proteins such as Tim23 and Tim22, which do not belong to the class of mitochondrial carriers (16Leuenberger D. Bally N.A. Schatz G. Koehler C.M. EMBO J. 1999; 18: 4816-4822Crossref PubMed Scopus (105) Google Scholar, 17Kaldi K. Bauer M.F. Sirrenberg C. Neupert W. Brunner M. EMBO J. 1998; 17: 1569-1576Crossref PubMed Scopus (59) Google Scholar, 18Kurz M. Martin H. Rassow J. Pfanner N. Ryan M.T. Mol. Biol. Cell. 1999; 10: 2461-2474Crossref PubMed Scopus (99) Google Scholar). Insertion of these precursors into the inner membrane is strictly dependent on Δψ but does not require ATP in the matrix.Proteins destined for the inner membrane require the help of small, structurally related Tim proteins in the intermembrane space. In the yeast Saccharomyces cerevisiae, five members of this protein family are expressed (19Bauer M.F. Rothbauer U. Muhlenbein N. Smith R.J. Gerbitz K. Neupert W. Brunner M. Hofmann S. FEBS Lett. 1999; 464: 41-47Crossref PubMed Scopus (65) Google Scholar). Of these, Tim9, Tim10, and Tim12 specifically support the import of mitochondrial carrier preproteins (11Koehler C.M. Jarosch E. Tokatlidis K. Schmid K. Schweyen R.J. Schatz G. Science. 1998; 279: 369-373Crossref PubMed Scopus (248) Google Scholar, 12Sirrenberg C. Bauer M.F. Guiard B. Neupert W. Brunner M. Nature. 1996; 384: 582-585Crossref PubMed Scopus (252) Google Scholar, 13Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 14Koehler C.M. Merchant S. Oppliger W. Schmid K. Jarosch E. Dolfini L. Junne T. Schatz G. Tokatlidis K. EMBO J. 1998; 17: 6477-6486Crossref PubMed Scopus (161) Google Scholar, 16Leuenberger D. Bally N.A. Schatz G. Koehler C.M. EMBO J. 1999; 18: 4816-4822Crossref PubMed Scopus (105) Google Scholar, 20Adam A. Endres M. Sirrenberg C. Lottspeich F. Neupert W. Brunner M. EMBO J. 1999; 18: 313-319Crossref PubMed Scopus (122) Google Scholar). They form two distinct hetero-oligomeric complexes of 70 kDa, which interact with the hydrophobic precursors thereby keeping them in an import competent conformation. The TIM9–10 complex interacts with the carrier proteins early in the import pathway when they are partially translocated across the TOM complex. The carrier proteins are then handed over to the TIM9–10-12 complex, which is tightly associated with the TIM22 complex and mediates their insertion into the inner membrane.Tim8 and Tim13 assist the import of a distinct subgroup of inner membrane proteins such as Tim23, the major component of the TIM23 translocase (16Leuenberger D. Bally N.A. Schatz G. Koehler C.M. EMBO J. 1999; 18: 4816-4822Crossref PubMed Scopus (105) Google Scholar, 21Paschen S.A. Rothbauer U. Kaldi K. Bauer M.F. Neupert W. Brunner M. EMBO J. 2000; 19: 6392-6400Crossref PubMed Scopus (116) Google Scholar, 22Davis A.J. Sepuri N.B. Holder J. Johnson A.E. Jensen R.E. J. Cell Biol. 2000; 150: 1271-1282Crossref PubMed Scopus (68) Google Scholar, 23Koehler C.M. Leuenberger D. Merchant S. Renold A. Junne T. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2141-2146Crossref PubMed Scopus (268) Google Scholar). Like the other members of the family, they exist as hetero-oligomeric 70-kDa complexes in the intermembrane space but, in contrast to Tim9, Tim10, and Tim12, are not essential for the cell viability in yeast. The TIM8–13 complex binds to the incoming Tim23 precursor still associated with the TOM complex. In yeast, it is only required when the membrane potential is low and the membrane insertion of Tim23 is inefficient (21Paschen S.A. Rothbauer U. Kaldi K. Bauer M.F. Neupert W. Brunner M. EMBO J. 2000; 19: 6392-6400Crossref PubMed Scopus (116) Google Scholar). Under these conditions the TIM8–13 complex is necessary to accumulate the Tim23 precursor and present it to the TIM22 complex thereby facilitating its insertion into the inner membrane.The human homologue of Tim8 is encoded by the DDP1 (deafness dystonia peptide 1) gene. Mutations in the DDP1/TIMM8A gene cause the Mohr-Tranebjaerg syndrome, a progressive, neurodegenerative disorder characterized by sensorineural hearing loss, dystonia, mental retardation, and blindness (24Jin H. May M. Tranebjaerg L. Kendall E. Fontan G. Jackson J. Subramony S.H. Arena F. Lubs H. Smith S. Stevenson R. Schwartz C. Vetrie D. Nat. Genet. 1996; 14: 177-180Crossref PubMed Scopus (212) Google Scholar, 25Tranebjaerg L. Schwartz C. Eriksen H. Andreasson S. Ponjavic V. Dahl A. Stevenson R.E. May M. Arena F. Barker D. J. Med. Genet. 1995; 32: 257-263Crossref PubMed Scopus (162) Google Scholar). Most of the patients harbor loss-of-function mutations leading to a complete absence of the DDP1 protein. In human mitochondria, DDP1 forms a hetero-oligomeric complex of 70 kDa together with hTim13 (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The DDP1·hTim13 complex specifically assists the import of the human Tim23 precursors into the inner membrane (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The human complex is able to complement the function of the TIM8–13 complex in yeast. Although import of yeast Tim23 does not require the TIM8–13 complex under normal conditions, import of human Tim23 appears to be dependent on the assistance of the DDP1·hTim13 complex under all conditions studied (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). It was therefore suggested that the pathomechanism underlying Mohr-Tranebjaerg syndrome might involve an impaired biogenesis of the human TIM23 complex.The small Tim proteins belong to an evolutionary conserved protein family characterized by a common Cys4 metal binding motif. Binding of Zn2+ ions was proposed to be required for the formation of typical zinc finger structures (13Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 19Bauer M.F. Rothbauer U. Muhlenbein N. Smith R.J. Gerbitz K. Neupert W. Brunner M. Hofmann S. FEBS Lett. 1999; 464: 41-47Crossref PubMed Scopus (65) Google Scholar, 26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). These zinc fingers may be crucial for the recognition and binding of translocation intermediates during their transfer through the aqueous environment of the intermembrane space.In this report, we analyzed the structural and functional consequences of a mutation (C66W), directly affecting the Cys4 metal binding motif. This cysteine to tryptophan exchange at amino acid position 66 is currently the only missense mutation known to cause Mohr-Tranebjaerg syndrome (27Tranebjaerg L. Hamel B.C. Gabreels F.J. Renier W.O. Van Ghelue M. Eur. J. Hum. Genet. 2000; 8: 464-467Crossref PubMed Scopus (65) Google Scholar). We show that the DDP1C66Wis efficiently imported into mitochondria and correctly sorted into the intermembrane space. However, the mutant protein is no more able to complement the function of Tim8 in yeast mitochondria lacking the endogenous TIM8–13 complex; in particular, the mutant DDP1 cannot restore import of Tim23. Analysis of purified recombinant DDP1 revealed that the C66W exchange impairs the ability of DDP1 to bind zinc. As a consequence, DDP1C66W does not fold properly and is no longer able to interact with its partner protein hTim13. Thus, the C66W substitution leads to a defect in the formation of a functional DDP1·hTim13 complex. This may interfere with the biogenesis of the TIM23 complex and an impaired biogenesis of the mitochondria might by the pathomechanistic basis of the Mohr-Tranebjaerg syndrome.DISCUSSIONDeletion of the DDP1/TIMM8A gene or loss-of-function mutations within the gene are associated with a severe disorder in humans, the Mohr-Tranebjaerg syndrome (Online Mendelian Inheritance in Man #304700). Despite some variability in its phenotypic expression, Mohr-Tranebjaerg syndrome is characterized by sensorineural hearing loss, dystonia, mental deterioration, and optic atrophy, indicating progressive neurodegeneration. Recently, a first case of Mohr-Tranebjaerg syndrome with a missense mutation inDDP1/TIMM8A was reported (27Tranebjaerg L. Hamel B.C. Gabreels F.J. Renier W.O. Van Ghelue M. Eur. J. Hum. Genet. 2000; 8: 464-467Crossref PubMed Scopus (65) Google Scholar). The mutated gene encodes a full-length protein; however, the clinical outcome of this patient is indistinguishable from published clinical courses of patients with loss-of-function mutations leading to absent or truncated DDP1 proteins. The missense mutation is an amino acid exchange from cysteine to tryptophan (C66W) affecting one of the four highly conserved cysteine residues that constitute a Cys4 zinc finger motif (13Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The precise functional role of the putative zinc fingers is currently unknown. They may be involved in folding or stability of the protein, in complex assembly, or in substrate recognition.In the present study, we generated DDP1 constructs carrying the C66W mutation to analyze which function of DDP1 is affected. We combinedin vitro analysis of purified recombinant protein within vivo functional analysis of the mutant DDP1 in the yeast system. The Δ8/Δ13 yeast strain, lacking both yTim8 and yTim13, enabled us to test the function of the mutated DDP1. As demonstrated previously, expression of wild-type DDP1 together with human Tim13 in Δ8/Δ13 cells rescues the cold-sensitive phenotype of the Δ8/Δ13 yeast strain (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In contrast, when DDP1C66W instead of wild-type DDP1 was introduced into Δ8/Δ13 cells, transformants failed to grow on glucose at 15 °C, indicating that the mutant DDP1 is non-functional in yeast. Furthermore, mutant DDP1 is apparently no more able to take over the function of its yeast homologue Tim8 during import of Tim23. We showed that the DDP1C66W precursor is efficiently imported into mitochondria and correctly sorted into the intermembrane space. Although the mutant DDP1 appears to be about 2-fold more susceptible to degradation by endogenous proteases, it accumulates in mitochondria to a level near that of wild-type DDP1. Thus, the inability of the mutant DDP1 to complement strain Δ8/Δ13 does not result from a reduced protein level in mitochondria.The C66W mutation affects a highly conserved element within DDP1 consisting of four cysteines, which likely form a metal binding site. We recently demonstrated that DDP1 and its partner protein hTim13 indeed bind zinc in a 1:1 stoichiometry and thus appear to form zinc fingers (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). We have shown that the C66W mutation impairs the ability of DDP1 to bind zinc ions. As a consequence, the C66W mutation leads to an incorrectly folded protein, as indicated by its increased susceptibility to externally added protease. Likewise, pretreatment of wild-type DDP1 with the metal-chelating agent EDTA/o-phe results in an increased sensitivity of the protein to trypsin. It is likely, therefore, that the C66W mutation alters the three-dimensional structure of the zinc binding domain thereby influencing the folding properties of DDP1.What are the functional consequences of an altered zinc finger structure? Zinc fingers are ubiquitous structural elements that are associated with protein·nucleic acid recognition or protein·protein interactions (31Leon O. Roth M. Biol. Res. 2000; 33: 21-30Crossref PubMed Scopus (78) Google Scholar). Our data provide evidence that a functional zinc finger is required for assembly of DDP1 into the 70-kDa complexes. Experiments in vitro using recombinant DDP1 and hTim13 showed that the interaction between these cognate partner proteins is completely abolished by the C66W mutation. Moreover, recombinant DDP1C66W had lost the ability to form higher molecular weight complexes. In contrast, purified wild-type DDP1 or hTim13 spontaneously assembled into hexameric complexes even in the absence of the cognate partner protein. Thus, the structural features of the zinc fingers are conserved between DDP1 and hTim13 allowing association into homo- or hetero-oligomeric complexes. In vivo, both partner proteins are present in comparable concentrations and assemble into hetero-oligomers (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar).Currently, it is unclear whether the zinc finger in DDP1 is also involved in substrate recognition and binding during import of human Tim23. The DDP1C66W construct, however, is not suitable to answer this question, because it cannot be excluded that a loss of substrate binding is secondary to its inability to form a complex with hTim13. Thus, to identify the substrate binding sites of DDP1, the generation of further mutant DDP1 constructs that harbor an intact Cys4 metal motif and are able to interact with hTim13 is needed.In summary, we showed that exchange of one of the conserved cysteine residues leads to impaired zinc binding and subsequent incorrect folding of the zinc finger domain. The functional consequence of the C66W mutation is primarily a deficiency of DDP1 to form a 70-kDa complex together with hTim13 in the mitochondrial intermembrane space. Thus, although the mutated DDP1 protein is imported into mitochondria and correctly sorted, it is non-functional and leads to a full-blown clinical phenotype in Mohr-Tranebjaerg patients. With the exception of a few components of the oxidative phosphorylation machinery, all mitochondrial proteins are encoded by nuclear genes and synthesized on cytosolic ribosomes. The import of such preproteins into mitochondria and the correct sorting into mitochondrial subcompartments is mediated by a set of import systems in the outer and inner mitochondrial membrane. Three distinct preprotein import systems have been described (1Herrmann J.M. Neupert W. Curr. Opin. Microbiol. 2000; 3: 210-214Crossref PubMed Scopus (120) Google Scholar, 2Bauer M.F. Hofmann I. Neupert I. Brunner I. Trends Cell Biol. 2000; 10: 25-31Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 3Pfanner N. Geissler A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 339-349Crossref PubMed Scopus (416) Google Scholar, 4Koehler C.M. FEBS Lett. 2000; 476: 27-31Crossref PubMed Scopus (91) Google Scholar, 5Jensen R.E. Johnson A.E. Nat. Struct. Biol. 2001; 8: 1008-1010Crossref PubMed Scopus (65) Google Scholar). All preproteins most likely use the general translocase in the outer membrane, the TOM1 complex. It mediates the recognition and binding of preproteins as well as their transfer across the outer membrane. Further movement of the translocation intermediates into and across the inner membrane is mediated by two distinct translocases in the inner membrane, the TIM23 and the TIM22 complexes. The TIM23 complex mediates import of preproteins with positively charged targeting signals at their N termini into the mitochondrial matrix space and into the inner membrane (6Berthold J. Bauer M.F. Schneider H.C. Klaus C. Dietmeier K. Neupert W. Brunner M. Cell. 1995; 81: 1085-1093Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 7Bauer M.F. Sirrenberg C. Neupert W. Brunner M. Cell. 1996; 87: 33-41Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 8Donzeau M. Kaldi K. Adam A. Paschen S. Wanner G. Guiard B. Bauer M.F. Neupert W. Brunner M. Cell. 2000; 101: 401-412Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The transfer of preproteins across the inner membrane strictly requires both an electrochemical potential (Δψ) across the inner membrane and the presence of ATP in the matrix as energy sources (9Moro F. Sirrenberg C. Schneider H.-C. Neupert W. Brunner M. EMBO J. 1999; 18: 3667-36745Crossref PubMed Scopus (83) Google Scholar). The TIM22 complex is used by a class of hydrophobic inner membrane proteins with internal and so far less characterized targeting signals (10Kerscher O. Holder J. Srinivasan M. Leung R.S. Jensen R.E. J. Cell Biol. 1997; 139: 1663-1675Crossref PubMed Scopus (171) Google Scholar, 11Koehler C.M. Jarosch E. Tokatlidis K. Schmid K. Schweyen R.J. Schatz G. Science. 1998; 279: 369-373Crossref PubMed Scopus (248) Google Scholar, 12Sirrenberg C. Bauer M.F. Guiard B. Neupert W. Brunner M. Nature. 1996; 384: 582-585Crossref PubMed Scopus (252) Google Scholar, 13Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 14Koehler C.M. Merchant S. Oppliger W. Schmid K. Jarosch E. Dolfini L. Junne T. Schatz G. Tokatlidis K. EMBO J. 1998; 17: 6477-6486Crossref PubMed Scopus (161) Google Scholar, 15Endres M. Neupert W. Brunner M. EMBO J. 1999; 18: 3214-3221Crossref PubMed Scopus (149) Google Scholar). Typical substrates are the members of the mitochondrial carrier family that are synthesized without a matrix-targeting signal. In addition, the TIM22 complex appears to mediate the import of precursors of other hydrophobic membrane proteins such as Tim23 and Tim22, which do not belong to the class of mitochondrial carriers (16Leuenberger D. Bally N.A. Schatz G. Koehler C.M. EMBO J. 1999; 18: 4816-4822Crossref PubMed Scopus (105) Google Scholar, 17Kaldi K. Bauer M.F. Sirrenberg C. Neupert W. Brunner M. EMBO J. 1998; 17: 1569-1576Crossref PubMed Scopus (59) Google Scholar, 18Kurz M. Martin H. Rassow J. Pfanner N. Ryan M.T. Mol. Biol. Cell. 1999; 10: 2461-2474Crossref PubMed Scopus (99) Google Scholar). Insertion of these precursors into the inner membrane is strictly dependent on Δψ but does not require ATP in the matrix. Proteins destined for the inner membrane require the help of small, structurally related Tim proteins in the intermembrane space. In the yeast Saccharomyces cerevisiae, five members of this protein family are expressed (19Bauer M.F. Rothbauer U. Muhlenbein N. Smith R.J. Gerbitz K. Neupert W. Brunner M. Hofmann S. FEBS Lett. 1999; 464: 41-47Crossref PubMed Scopus (65) Google Scholar). Of these, Tim9, Tim10, and Tim12 specifically support the import of mitochondrial carrier preproteins (11Koehler C.M. Jarosch E. Tokatlidis K. Schmid K. Schweyen R.J. Schatz G. Science. 1998; 279: 369-373Crossref PubMed Scopus (248) Google Scholar, 12Sirrenberg C. Bauer M.F. Guiard B. Neupert W. Brunner M. Nature. 1996; 384: 582-585Crossref PubMed Scopus (252) Google Scholar, 13Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 14Koehler C.M. Merchant S. Oppliger W. Schmid K. Jarosch E. Dolfini L. Junne T. Schatz G. Tokatlidis K. EMBO J. 1998; 17: 6477-6486Crossref PubMed Scopus (161) Google Scholar, 16Leuenberger D. Bally N.A. Schatz G. Koehler C.M. EMBO J. 1999; 18: 4816-4822Crossref PubMed Scopus (105) Google Scholar, 20Adam A. Endres M. Sirrenberg C. Lottspeich F. Neupert W. Brunner M. EMBO J. 1999; 18: 313-319Crossref PubMed Scopus (122) Google Scholar). They form two distinct hetero-oligomeric complexes of 70 kDa, which interact with the hydrophobic precursors thereby keeping them in an import competent conformation. The TIM9–10 complex interacts with the carrier proteins early in the import pathway when they are partially translocated across the TOM complex. The carrier proteins are then handed over to the TIM9–10-12 complex, which is tightly associated with the TIM22 complex and mediates their insertion into the inner membrane. Tim8 and Tim13 assist the import of a distinct subgroup of inner membrane proteins such as Tim23, the major component of the TIM23 translocase (16Leuenberger D. Bally N.A. Schatz G. Koehler C.M. EMBO J. 1999; 18: 4816-4822Crossref PubMed Scopus (105) Google Scholar, 21Paschen S.A. Rothbauer U. Kaldi K. Bauer M.F. Neupert W. Brunner M. EMBO J. 2000; 19: 6392-6400Crossref PubMed Scopus (116) Google Scholar, 22Davis A.J. Sepuri N.B. Holder J. Johnson A.E. Jensen R.E. J. Cell Biol. 2000; 150: 1271-1282Crossref PubMed Scopus (68) Google Scholar, 23Koehler C.M. Leuenberger D. Merchant S. Renold A. Junne T. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2141-2146Crossref PubMed Scopus (268) Google Scholar). Like the other members of the family, they exist as hetero-oligomeric 70-kDa complexes in the intermembrane space but, in contrast to Tim9, Tim10, and Tim12, are not essential for the cell viability in yeast. The TIM8–13 complex binds to the incoming Tim23 precursor still associated with the TOM complex. In yeast, it is only required when the membrane potential is low and the membrane insertion of Tim23 is inefficient (21Paschen S.A. Rothbauer U. Kaldi K. Bauer M.F. Neupert W. Brunner M. EMBO J. 2000; 19: 6392-6400Crossref PubMed Scopus (116) Google Scholar). Under these conditions the TIM8–13 complex is necessary to accumulate the Tim23 precursor and present it to the TIM22 complex thereby facilitating its insertion into the inner membrane. The human homologue of Tim8 is encoded by the DDP1 (deafness dystonia peptide 1) gene. Mutations in the DDP1/TIMM8A gene cause the Mohr-Tranebjaerg syndrome, a progressive, neurodegenerative disorder characterized by sensorineural hearing loss, dystonia, mental retardation, and blindness (24Jin H. May M. Tranebjaerg L. Kendall E. Fontan G. Jackson J. Subramony S.H. Arena F. Lubs H. Smith S. Stevenson R. Schwartz C. Vetrie D. Nat. Genet. 1996; 14: 177-180Crossref PubMed Scopus (212) Google Scholar, 25Tranebjaerg L. Schwartz C. Eriksen H. Andreasson S. Ponjavic V. Dahl A. Stevenson R.E. May M. Arena F. Barker D. J. Med. Genet. 1995; 32: 257-263Crossref PubMed Scopus (162) Google Scholar). Most of the patients harbor loss-of-function mutations leading to a complete absence of the DDP1 protein. In human mitochondria, DDP1 forms a hetero-oligomeric complex of 70 kDa together with hTim13 (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The DDP1·hTim13 complex specifically assists the import of the human Tim23 precursors into the inner membrane (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The human complex is able to complement the function of the TIM8–13 complex in yeast. Although import of yeast Tim23 does not require the TIM8–13 complex under normal conditions, import of human Tim23 appears to be dependent on the assistance of the DDP1·hTim13 complex under all conditions studied (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). It was therefore suggested that the pathomechanism underlying Mohr-Tranebjaerg syndrome might involve an impaired biogenesis of the human TIM23 complex. The small Tim proteins belong to an evolutionary conserved protein family characterized by a common Cys4 metal binding motif. Binding of Zn2+ ions was proposed to be required for the formation of typical zinc finger structures (13Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 19Bauer M.F. Rothbauer U. Muhlenbein N. Smith R.J. Gerbitz K. Neupert W. Brunner M. Hofmann S. FEBS Lett. 1999; 464: 41-47Crossref PubMed Scopus (65) Google Scholar, 26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). These zinc fingers may be crucial for the recognition and binding of translocation intermediates during their transfer through the aqueous environment of the intermembrane space. In this report, we analyzed the structural and functional consequences of a mutation (C66W), directly affecting the Cys4 metal binding motif. This cysteine to tryptophan exchange at amino acid position 66 is currently the only missense mutation known to cause Mohr-Tranebjaerg syndrome (27Tranebjaerg L. Hamel B.C. Gabreels F.J. Renier W.O. Van Ghelue M. Eur. J. Hum. Genet. 2000; 8: 464-467Crossref PubMed Scopus (65) Google Scholar). We show that the DDP1C66Wis efficiently imported into mitochondria and correctly sorted into the intermembrane space. However, the mutant protein is no more able to complement the function of Tim8 in yeast mitochondria lacking the endogenous TIM8–13 complex; in particular, the mutant DDP1 cannot restore import of Tim23. Analysis of purified recombinant DDP1 revealed that the C66W exchange impairs the ability of DDP1 to bind zinc. As a consequence, DDP1C66W does not fold properly and is no longer able to interact with its partner protein hTim13. Thus, the C66W substitution leads to a defect in the formation of a functional DDP1·hTim13 complex. This may interfere with the biogenesis of the TIM23 complex and an impaired biogenesis of the mitochondria might by the pathomechanistic basis of the Mohr-Tranebjaerg syndrome. DISCUSSIONDeletion of the DDP1/TIMM8A gene or loss-of-function mutations within the gene are associated with a severe disorder in humans, the Mohr-Tranebjaerg syndrome (Online Mendelian Inheritance in Man #304700). Despite some variability in its phenotypic expression, Mohr-Tranebjaerg syndrome is characterized by sensorineural hearing loss, dystonia, mental deterioration, and optic atrophy, indicating progressive neurodegeneration. Recently, a first case of Mohr-Tranebjaerg syndrome with a missense mutation inDDP1/TIMM8A was reported (27Tranebjaerg L. Hamel B.C. Gabreels F.J. Renier W.O. Van Ghelue M. Eur. J. Hum. Genet. 2000; 8: 464-467Crossref PubMed Scopus (65) Google Scholar). The mutated gene encodes a full-length protein; however, the clinical outcome of this patient is indistinguishable from published clinical courses of patients with loss-of-function mutations leading to absent or truncated DDP1 proteins. The missense mutation is an amino acid exchange from cysteine to tryptophan (C66W) affecting one of the four highly conserved cysteine residues that constitute a Cys4 zinc finger motif (13Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The precise functional role of the putative zinc fingers is currently unknown. They may be involved in folding or stability of the protein, in complex assembly, or in substrate recognition.In the present study, we generated DDP1 constructs carrying the C66W mutation to analyze which function of DDP1 is affected. We combinedin vitro analysis of purified recombinant protein within vivo functional analysis of the mutant DDP1 in the yeast system. The Δ8/Δ13 yeast strain, lacking both yTim8 and yTim13, enabled us to test the function of the mutated DDP1. As demonstrated previously, expression of wild-type DDP1 together with human Tim13 in Δ8/Δ13 cells rescues the cold-sensitive phenotype of the Δ8/Δ13 yeast strain (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In contrast, when DDP1C66W instead of wild-type DDP1 was introduced into Δ8/Δ13 cells, transformants failed to grow on glucose at 15 °C, indicating that the mutant DDP1 is non-functional in yeast. Furthermore, mutant DDP1 is apparently no more able to take over the function of its yeast homologue Tim8 during import of Tim23. We showed that the DDP1C66W precursor is efficiently imported into mitochondria and correctly sorted into the intermembrane space. Although the mutant DDP1 appears to be about 2-fold more susceptible to degradation by endogenous proteases, it accumulates in mitochondria to a level near that of wild-type DDP1. Thus, the inability of the mutant DDP1 to complement strain Δ8/Δ13 does not result from a reduced protein level in mitochondria.The C66W mutation affects a highly conserved element within DDP1 consisting of four cysteines, which likely form a metal binding site. We recently demonstrated that DDP1 and its partner protein hTim13 indeed bind zinc in a 1:1 stoichiometry and thus appear to form zinc fingers (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). We have shown that the C66W mutation impairs the ability of DDP1 to bind zinc ions. As a consequence, the C66W mutation leads to an incorrectly folded protein, as indicated by its increased susceptibility to externally added protease. Likewise, pretreatment of wild-type DDP1 with the metal-chelating agent EDTA/o-phe results in an increased sensitivity of the protein to trypsin. It is likely, therefore, that the C66W mutation alters the three-dimensional structure of the zinc binding domain thereby influencing the folding properties of DDP1.What are the functional consequences of an altered zinc finger structure? Zinc fingers are ubiquitous structural elements that are associated with protein·nucleic acid recognition or protein·protein interactions (31Leon O. Roth M. Biol. Res. 2000; 33: 21-30Crossref PubMed Scopus (78) Google Scholar). Our data provide evidence that a functional zinc finger is required for assembly of DDP1 into the 70-kDa complexes. Experiments in vitro using recombinant DDP1 and hTim13 showed that the interaction between these cognate partner proteins is completely abolished by the C66W mutation. Moreover, recombinant DDP1C66W had lost the ability to form higher molecular weight complexes. In contrast, purified wild-type DDP1 or hTim13 spontaneously assembled into hexameric complexes even in the absence of the cognate partner protein. Thus, the structural features of the zinc fingers are conserved between DDP1 and hTim13 allowing association into homo- or hetero-oligomeric complexes. In vivo, both partner proteins are present in comparable concentrations and assemble into hetero-oligomers (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar).Currently, it is unclear whether the zinc finger in DDP1 is also involved in substrate recognition and binding during import of human Tim23. The DDP1C66W construct, however, is not suitable to answer this question, because it cannot be excluded that a loss of substrate binding is secondary to its inability to form a complex with hTim13. Thus, to identify the substrate binding sites of DDP1, the generation of further mutant DDP1 constructs that harbor an intact Cys4 metal motif and are able to interact with hTim13 is needed.In summary, we showed that exchange of one of the conserved cysteine residues leads to impaired zinc binding and subsequent incorrect folding of the zinc finger domain. The functional consequence of the C66W mutation is primarily a deficiency of DDP1 to form a 70-kDa complex together with hTim13 in the mitochondrial intermembrane space. Thus, although the mutated DDP1 protein is imported into mitochondria and correctly sorted, it is non-functional and leads to a full-blown clinical phenotype in Mohr-Tranebjaerg patients. Deletion of the DDP1/TIMM8A gene or loss-of-function mutations within the gene are associated with a severe disorder in humans, the Mohr-Tranebjaerg syndrome (Online Mendelian Inheritance in Man #304700). Despite some variability in its phenotypic expression, Mohr-Tranebjaerg syndrome is characterized by sensorineural hearing loss, dystonia, mental deterioration, and optic atrophy, indicating progressive neurodegeneration. Recently, a first case of Mohr-Tranebjaerg syndrome with a missense mutation inDDP1/TIMM8A was reported (27Tranebjaerg L. Hamel B.C. Gabreels F.J. Renier W.O. Van Ghelue M. Eur. J. Hum. Genet. 2000; 8: 464-467Crossref PubMed Scopus (65) Google Scholar). The mutated gene encodes a full-length protein; however, the clinical outcome of this patient is indistinguishable from published clinical courses of patients with loss-of-function mutations leading to absent or truncated DDP1 proteins. The missense mutation is an amino acid exchange from cysteine to tryptophan (C66W) affecting one of the four highly conserved cysteine residues that constitute a Cys4 zinc finger motif (13Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The precise functional role of the putative zinc fingers is currently unknown. They may be involved in folding or stability of the protein, in complex assembly, or in substrate recognition. In the present study, we generated DDP1 constructs carrying the C66W mutation to analyze which function of DDP1 is affected. We combinedin vitro analysis of purified recombinant protein within vivo functional analysis of the mutant DDP1 in the yeast system. The Δ8/Δ13 yeast strain, lacking both yTim8 and yTim13, enabled us to test the function of the mutated DDP1. As demonstrated previously, expression of wild-type DDP1 together with human Tim13 in Δ8/Δ13 cells rescues the cold-sensitive phenotype of the Δ8/Δ13 yeast strain (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In contrast, when DDP1C66W instead of wild-type DDP1 was introduced into Δ8/Δ13 cells, transformants failed to grow on glucose at 15 °C, indicating that the mutant DDP1 is non-functional in yeast. Furthermore, mutant DDP1 is apparently no more able to take over the function of its yeast homologue Tim8 during import of Tim23. We showed that the DDP1C66W precursor is efficiently imported into mitochondria and correctly sorted into the intermembrane space. Although the mutant DDP1 appears to be about 2-fold more susceptible to degradation by endogenous proteases, it accumulates in mitochondria to a level near that of wild-type DDP1. Thus, the inability of the mutant DDP1 to complement strain Δ8/Δ13 does not result from a reduced protein level in mitochondria. The C66W mutation affects a highly conserved element within DDP1 consisting of four cysteines, which likely form a metal binding site. We recently demonstrated that DDP1 and its partner protein hTim13 indeed bind zinc in a 1:1 stoichiometry and thus appear to form zinc fingers (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). We have shown that the C66W mutation impairs the ability of DDP1 to bind zinc ions. As a consequence, the C66W mutation leads to an incorrectly folded protein, as indicated by its increased susceptibility to externally added protease. Likewise, pretreatment of wild-type DDP1 with the metal-chelating agent EDTA/o-phe results in an increased sensitivity of the protein to trypsin. It is likely, therefore, that the C66W mutation alters the three-dimensional structure of the zinc binding domain thereby influencing the folding properties of DDP1. What are the functional consequences of an altered zinc finger structure? Zinc fingers are ubiquitous structural elements that are associated with protein·nucleic acid recognition or protein·protein interactions (31Leon O. Roth M. Biol. Res. 2000; 33: 21-30Crossref PubMed Scopus (78) Google Scholar). Our data provide evidence that a functional zinc finger is required for assembly of DDP1 into the 70-kDa complexes. Experiments in vitro using recombinant DDP1 and hTim13 showed that the interaction between these cognate partner proteins is completely abolished by the C66W mutation. Moreover, recombinant DDP1C66W had lost the ability to form higher molecular weight complexes. In contrast, purified wild-type DDP1 or hTim13 spontaneously assembled into hexameric complexes even in the absence of the cognate partner protein. Thus, the structural features of the zinc fingers are conserved between DDP1 and hTim13 allowing association into homo- or hetero-oligomeric complexes. In vivo, both partner proteins are present in comparable concentrations and assemble into hetero-oligomers (26Rothbauer U. Hofmann S. Muhlenbein N. Paschen S.A. Gerbitz K.D. Neupert W. Brunner M. Bauer M.F. J. Biol. Chem. 2001; 276: 37327-37334Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Currently, it is unclear whether the zinc finger in DDP1 is also involved in substrate recognition and binding during import of human Tim23. The DDP1C66W construct, however, is not suitable to answer this question, because it cannot be excluded that a loss of substrate binding is secondary to its inability to form a complex with hTim13. Thus, to identify the substrate binding sites of DDP1, the generation of further mutant DDP1 constructs that harbor an intact Cys4 metal motif and are able to interact with hTim13 is needed. In summary, we showed that exchange of one of the conserved cysteine residues leads to impaired zinc binding and subsequent incorrect folding of the zinc finger domain. The functional consequence of the C66W mutation is primarily a deficiency of DDP1 to form a 70-kDa complex together with hTim13 in the mitochondrial intermembrane space. Thus, although the mutated DDP1 protein is imported into mitochondria and correctly sorted, it is non-functional and leads to a full-blown clinical phenotype in Mohr-Tranebjaerg patients. We thank Bettina Treske for excellent technical assistance. We are grateful to Dr. P. Klüfers and Helmuth Hartl for use of the ICP equipment.
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