No abstract

Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder. We show that DDP1 acts with human Tim13 in a complex in the intermembrane space. The DDP1⅐hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria. The human DDP1⅐hTim13 complex complements the function of the TIM8⅐13 complex in yeast and facilitates import of yeast and human Tim23. Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction.The vast majority of mitochondrial proteins are encoded as precursors in the nuclear genome. Mitochondrial biogenesis is, therefore, dependent on the import and sorting of the nuclear encoded precursor proteins into mitochondrial subcompartments. In eukaryotes three distinct preprotein import systems located in the mitochondrial outer and inner membrane have been described (1-5). The outer membrane contains a general preprotein translocase, the TOM 1 complex, which mediates the recognition and binding of preproteins and their transfer across the outer membrane. This complex is most likely used by all nuclear encoded precursors. Import into and across the inner membrane is mediated by two distinct inner membrane translocases, the TIM22 and the TIM23 complexes. Both TIM complexes cooperate with the TOM complex but differ in their substrate specificity (6 -11). The TIM23 complex mediates import of preproteins with a positively charged matrix targeting signal into the mitochondrial matrix space and into the inner membrane (6, 12, 13). The translocation of such precursors into the matrix requires the membrane potential ⌬ across the inner membrane and ATP in the matrix. The ⌬ drives the translocation of the presequences through the protein-conducting channel of the TIM23 complex which is formed by the membrane-integrated proteins Tim23 and Tim17 (6,12). A molecular motor that is attached to the inner side of this channel then promotes further translocation of the mature portion of the preproteins into the matrix. This motor consists of the peripheral membrane protein Tim44, the mitochondrial Hsp70, and the nucleotide exchange factor Mge1. Together, these components in repeated ATP-dependent reaction cycles facilitate the vectorial translocation into the matrix in a stepwise manner (14).The TIM22 complex mediates the insertion of a class of hydrophobic proteins with internal targeting signals into the inner membrane (7)(8)(9)(10)(11)(15)(16)(17). Typical substrates are members of the mitochondrial carrier family and other integral inner membrane proteins that are synthesized without a matrix-targeting signal. Insertion of these precursors into the inner membrane is s...

The Mohr^Tranebjaerg syndrome (MTS), a neurodegenerative syndrome characterized by progressive sensorineural hearing loss, dystonia, mental retardation and blindness, is a mitochondrial disease caused by mutations in the deafness/ dystonia peptide 1 (DDP1) gene. DDP1 shows similarity to the yeast proteins Tim9, Tim10 and Tim12, components of the mitochondrial import machinery for carrier proteins. Here, we show that DDP1 belongs to a large family of evolutionarily conserved proteins. We report the identification, chromosomal localization and expressional analysis of six human family members which represent further candidate genes for neurodegenerative diseases.z 1999 Federation of European Biochemical Societies.

Mohr-Tranebjaerg syndrome is a progressive, neurodegenerative disorder caused by loss-of-function mutations in the DDP1/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 Cys 4 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 Cys 4 motif. 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 Zn 2؉ ions via the Cys 4 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.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.

Tim9, Tim10a, and Tim10b are members of the family of small Tim proteins located in the intermembrane space of mammalian mitochondria. In yeast, members of this family act along the TIM22 import pathway during import of metabolite carriers and other integral inner membrane proteins. Here, we show that the human small proteins form two distinct hetero-oligomeric complexes. A 70-kDa complex that contains Tim9 and Tim10a and a Tim9-10a-10b that is part of a higher molecular weight assembly of 450 kDa. This distribution among two complexes suggests Tim10b to be the functional homologue of yeast Tim12. Both human complexes are tightly associated with the inner membrane and, compared with yeast, soluble 70-kDa complexes appear to be completely absent in the intermembrane space. Thus, the function of soluble 70-kDa complexes as trans-site receptors for incoming carrier proteins is not conserved from lower to higher eukaryotes. During import, the small Tim complexes directly interact with human adenine nucleotide translocator (ANT) in transit in a metal-dependent manner. For insertion of carrier preproteins into the inner membrane, the human small Tim proteins directly interact with human Tim22, the putative insertion pore of the TIM22 translocase. However, in contrast to yeast, only a small fraction of Tim9-Tim10a-Tim10b complex is in a stable association with Tim22. We conclude that different mechanisms and specific requirements for import and insertion of mammalian carrier preproteins have evolved in higher eukaryotes.Most mitochondrial proteins are encoded by nuclear genes, translated at cytosolic ribosomes, and imported to their final destination in the mitochondrion. Import of nuclear-encoded mitochondrial preproteins is a multistep process mediated by a common translocase in the outer membrane, the TOM 1 complex, and two distinct translocases in the inner membrane, named TIM23 and TIM22 complex (1-4). The TOM complex is used by all known nuclear-encoded precursors and translocates them into and across the outer membrane. The TIM23 and the TIM22 complex show different substrate specificities and energy requirements. Presequence-containing proteins are translocated across the inner membrane into the matrix via the TIM23 complex; whereas polytopic inner membrane proteins without presequences but with internal targeting signals use the TIM22 complex. The mitochondrial import pathways are evolutionary conserved and many components of the mitochondrial import machineries characterized in the fungal system have counterparts in mammalian mitochondria (5). The TIM23 complex consists of the integral membrane components Tim23, Tim17, and Tim50. Tim23 and Tim17 constitute the protein-conducting channel and act in cooperation to form a receptor for presequences in the intermembrane space (6 -8). Tim50 exposing its large C-terminal domain to the intermembrane space was proposed to facilitate the transfer of precursor proteins to the translocation channel (9 -11). Translocation across the inner membrane is initially driven by t...