The human deafness dystonia syndrome results from the mutation of a protein (DDP) of unknown function. We show now that DDP is a mitochondrial protein and similar to five small proteins (Tim8p, Tim9p, Tim10p, Tim12p, and Tim13p) of the yeast mitochondrial intermembrane space. Tim9p, Tim10p, and Tim12p mediate the import of metabolite transporters from the cytoplasm into the mitochondrial inner membrane and interact structurally and functionally with Tim8p and Tim13p. DDP is most similar to Tim8p. Tim8p exists as a soluble 70-kDa complex with Tim13p and Tim9p, and deletion of Tim8p is synthetically lethal with a conditional mutation in Tim10p. The deafness dystonia syndrome thus is a novel type of mitochondrial disease that probably is caused by a defective mitochondrial proteinimport system.Most mitochondrial proteins are synthesized with a cleavable NH 2 -terminal targeting sequence and imported into mitochondria by a general import pathway composed of cytosolic chaperones and two heterooligomeric membrane complexes: a TOM (translocase of the outer membrane) complex in the outer membrane and a TIM complex in the inner membrane (1-4). Recently, another translocase of the mitochondrial inner membrane specific for the import and insertion of multispanning carrier proteins has been identified. This new TIM machinery includes soluble components, Tim9p and Tim10p, in the mitochondrial intermembrane space, and Tim12p, Tim22p, and Tim54p of the mitochondrial inner membrane (5-9). Tim9p and Tim10p are partner proteins in a 70-kDa complex that transfers the carriers from the outer membrane to the inner membrane. A 300-kDa complex consisting of Tim12p, Tim22p, Tim54p, and a fraction of the Tim9p and Tim10p mediates insertion of the carrier into the inner membrane.Because the mitochondrion is essential for energy production in higher eukaryotes and many diseases already have been linked to errors in mitochondrial metabolism (10, 11), we reasoned that mitochondrial protein import defects potentially could lead to disease. The human deafness dystonia syndrome (MTS͞DFN-1) is a recessive, X-linked neurodegenerative disorder characterized by progressive sensorineural deafness, cortical blindness, dystonia, dysphagia, and paranoia (12). It is caused by truncation or deletion of an 11-kDa protein (referred to as DDP1) whose intracellular location and function are unknown (13). DDP1 is closely related to the ORF YJR135w-a in Saccharomyces cerevisiae (14).By searching various sequence databases, we now report that DDP and YGR135w-a (referred to as Tim8p) are similar to Tim9p, Tim10p, and Tim12p, which mediate the import of mitochondrial carrier proteins. In addition, we have identified another similar protein in S. cerevisiae (YGR181w, referred to as Tim13p) and another in humans termed DDP2. DDP1, Tim8p, and Tim13p are localized to the mitochondrial intermembrane space, and the yeast homolog of DDP1, Tim8p, interacts with the import system for multispanning inner membrane proteins. The deafness dystonia syndrome thus is a ...

Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane. We now identify Tim9p, another essential component of this import pathway. Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and Tim10p copurify in successive chromatographic fractionations and co-immunoprecipitated with each other. Tim9p can be cross-linked to a partly translocated carrier protein. A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p. The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p. A Ser 67 →Cys 67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants. Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.

Mitochondrial precursor proteins made in the cytosol bind to a hetero‐oligomeric protein import receptor on the mitochondrial surface and then pass through the translocation channel across the outer membrane. This translocation step is accelerated by an acidic domain of the receptor subunit Mas22p, which protrudes into the intermembrane space. This ‘trans’ domain of Mas22p specifically binds functional mitochondrial targeting peptides with a Kd of < 1 microM and is required to anchor the N‐terminal targeting sequence of a translocation‐arrested precursor in the intermembrane space. If this Mas22p domain is deleted, respiration‐driven growth of the cells is compromised and import of different precursors into isolated mitochondria is inhibited 3‐ to 8‐fold. Binding of precursors to the mitochondrial surface appears to be mediated by cytosolically exposed acidic domains of the receptor subunits Mas20p and Mas22p. Translocation of a precursor across the outer membrane thus appears to involve sequential binding of the precursor's basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane.

We have identified a 20 kDa yeast mitochondrial outer membrane protein (termed MAS20) which appears to function as a protein import receptor. We cloned, sequenced and physically mapped the MAS20 gene and found that the protein is homologous to the MOM19 import receptor from Neurospora crassa. MAS20 and MOM19 contain the sequence motif F‐X‐K‐A‐L‐X‐V/L, which is repeated several times with minor variations in the MAS70/MOM72 receptors. To determine how MAS20 functions together with the previously identified yeast receptor MAS70, we constructed yeast mutants lacking either one or both of the receptors. Deletion of either receptor alone had little or no effect on fermentative growth and only partially inhibited mitochondrial protein import in vivo. Deletion of both receptors was lethal. Deleting only MAS70 did not affect respiration; deleting only MAS20 caused loss of respiration, but respiration could be restored by overexpressing MAS70. Import of the F1‐ATPase beta‐subunit into isolated mitochondria was only partly inhibited by IgGs against either MAS20 or MAS70, but both IgGs inhibited import completely. We conclude that the two receptors have overlapping specificities for mitochondrial precursor proteins and that neither receptor is by itself essential.

We have cloned the gene encoding the protein Mas22p, which spans the outer membrane of yeast mitochondria. Cells that completely lack Mas22p are inviable. The plasmid-borne MAS22 gene suppresses several defects resulting from the deletion of one or more of the mitochondrial protein import receptors. Defects of Mas2Op-deficient cells are explained by the reduced level of Mas22p in these mutants. Mas22p has one acidic domain in the cytosol and a second acidic domain in the mitochondrial intermembrane space. We suggest that these domains of Mas22p on either side of the outer membrane function as a relay system for transferring the basic targeting sequences of precursor proteins into the mitochondria.