Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria, and sorted to one of the four mitochondrial subcompartments. Here we identified a new inner membrane protein, Tim40, that mediates sorting of small Tim proteins to the intermembrane space. Tim40 is essential for yeast cell growth, and its function in vivo requires six conserved Cys residues but not anchoring of the protein to the inner membrane by its N-terminal hydrophobic segment. Depletion of Tim40 impairs the import of small Tim proteins into mitochondria both in vivo and in vitro. In wild-type mitochondria, Tim40 forms a translocation intermediate with small Tim proteins prior to their assembly in the intermembrane space in vitro. These results suggest the essential role of Tim40 in sorting/assembly of small Tim proteins.Mitochondria are essential organelles in eukaryotic cells that consist of four compartments, the outer membrane, intermembrane space (IMS), 1 inner membrane, and matrix. Since most mitochondrial proteins are encoded by the nuclear genome and synthesized in the cytosol, mitochondria contain an elaborate system to take up these proteins from the cytosol and to sort them to specific intramitochondrial compartments. Recently, evidence has accumulated that the import/sorting pathways for mitochondrial proteins are much more complex than previously envisaged and involve the TOM40 (TOM, the translocase of the mitochondrial outer membrane) and SAM (protein sorting and assembly machinery) complexes in the outer membrane, the TIM23 (TIM, the translocase of the mitochondrial inner membrane) and the TIM22 complexes in the inner membrane, small Tim proteins in the IMS, and the mitochondrial Hsp70 system in the matrix (1-3).The mitochondrial IMS contains many soluble, small size proteins including small Tim proteins and cytochrome c. They are synthesized without a cleavable presequence and enter the IMS with the aid of the TOM40 complex but independently of the TIM23 or TIM22 complex. Since there is no membrane potential across the outer membrane and the IMS lacks an ATP-dependent chaperone system, vectorial import of small IMS proteins should be driven by a unique mechanism (2, 4). One possible scenario to achieve this is the attachment of ligands such as heme (for cytochrome c) or zinc ion (for small Tim proteins) to the imported proteins in the IMS (5, 6). This will result in their folding/assembly preferentially in the IMS so that their translocation back to the cytosol will be prevented, leading to their accumulation in the IMS. However, it is still unclear whether proteinaceous factors in the IMS are further required for the small IMS protein biogenesis, which is discharged from the TOM40 complex, specific ligand binding, and assembly in the IMS etc.In the present study, we looked for a component, if any, that mediates protein sorting to the mitochondrial IMS in yeast. Our approach relied on the fact that many mitochondrial proteins mediating mitochondrial protein assembly/import are essential or important for y...

Here, we report the identification of yeast 15-kD Tim15/Zim17, a new member of mitochondrial Hsp70 (mtHsp70)-associated motor and chaperone (MMC) proteins. The 15-kD MMC protein is a peripheral inner membrane protein with a zinc-finger motif. Depletion of the 15-kD protein led to impaired import of presequence-containing proteins into the matrix in vivo and in vitro. Overexpression of the 15-kD protein rescued the functional defects of mtHsp70 in ssc1-3 cells, and a fusion protein containing the 15-kD protein physically interacts with purified mtHsp70. Tim15/Zim17 therefore cooperates with mtHsp70 to facilitate import of presequence-containing proteins into the matrix.