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...

Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial β-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial β-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the β-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of β-barrel proteins in the outer membrane.

Newly synthesized mitochondrial proteins are imported into mitochondria with the aid of protein translocator complexes in the outer and inner mitochondrial membranes. We report the identification of yeast Tam41, a new member of mitochondrial protein translocator systems. Tam41 is a peripheral inner mitochondrial membrane protein facing the matrix. Disruption of the TAM41 gene led to temperature-sensitive growth of yeast cells and resulted in defects in protein import via the TIM23 translocator complex at elevated temperature both in vivo and in vitro. Although Tam41 is not a constituent of the TIM23 complex, depletion of Tam41 led to a decreased molecular size of the TIM23 complex and partial aggregation of Pam18 and -16. Import of Pam16 into mitochondria without Tam41 was retarded, and the imported Pam16 formed aggregates in vitro. These results suggest that Tam41 facilitates mitochondrial protein import by maintaining the functional integrity of the TIM23 protein translocator complex from the matrix side of the inner membrane.

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.

Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria via the TOM40 (translocase of the mitochondrial outer membrane 40) complex, and follow several distinct sorting pathways to reach their destination submitochondrial compartments. Phosphate carrier (PiC) is an inner membrane protein with 6 transmembrane segments (TM1-TM6) and requires, after translocation across the outer membrane, the Tim9-Tim10 complex and the TIM22 complex to be inserted into the inner membrane. Here we analyzed an in vitro import of fusion proteins between various PiC segments and mouse dihydrofolate reductase. The fusion protein without TM1 and TM2 was translocated across the outer membrane but was not inserted into the inner membrane. The fusion proteins without TM1-TM4 were not inserted into the inner membrane but instead translocated across the inner membrane. Functional defects of Tim50 of the TIM23 complex caused either by depletion of the protein or the addition of anti-Tim50 antibodies blocked translocation of the fusion proteins without TM1-TM4 across the inner membrane, suggesting that lack of TM1-TM4 led to switch of its sorting pathway from the TIM22 pathway to the TIM23 pathway. PiC thus appears to have a latent signal for sorting to the TIM23 pathway, which is exposed by reduced interactions with the Tim9-Tim10 complex and maintenance of the import competence.Newly synthesized organellar and secretory proteins are translocated across or inserted into biological membranes before they become functional. Among several different eukaryotic organelles, mitochondria are unique in being bounded by two biological membranes, the outer and inner mitochondrial membranes, rendering delivery of mitochondrial proteins to their destination suborganellar compartments highly complex. Mitochondria have translocators, membrane protein complexes that recognize targeting/sorting signals of mitochondrial proteins and facilitate their translocation across and/or insertion into the membranes. The outer membrane contains two TOM 1 (translocase of the mitochondrial outer membrane) complexes as translocators, the TOM40 complex and TOB/SAM complex, and the inner membrane contains two TIM (translocase of the mitochondrial inner membrane) complexes as translocators, the TIM23 complex and TIM22 complex (1-3).The TOM40 complex mediates translocation across or insertion into the outer membrane for nearly all mitochondrial proteins. Most matrix proteins and some inner membrane proteins are synthesized in the cytosol as precursor proteins with an N-terminal cleavable presequence. After translocation through the TOM40 channel in the outer membrane, the proteins are passed onto the TIM23 complex and move through the TIM23 channel with the aid of MMC (mitochondrial Hsp70-associated motor and chaperone) proteins, i.e. mitochondrial Hsp70 and its partner proteins (4, 5). Polytopic inner membrane proteins, including metabolic carrier proteins, are synthesized in the cytosol without a presequence. After crossing the outer membrane via...