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...

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.

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.

The splicing of nuclear encoded RNAs, including tRNAs, has been widely believed to occur in the nucleus. However, we recently found that one of the tRNA splicing enzymes, splicing endonuclease, is localized to the outer surface of mitochondria in Saccharomyces cerevisiae . These results suggested the unexpected possibility of tRNA splicing in the cytoplasm. To investigate this possibility, we examined whether cytoplasmic pre-tRNAs are bona fide intermediates for tRNA maturation in vivo . We isolated a new reversible allele of temperature-sensitive (ts) sen2 ( HA-sen2-42 ), which encodes a mutant form of one of the catalytic subunits of yeast splicing endonuclease. The HA-sen2-42 cells accumulated large amounts of pre-tRNAs in the cytoplasm at a restrictive temperature, but the pre-tRNAs were diminished when the cells were transferred to a permissive temperature. Using pulse-chase/hybrid-precipitation techniques, we showed that the pre-tRNAs were not degraded but rather converted into mature tRNAs during incubation at the permissive temperature. These and other results indicate that, in S. cerevisiae , pre-tRNAs in the cytoplasm are genuine substrates for splicing, and that the splicing is indeed carried out in the cytoplasm.

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.