Identification of Tim40 That Mediates Protein Sorting to the Mitochondrial Intermembrane Space

Abstract

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...

Mia40 targets cysteines in a hydrophobic environment to direct oxidative protein folding in the mitochondria

“…To examine whether this modification influenced binding, we performed experiments in which the energy donor Trp was placed in Mia40 and the acceptor AEDANS in Cox17*. Four single-cysteine variants of Cox17* were produced and labelled with AEDANS (variants [8][9][10][11]. Their interaction with the wild-type form of Mia40 was then measured in equilibrium titrations, and its kinetics was followed in the stopped-flow instrument by the FRET between the single Trp (W294) of Mia40 and the AEDANS groups of the four Cox17* variants ( Supplementary Fig.…”

“…We therefore exchanged the entire sequence between Cys26 and Cys36 of the fist CX 9 C motif with the sequence between Cys47 and Cys57 of the second CX 9 C motif (that contains the MISS/ITS sequence). The MISS/ITS sequence ARTICLE was thus moved from the vicinity of Cys57 to the vicinity of Cys26 in the two variants that contain Cys26 and Cys57, respectively (variants 28-31).…”

“…Similar to Mia40 itself, these proteins are composed of two short antiparallel helices that are covalently linked by two disulphide bonds. This is shown for the CX 9 C protein Cox17 (ref. 17) in Fig.…”

“…T he intermembrane space (IMS) of the mitochondria was long believed to provide a reducing environment, similar to the cytosol, but recently proteins with disulphide bonds and subsequently a thiol oxidase (Mia40, mitochondrial import and assembly) were discovered in the IMS [1][2][3][4][5][6][7][8][9][10][11][12] . Unlike the other known thiol oxidases, Mia40 does not belong to the thioredoxin family, and its catalytic disulphide is arranged in a unique Cys-Pro-Cys motif.…”

“…Sulphur atoms of the structural disulphides are coloured yellow, cysteines are numbered according to the amino-acid sequence. The first CX 9 C motif is coloured purple (residues [26][27][28][29][30][31][32][33], the second CX 9 C motif (residues 47-57) is magenta and the hydrophobic residues 37-39 following Cys36 are blue. The residues of the MISS/ITS sequence (F50, I51, Y54) and I37, L38, F39 are shown in ball and sticks representation.…”