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The TIM10 chaperone facilitates the insertion of hydrophobic proteins at the mitochondrial inner membrane. Here we report the novel molecular mechanism of TIM10 assembly. This process crucially depends on oxidative folding in mitochondria and involves: (i) import of the subunits in a Cys-reduced and unfolded state; (ii) folding to an assembly-competent(More)
The TIM10 complex is localized in the mitochondrial intermembrane space and mediates insertion of hydrophobic proteins at the inner membrane. We have characterized TIM10 assembly and analyzed the structural properties of its subunits, Tim9 and Tim10. Both proteins are alpha-helical with a protease-resistant central domain, and each self-associates to form(More)
Erv1 (essential for respiration and viability 1) is an FAD-dependent thiol oxidase of the Erv/ALR (augmenter of liver regeneration) sub-family. It is an essential component of the mitochondrial import and assembly (MIA) pathway, playing an important role in the oxidative folding of the mitochondrial intermembrane space (IMS) proteins and linking the MIA(More)
Tim9 and Tim10 are essential components of the "small Tim" family of proteins that facilitate insertion of polytopic proteins at the inner mitochondrial membrane. The small Tims are themselves imported from the cytosol and are organized in specific translocation assemblies in the intermembrane space. Their conformational properties and how these influence(More)
Erv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX(16)C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected(More)
Tim10 and all the small Tim proteins of the mitochondrial intermembrane space contain a consensus twin CX3C Zn2+-finger motif. While disulphide bond formation between the Cys residues of this motif is essential for complex formation by the small Tim proteins, the specific role of Zn2+-binding during the import and assembly of these proteins is not clear. In(More)
Zinc is an essential cofactor required for the function of approximately 8% of the yeast and 10% of the human proteome. All of the "small Tim" proteins of the mitochondrial intermembrane space contain a strictly conserved "twin CX(3)C" zinc finger motif, which can bind zinc ions in the Cys-reduced form. We have shown previously that although disulfide bond(More)
The TIM10 complex, composed of the homologous proteins Tim10 and Tim9, chaperones hydrophobic proteins inserted at the mitochondrial inner membrane. A salient feature of the TIM10 complex subunits is their conserved "twin CX3C" motif. Systematic mutational analysis of all cysteines of Tim10 showed that their underlying molecular defect is impaired folding(More)
Mia40 is a highly conserved mitochondrial protein that plays an essential role in the import and oxidative folding of many proteins of the mitochondrial intermembrane space. Mia40 uses its redox active CPC motif to shuttle disulfides between its client proteins (newly imported proteins) and the thiol oxidase Erv1. As a thiol oxidoreductase, no cofactor was(More)
Thiol-disulphide redox regulation has a key role during the biogenesis of mitochondrial intermembrane space (IMS) proteins. Only the Cys-reduced form of precursor proteins can be imported into mitochondria, which is followed by disulphide bond formation in the mitochondrial IMS. In contrast to the wealth of knowledge on the oxidation process inside(More)