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Biogenesis of proteins of the mitochondrial intermembrane space. Identification and characterization of Mia40 in Saccharomyces cerevisiae
Biogenesis of proteins of the mitochondrial intermembrane space. Identification and characterization of Mia40 in Saccharomyces cerevisiae
All intermembrane space (IMS) proteins are synthesized in the cytosol and have to be imported into mitochondria. Many proteins of the IMS lack typical N-terminal targeting signals and are characterized by a small molecular mass and highly conserved cysteine residues present in characteristic patterns. These proteins cross the outer membrane of mitochondria via the TOM complex and need their cysteine residues for the efficient retention in the IMS. The aim of this study was to analyse whether specific factors are required for the import of these proteins into the mitochondrial IMS. The candidate protein, later termed Mia40 (mitochondrial intermembrane space import and assembly), was structurally and functionally characterized. The experiments presented here confirmed the mitochondrial location of Mia40 and determined its topology. Mia40 contains a classical N-terminal mitochondrial targeting signal followed by a hydrophobic segment. It is anchored in the inner membrane by a hydrophobic stretch and exposes a large C-terminal domain to the IMS. This domain harbours six highly conserved cysteine residues forming a CXC-CX9C-CX9C- motif (X represents non-cysteine amino acid residues). Since Mia40 is essential for viability of yeast, a strain harbouring the MIA40 gene under control of the glucose-repressible GAL10 promoter was used to study the function of Mia40 in mitochondria. Depletion of Mia40 resulted in strongly reduced levels of Tim13, Cox17 and of other IMS proteins with cysteine motifs, which was due to the impairment of their import into mitochondria. Mia40 is directly involved in the translocation of the small IMS proteins with conserved cysteine motifs: the newly imported IMS proteins form mixed disulfide intermediates with Mia40. In mitochondria, the majority of Mia40 is present in the oxidized state, thus allowing the formation of the mixed disulfide intermediates in an isomerization reaction. Subsequently, Mia40 transfers the disulfide bond from the mixed disulfide to the substrate proteins and thereby triggers the folding and the trapping of these proteins in the IMS. Mia40 is left in a partially reduced state and a reoxidation step is required for the next round of import. Erv1 is an essential FAD-containing sulfhydryl oxidase present in the IMS of fungi, plants and animals. The import of Tim13 was less efficient in mitochondria depleted of Erv1 and Mia40 interacted directly with Erv1 via disulfide bonds. In addition, the depletion of Erv1 affected the redox state of Mia40, which accumulated in a partially reduced state, suggesting that Erv1 is required for the recovery of the oxidized state of Mia40. Thus, Mia40 and Erv1 form a disulfide relay system mediating the import of small cysteine-rich proteins into the IMS. Erv1 passes its electrons further to cytochrome c, linking the import of small IMS proteins to the respiratory chain activity. Notably, Erv1 is not only a component but also a substrate of the disulfide relay system. It represents a novel type of substrate of the Mia40-mediated pathway. Thus, this pathway appears to be quite versatile and not limited to proteins with twin CX3C or twin CX9C motifs. The conserved cysteine residues in Mia40 are crucial for its function. Using single and double cysteine mutants of Mia40, it was possible to assign specific roles to each cysteine residue. In the oxidized state of Mia40 all cysteine residues form intramolecular disulfide bonds. The first two cysteine residues in the CPC motif compose a redox-sensitive disulfide bridge and breaking of this disulfide leads to Mia40 in the partially reduced state. The disulfide bond formed by the first two cysteine residues in Mia40 seems to be involved in the interaction with Erv1 and the substrate proteins, suggesting that it is essential for the catalysis of redox reactions of Mia40. The two other disulfide bonds connect the two CX9C fragments in Mia40 and most likely play a structural role. Taken together, the essential protein Mia40 is the central component of a novel translocation pathway. Mia40 together with Erv1 forms a disulfide relay system required for the import of small cysteine-rich proteins into the IMS of mitochondria.
mitochondria, intermembrane space, Mia40, disulfide bonds
Terziyska, Nadia
2008
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Terziyska, Nadia (2008): Biogenesis of proteins of the mitochondrial intermembrane space: Identification and characterization of Mia40 in Saccharomyces cerevisiae. Dissertation, LMU München: Faculty of Biology
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Abstract

All intermembrane space (IMS) proteins are synthesized in the cytosol and have to be imported into mitochondria. Many proteins of the IMS lack typical N-terminal targeting signals and are characterized by a small molecular mass and highly conserved cysteine residues present in characteristic patterns. These proteins cross the outer membrane of mitochondria via the TOM complex and need their cysteine residues for the efficient retention in the IMS. The aim of this study was to analyse whether specific factors are required for the import of these proteins into the mitochondrial IMS. The candidate protein, later termed Mia40 (mitochondrial intermembrane space import and assembly), was structurally and functionally characterized. The experiments presented here confirmed the mitochondrial location of Mia40 and determined its topology. Mia40 contains a classical N-terminal mitochondrial targeting signal followed by a hydrophobic segment. It is anchored in the inner membrane by a hydrophobic stretch and exposes a large C-terminal domain to the IMS. This domain harbours six highly conserved cysteine residues forming a CXC-CX9C-CX9C- motif (X represents non-cysteine amino acid residues). Since Mia40 is essential for viability of yeast, a strain harbouring the MIA40 gene under control of the glucose-repressible GAL10 promoter was used to study the function of Mia40 in mitochondria. Depletion of Mia40 resulted in strongly reduced levels of Tim13, Cox17 and of other IMS proteins with cysteine motifs, which was due to the impairment of their import into mitochondria. Mia40 is directly involved in the translocation of the small IMS proteins with conserved cysteine motifs: the newly imported IMS proteins form mixed disulfide intermediates with Mia40. In mitochondria, the majority of Mia40 is present in the oxidized state, thus allowing the formation of the mixed disulfide intermediates in an isomerization reaction. Subsequently, Mia40 transfers the disulfide bond from the mixed disulfide to the substrate proteins and thereby triggers the folding and the trapping of these proteins in the IMS. Mia40 is left in a partially reduced state and a reoxidation step is required for the next round of import. Erv1 is an essential FAD-containing sulfhydryl oxidase present in the IMS of fungi, plants and animals. The import of Tim13 was less efficient in mitochondria depleted of Erv1 and Mia40 interacted directly with Erv1 via disulfide bonds. In addition, the depletion of Erv1 affected the redox state of Mia40, which accumulated in a partially reduced state, suggesting that Erv1 is required for the recovery of the oxidized state of Mia40. Thus, Mia40 and Erv1 form a disulfide relay system mediating the import of small cysteine-rich proteins into the IMS. Erv1 passes its electrons further to cytochrome c, linking the import of small IMS proteins to the respiratory chain activity. Notably, Erv1 is not only a component but also a substrate of the disulfide relay system. It represents a novel type of substrate of the Mia40-mediated pathway. Thus, this pathway appears to be quite versatile and not limited to proteins with twin CX3C or twin CX9C motifs. The conserved cysteine residues in Mia40 are crucial for its function. Using single and double cysteine mutants of Mia40, it was possible to assign specific roles to each cysteine residue. In the oxidized state of Mia40 all cysteine residues form intramolecular disulfide bonds. The first two cysteine residues in the CPC motif compose a redox-sensitive disulfide bridge and breaking of this disulfide leads to Mia40 in the partially reduced state. The disulfide bond formed by the first two cysteine residues in Mia40 seems to be involved in the interaction with Erv1 and the substrate proteins, suggesting that it is essential for the catalysis of redox reactions of Mia40. The two other disulfide bonds connect the two CX9C fragments in Mia40 and most likely play a structural role. Taken together, the essential protein Mia40 is the central component of a novel translocation pathway. Mia40 together with Erv1 forms a disulfide relay system required for the import of small cysteine-rich proteins into the IMS of mitochondria.