Functional Characterization of Mia40p, the Central Component of the Disulfide Relay System of the Mitochondrial Intermembrane Space

Grumbt, Barbara; Stroobant, Vincent; Terziyska, Nadia; Israel, Lars; Hell, Kai

doi:10.1074/jbc.m707439200

Cited by 84 publications

(135 citation statements)

References 52 publications

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“…However, the C3-C6 and C4-C5 disulfide bonds, which involve the twin CX 9 C motif, are sandwiched by the two helices, and the accessible surface areas of S ␥ atoms for Cys307, Cys340, Cys317, and Cys330 are 0, 11.60, 8.5, and 0 Å 2 , respectively. Those results are consistent with the previous report that the C1-C2 bond is more redox-sensitive than the two others (11,14). Because Mia40 with the C3S, C6S, or C3,6S mutation becomes sensitive to protease digestion, the C3-C6 disulfide bond was proposed to have a structural role in Mia40 (14).…”

Section: (5)supporting

confidence: 82%

“…Yeast Mia40 contains six conserved cysteine residues (C1-C6) that can form three disulfide pairs in its oxidized form: the C1-C2 disulfide pair in the CPC motif, and the C3-C6 and C4-C5 disulfide pairs (11). Replacement of one of the three cysteine pairs is lethal (4), suggesting the importance of the three cysteine pairs in Mia40 functions.…”

Section: Resultsmentioning

confidence: 99%

“…The IMS domain of Mia40 contains six conserved Cys residues (C1-C6) at positions 296, 298, 307, 317, 330, and 340, which form a CPC motif (C1 and C2) and twin CX 9 C motifs (C3-X 9 -C4 and C5-X 9 -C6). These invariant Cys residues are essential for the Mia40 function (4), and form three disulfide bonds (C1-C2, C3-C6, and C4-C5) in the oxidized state (11). After crossing the outer membrane via the TOM40 complex, substrate proteins for the Mia40 pathway bind to Mia40 transiently, suggesting the role of Mia40 in recognition and trapping of the substrates to prevent their retrotranslocation (4,5).…”

Section: Structural Basis Of Yeast Tim40/mia40 As An Oxidative Translmentioning

confidence: 99%

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Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space

Kawano

Yamano²,

Naoé³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The mitochondrial intermembrane space (IMS) contains many small cysteine-bearing proteins, and their passage across the outer membrane and subsequent folding require recognition and disulfide bond transfer by an oxidative translocator Tim40/Mia40 in the inner membrane facing the IMS. Here we determined the crystal structure of the core domain of yeast Mia40 (Mia40C4) as a fusion protein with maltose-binding protein at a resolution of 3 Å. The overall structure of Mia40C4 is a fruit-dish-like shape with a hydrophobic concave region, which accommodates a linker segment of the fusion protein in a helical conformation, likely mimicking a bound substrate. Replacement of the hydrophobic residues in this region resulted in growth defects and impaired assembly of a substrate protein. The Cys296-Cys298 disulfide bond is close to the hydrophobic concave region or possible substrate-binding site, so that it can mediate disulfide bond transfer to substrate proteins. These results are consistent with the growth phenotypes of Mia40 mutant cells containing Ser replacement of the conserved cysteine residues.

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Section: (5)supporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Structural Basis Of Yeast Tim40/mia40 As An Oxidative Translmentioning

confidence: 99%

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Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space

Kawano

Yamano²,

Naoé³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Considering that (i) the latter interaction is based on hydrophobic contacts established through an amphipathic α-helix of the substrate (23,27), and (ii) the residues close to CRAC motif show a tendency to form an α-helical amphipatic turn exposing hydrophobic residues to the solvent, we investigated the effect of these hydrophobic residues in complex formation with MIA40 through mutagenesis. MIA40/Mia40 interacts with ALR/Erv1 in two ways: (i) with unfolded and reduced Erv1 during its import (28,29), and (ii) with folded and oxidized ALR/Erv1 after its import to reoxidize the CPC motif of MIA40/ Mia40 (13,30). Our analysis focused so far exclusively on the second interaction; i.e., between folded/oxidized ALR and MIA40.…”

Section: Resultsmentioning

confidence: 99%

Molecular recognition and substrate mimicry drive the electron-transfer process between MIA40 and ALR

Banci

Bertini

Calderone

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

115

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Oxidative protein folding in the mitochondrial intermembrane space requires the transfer of a disulfide bond from MIA40 to the substrate. During this process MIA40 is reduced and regenerated to a functional state through the interaction with the flavin-dependent sulfhydryl oxidase ALR. Here we present the mechanistic basis of ALR-MIA40 interaction at atomic resolution by biochemical and structural analyses of the mitochondrial ALR isoform and its covalent mixed disulfide intermediate with MIA40. This ALR isoform contains a folded FAD-binding domain at the C-terminus and an unstructured, flexible N-terminal domain, weakly and transiently interacting one with the other. A specific region of the N-terminal domain guides the interaction with the MIA40 substrate binding cleft (mimicking the interaction of the substrate itself), without being involved in the import of ALR. The hydrophobicity-driven binding of this region ensures precise protein-protein recognition needed for an efficient electron transfer process.

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“…Upon import of a Cys-reduced substrate, Mia40 interacts with the substrate via intermolecular disulfide bond and shuttles a disulfide to its substrate. Although oxidized Mia40 promotes disulfide bond formation in the substrates, Erv1p functions in catalyzing reoxidation of the reduced Mia40 and/or release of the substrate (11)(12)(13).…”

mentioning

confidence: 99%

Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

Ang

2009

Journal of Biological Chemistry

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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 systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX 16 C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur.Disulfide bonds play very important roles in the structure and function of many proteins by stabilizing protein folding and/or acting as thiol/disulfide redox switches. The process of disulfide formation is catalyzed by dedicated enzymes in vivo (1-4). Erv1p is a FAD-dependent sulfhydryl oxidase located in the Saccharomyces cerevisiae mitochondrial intermembrane space (4 -6). It is an essential component of the redox regulated Mia40/Erv1 import and assembly pathway used by many of the cysteine-containing intermembrane space proteins, such as members of the "small Tim" and Cox17 families (7-10). Upon import of a Cys-reduced substrate, Mia40 interacts with the substrate via intermolecular disulfide bond and shuttles a disulfide to its substrate. Although oxidized Mia40 promotes disulfide bond formation in the substrates, Erv1p functions in catalyzing reoxidation of the reduced Mia40 and/or release of the substrate (11-13).The common features for the FAD-dependent sulfhydryl oxidases are that the enzymes can catalyze the electron transfer from substrate molecules (e.g. protein thiols) through the noncovalent bound FAD cofactor to molecular oxygen or oxidized cytochrome c (14). The sulfhydryl oxidases can be divided into three groups: Ero1 enzymes, multidomain quiesin sulfhydryl oxidases, and single domain Erv (essential for respiration and vegetative growth)/ALR proteins. The yeast Ero1p and the mammalian homologues (Ero1␣ and Ero1␤) are large flavoenzymes present in the ER with...

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Functional Characterization of Mia40p, the Central Component of the Disulfide Relay System of the Mitochondrial Intermembrane Space

Cited by 84 publications

References 52 publications

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space

Molecular recognition and substrate mimicry drive the electron-transfer process between MIA40 and ALR

Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

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