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, 48 (8), 2191-9

The MIA Pathway: A Key Regulator of Mitochondrial Oxidative Protein Folding and Biogenesis


The MIA Pathway: A Key Regulator of Mitochondrial Oxidative Protein Folding and Biogenesis

Amelia Mordas et al. Acc Chem Res.


Mitochondria are fundamental intracellular organelles with key roles in important cellular processes like energy production, Fe/S cluster biogenesis, and homeostasis of lipids and inorganic ions. Mitochondrial dysfunction is consequently linked to many human pathologies (cancer, diabetes, neurodegeneration, stroke) and apoptosis. Mitochondrial biogenesis relies on protein import as most mitochondrial proteins (about 10-15% of the human proteome) are imported after their synthesis in the cytosol. Over the last several years many mitochondrial translocation pathways have been discovered. Among them, the import pathway that targets proteins to the intermembrane space (IMS) stands out as it is the only one that couples import to folding and oxidation and results in the covalent modification of the incoming precursor that adopt internal disulfide bonds in the process (the MIA pathway). The discovery of this pathway represented a significant paradigm shift as it challenged the prevailing dogma that the endoplasmic reticulum is the only compartment of eukaryotic cells where oxidative folding can occur. The concept of the oxidative folding pathway was first proposed on the basis of folding and import data for the small Tim proteins that have conserved cysteine motifs and must adopt intramolecular disulfides after import so that they are retained in the organelle. The introduction of disulfides in the IMS is catalyzed by Mia40 that functions as a chaperone inducing their folding. The sulfhydryl oxidase Erv1 generates the disulfide pairs de novo using either molecular oxygen or, cytochrome c and other proteins as terminal electron acceptors that eventually link this folding process to respiration. The solution NMR structure of Mia40 (and supporting biochemical experiments) showed that Mia40 is a novel type of disulfide donor whose recognition capacity for its substrates relies on a hydrophobic binding cleft found adjacent to a thiol active CPC motif. Targeting of the substrates to this pathway is guided by a novel type of IMS targeting signal called ITS or MISS. This consists of only 9 amino acids, found upstream or downstream of a unique Cys that is primed for docking to Mia40 when the substrate is accommodated in the Mia40 binding cleft. Different routes exist to complete the folding of the substrates and their final maturation in the IMS. Identification of new Mia40 substrates (some even without the requirement of their cysteines) reveals an expanded chaperone-like activity of this protein in the IMS. New evidence on the targeting of redox active proteins like thioredoxin, glutaredoxin, and peroxiredoxin into the IMS suggests the presence of redox-dependent regulatory mechanisms of the protein folding and import process in mitochondria. Maintenance of redox balance in mitochondria is crucial for normal cell physiology and depends on the cross-talk between the various redox signaling processes and the mitochondrial oxidative folding pathway.


Figure 1
Figure 1
Electron transfer across the MIA pathway in the mitochondrial IMS. Precursors that have been synthesized on cytosolic ribosomes enter the mitochondria through the TOM complex, in a reduced and unfolded state. Those destined for detainment in the IMS by oxidative folding follow the MIA pathway. Electron flow begins from the reduced precursor to the redox active cysteine-proline-cysteine (CPC) motif of Mia40/MIA40, to the N-terminus of one Erv1/ALR subunit, to the Erv1/ALR core FAD domain of the C-terminus of the other Erv1/ALR subunit, to cytochrome c (Cyt c), to cytochrome c oxidase, and last to oxygen (O2). Alternatively, electrons can flow from Erv1/ALR directly to O2, and in yeast from Cyt c to cytochrome c peroxidase (Ccp). Note that, in mammalian cells, MIA40 is soluble in the IMS.
Figure 2
Figure 2
Solution structures of human MIA40 alone and in complex with human COX17. (A) Solution NMR structure of human MIA40 (hMIA40) (PDB ID: 2K3J). The redox active cysteine-proline-cysteine (CPC, yellow, green, yellow) motif is located within a one and a half turn, flexible helical structure (α1, red). Two other helices are present within the core domain (α2, blue; α3, cyan), creating a hydrophobic cleft which is connected by two intramolecular disulfide bonds (yellow). (B) Solution NMR structure of core hMIA40 (cyan) in complex with the MIA40-induced α-helix in hCOX17 (magenta) (PDB ID: 2L0Y). hCOX17 sits on top of the hydrophobic cleft of hMIA40 adjacent to the CPC motif. This interaction is covalent via an intermolecular disulfide bond but the initial recognition of hMIA40 and hCOX17 occurs via noncovalent interactions mediated by hydrophobic residues present in both hMIA40 (red) and hCOX17 (blue).
Figure 3
Figure 3
Molecular recognition of substrates containing twin CX3C or CX9C motifs by Mia40. Precursors containing IMS-targeting signals (ITS/MISS) “slide” onto the hydrophobic binding cleft of Mia40 via hydrophobic interactions. This allows docking of its active cysteine to the CPC motif of Mia40 intermolecularly. The substrate is released when the “resolving” cysteine forms an intramolecular disulfide with the “docking” cysteine.
Figure 4
Figure 4
Possible mechanisms for completion of substrate oxidation. Sliding of the substrate onto Mia40 via hydrophobic interactions results in the nucleophilic attack of the “docking” cysteine of the substrate on the first cysteine of the CPC motif of Mia40 (red line). This forms a mixed, covalent intermediate which is coupled to folding of the substrates first coiled-coil helix. After this, the substrate is thought to follow one of three possible scenarios. (A) Substrate release after initial oxidation is coupled to the folding of the second coiled-coil helix which induces formation of the second disulfide bond by an unknown oxidant. Or (B) the partially oxidized released substrate slides onto another oxidized Mia40 nearby and the reaction occurs again, releasing the fully oxidized substrate. Or (C) the full reaction occurs sequentially at the same site in a ternary complex between Mia40, Erv1, and the substrate due to the reoxidation of Mia40 by Erv1.

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