Molecular chaperone function of Mia40 triggers consecutive induced folding steps of the substrate in mitochondrial protein import

Abstract

Several proteins of the mitochondrial intermembrane space are targeted by internal targeting signals. A class of such proteins with α-helical hairpin structure bridged by two intramolecular disulfides is trapped by a Mia40-dependent oxidative process. Here, we describe the oxidative folding mechanism underpinning this process by an exhaustive structural characterization of the protein in all stages and as a complex with Mia40. Two consecutive induced folding steps are at the basis of the protein-trapping process. In the first one, Mia40 functions as a molecular chaperone assisting α-helical folding of the internal targeting signal of the substrate. Subsequently, in a Mia40-independent manner, folding of the second substrate helix is induced by the folded targeting signal functioning as a folding scaffold. The Mia40-induced folding pathway provides a proof of principle for the general concept that internal targeting signals may operate as a folding nucleus upon compartment-specific activation.

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

“…These hydrophobic residues are essentially the same ones that are involved in protein recognition of the CX 9 C or CX 3 C substrates (23), suggesting that the recognition process between MIA40 and ALR is based on the same molecular grounds found in the MIA40∕CX 9 C or CX 3 C substrate interaction. 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).…”

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

“…Protein folding is often retarded by unproductive intermediates. The role of the environment of Cys36 has not been analysed in import experiments with isolated mitochondria or in binding experiments 25 .…”

“…22). This model could not explain how the other disulphide is formed in the yeast protein 23,25 , because the (b) Surface representation of Mia40, colouring and orientation is as in (a). Panels a and b were created using Protein Data Bank files 2ZXT (Mia40) 14 and IU97 (Cox17) 17 and the programme PyMOL.…”

Human anamorsin binds [2Fe–2S] clusters with unique electronic properties

“…This suggests that the two cluster sites are functionally independent and that the M2 motif is not essential in the electron transfer process required for cytosolic Fe/S protein biogenesis. The M2 motif has been previously [9] found to modulate the subcellular localization of anamorsin in the IMS, being imported through the mitochondrial Mia40-dependent disulfide relay system [10,11,43,44]. In such a process, anamorsin is trapped in the IMS through the oxidoreductase Mia40, which specifically forms two disulfide bonds in the M2 motif.…”

“…This motif is a typical feature of the socalled CIAPIN1 domain, located at the C-terminus of more than 300 amino acid sequences of the eukaryotic proteins constituting the anamorsin protein family, which has protein members ranging from basal protozoa to fungi, higher plants and animals, up to humans. All CIAPIN1 domains have an additional well-conserved cysteine-rich CX 2 CX 7 CX 2 C (M2) motif, which forms in vitro two disulfide bonds upon interaction of anamorsin with the oxidoreductase Mia40 [9], an essential protein responsible for protein import and folding in the mitochondrial intermembrane space (IMS) [10][11][12]. Anamorsin can also be imported into the IMS of isolated mitochondria, where it interacts with Mia40, forming an intermolecular disulfide-bonded intermediate [9].…”