Biogenesis of yeast Mia40 – uncoupling folding from import and atypical recognition features

The discovery of the mitochondrial intermembrane space assembly (MIA) pathway was followed by studies that focused mainly on the typical small substrates of this disulfide relay system and the interactions between its two central partners: the oxidoreductase Mia40 and the FAD‐protein Erv1. Recent studies have revealed that more complex proteins utilize this pathway, including Mia40 itself. In the present study, we dissect the Mia40 biogenesis in distinct stages, supporting a kinetically coordinated sequence of events, starting with (a) import and insertion through the Tim23 translocon, followed by (b) folding of the core of imported Mia40 assisted by the endogenous Mia40 and (c) final interaction with Erv1. The interaction with endogenous Mia40 and the subsequent interaction with Erv1 represent kinetically distinguishable steps that rely on completely different determinants. Interaction with Mia40 proceeds very early (within 30 s) and is characterized by no Cys‐specificity, an increased tolerance to mutations of the hydrophobic substrate‐binding cleft and no apparent dependence on glutathione as a proofreading mechanism. All of these features illustrate a very atypical behaviour for the Mia40 precursor compared to other substrates of the MIA pathway. By contrast, interaction with Erv1 occurs after 5 min of import and relies on a more stringent specificity. A stepwise model for the yeast Mia40 biogenesis in mitochondria. The mechanism requires crossing via the TOM complex, followed by insertion through the TIM23 translocon. The interaction with endogenous Mia40 drives folding of the protein core and allows oxidation of the CPC motif by Erv1. Different determinants guide each step to complete the assembly of Mia40 in the IMS.