Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding.

Valentina Peleh,Johannes M Herrmann,Emmanuelle Cordat

doi:10.7554/elife.16177

Valentina Peleh, Johannes M Herrmann + Show 1 more

Open Access

https://doi.org/10.7554/elife.16177

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Journal: eLife	Publication Date: Jun 25, 2016
Citations: 63	License type: CC BY 4.0

Affiliation: University of Kaiserslautern, University of Alberta

Abstract

Many proteins of the mitochondrial IMS contain conserved cysteines that are oxidized to disulfide bonds during their import. The conserved IMS protein Mia40 is essential for the oxidation and import of these proteins. Mia40 consists of two functional elements: an N-terminal cysteine-proline-cysteine motif conferring substrate oxidation, and a C-terminal hydrophobic pocket for substrate binding. In this study, we generated yeast mutants to dissect both Mia40 activities genetically and biochemically. Thereby we show that the substrate-binding domain of Mia40 is both necessary and sufficient to promote protein import, indicating that trapping by Mia40 drives protein translocation. An oxidase-deficient Mia40 mutant is inviable, but can be partially rescued by the addition of the chemical oxidant diamide. Our results indicate that Mia40 predominantly serves as a trans-site receptor of mitochondria that binds incoming proteins via hydrophobic interactions thereby mediating protein translocation across the outer membrane by a 'holding trap' rather than a 'folding trap' mechanism.

Highlights

In most cellular compartments, cysteine residues are predominantly present in the reduced state
Our observations suggest that trapping activity of Mia40 is essential since Mia40 serves as a ‘trans-site receptor’ whose hydrophobic binding to incoming polypeptides drives protein import into the mitochondrial IMS
We constructed Mia40 variants in which either the CPC motif was mutated to a redox-inactive SPS motif (Figure 1B, Mia40-SPS) or the hydrophobic binding region was compromised by replacement of two conserved phenylalanine residues at positions 315 and 318 by glutamate residues (Figure 1B, Mia40-FE)

Summary

Introduction

Cysteine residues are predominantly present in the reduced state. While oxidative protein folding in the periplasm and the ER is well characterized, the details of the mitochondrial disulfide bond formation are still elusive. Mitochondria consist of about 600 (yeast) to 1500 (humans) nuclear encoded proteins (Pagliarini et al, 2008; Vogtle et al, 2009). Following their synthesis on cytosolic ribosomes, these proteins are recognized by receptors on the mitochondrial surface and threaded through mitochondrial protein translocases (Schulz et al, 2015; Harbauer et al, 2014; Endo et al, 2011; Chacinska et al, 2009; Neupert and Herrmann, 2007). After import into the mitochondria the presequences are proteolytically removed by the matrix processing peptidase MPP (von Heijne, 1986; Vogtle et al, 2009)

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