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

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.DOI: http://dx.doi.org/10.7554/eLife.16177.001

Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7

“…The mitochondrial intermembrane space import and assembly (MIA) pathway is responsible for the import and stable accumulation of cysteine-rich proteins in the IMS (Chacinska et al, 2004;Naoe et al, 2004;Terziyska et al, 2005). The MIA pathway includes oxidoreductase MIA40/CHCHD4 (Mia40 in yeast), which recognizes and oxidizes substrate proteins, and ALR/GFER (Erv1 in yeast), which receives electrons from oxidation of a substrate (Hofmann et al, 2005;Mesecke et al, 2005;Muller et al, 2008;Banci et al, 2009Banci et al, , 2010Sztolsztener et al, 2013;Koch & Schmid, 2014;Peleh et al, 2016). The classic MIA40 substrates are mostly small proteins (< 20 kDa), such as TIMM8A and COX6B, with a specific arrangement of cysteine residues, such as CX 3 C or CX 9 C (Koehler, 2004;Milenkovic et al, 2009;Sideris et al, 2009;Bourens et al, 2012;Fischer et al, 2013).…”

Cysteine residues in mitochondrial intermembrane space proteins: more than just import

“…In yeast, substrates do not rely on typical TOM receptor subunits such as Tom22 for recognition by the TOM complex (Gornicka et al ., ). To interact with substrates, CHCHD4 is equipped with two functional parts, a redox‐active cysteine‐proline‐cysteine (CPC) motif in a flexible N‐terminal region and a hydrophobic patch in a central inflexible core domain (Banci et al ., ; Banci et al ., ; Weckbecker et al ., ; Koch and Schmid, ; Koch and Schmid, ; Peleh et al ., ). During its covalent (formation of a mixed disulfide bond between CHCHD4 and substrate) and non‐covalent (interaction of hydrophobic patches in substrate and CHCHD4) interactions with its substrates, CHCHD4 guides substrates into the IMS, oxidizes and folds them and possibly even helps to assemble them into larger complexes if required.…”

Cytosolic redox components regulate protein homeostasis via additional localisation in the mitochondrial intermembrane space

“…It shares with its homologues a characteristic domain about 60 amino acids in length that contains six cysteine residues organised as one CPC (cysteine–proline–cysteine) and two CX 9 C motifs . The CX 9 C motifs stabilise the core of the protein and form a hydrophobic cleft that serves to bind and hold substrates, whereas the active CPC motif is the acceptor of e − shuttled from the incoming protein . In the same way as the periplasm and ER oxidative pathways, Mia40 is recycled back to an oxidised state by a sulfhydryl oxidase after it oxidises its substrates.…”

“…Examples are the copper chaperone for superoxide dismutase (Ccs) 1 , the Fe‐S cluster protein Dre2 and the inner membrane protease Atp23 . The identification of these proteins leads to the conclusion that Mia40 has a wider role in protein import, not only as an oxidative molecule but also as a receptor . Mia40 itself is not a conventional substrate of Mia40 and in fact its import has been found to occur in three stages.…”