Human, yeast and other eukaryotic cells contain compartments called mitochondria that perform several vital tasks, including supplying the cell with energy. Each mitochondrion is surrounded by an inner and an outer membrane, which are separated by an intermembrane space that contains a host of molecules, including proteins.

Intermembrane space proteins are made in the cytosol before being transported into the intermembrane space through pores in the mitochondrion’s outer membrane. Many of these proteins have the ability to form disulfide bonds within their structures, which help the proteins to fold and assemble correctly, but they only acquire these bonds once they have entered the intermembrane space.

An enzyme called Mia40 sits inside the intermembrane space and helps other proteins to fold correctly. This Mia40-induced folding had been suggested to help proteins to move into the intermembrane space.

Mia40 contains two important regions: one region acts as an enzyme and adds disulfide bonds to other proteins, and the other region binds to the intermembrane space proteins. Peleh et al. have now generated versions of Mia40 that lack one or the other of these regions in yeast cells, and then tested to see if these mutants could drive proteins across the outer membrane of mitochondria. The results show that it is the ability of Mia40 to bind proteins – and not its enzyme activity – that is essential for importing proteins into the intermembrane space.

As disulfide bond formation is not critical for importing proteins into the intermembrane space, future studies could test whether Mia40 also helps to transport proteins that cannot form disulfide bonds. Presumably, Mia40 has a much broader relevance for importing mitochondrial proteins than was previously thought.

In most cellular compartments, cysteine residues are predominantly present in the reduced state. In contrast, the bacterial periplasm, the endoplasmic reticulum (ER) and the IMS of mitochondria contain dedicated oxidation machineries (disulfide relays) to introduce disulfide bonds into a broad range of substrate proteins (Riemer et al., 2009; Kadokura et al., 2003; Bulleid and Ellgaard, 2011; Stojanovski et al., 2012; Modjtahedi et al., 2016; Riemer et al., 2015). At least in the case of the periplasm and the ER, disulfide bond formation presumably serves the function of stabilizing the structures of (secretory) proteins. 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; Vögtle 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). Proteins destined for the matrix of mitochondria contain presequences (or matrix-targeting signals) at their N termini that target these proteins through the protein-conducting channels of the translocase of the outer membrane (TOM complex) and the inner membrane (TIM23 complex). After import into the mitochondria the presequences are proteolytically removed by the matrix processing peptidase MPP (von Heijne, 1986; Vögtle et al., 2009).

The targeting of proteins into the IMS, the compartment between the outer and the inner membrane of mitochondria, is less well understood. Some IMS proteins also contain N-terminal signals in the form of bipartite presequences which consist of a matrix-targeting signal followed by a hydrophobic stop-transfer domain. These proteins are arrested at the inner membrane before their mature domains are released into the IMS by proteolytic cleavage (Glick et al., 1992; Herlan et al., 2004; Rojo et al., 1998). The cytochrome b₂ protein of Saccharomyces cerevisiae is the best studied example for this stop-transfer targeting (Glick et al., 1992; Hartl et al., 1987; Gärtner et al., 1995).

Most IMS proteins do not contain N-terminal targeting signals but instead contain patterns of cysteine residues within their mature sequence that serve as targeting signals (Koehler, 2004; Sideris and Tokatlidis, 2007; Sideris et al., 2009; Milenkovic et al., 2009). In most cases, these proteins contain two pairs of cysteine residues that are either spaced by three or nine amino acid residues, therefore referred to as ’twin Cx₃C’ and ’twin Cx₉C’ proteins, respectively. In yeast, five ’twin Cx₃C’ (also called small Tim proteins) play a role as chaperones for carrier proteins in the IMS (Curran et al., 2002b; 2002a; Koehler et al., 1998; Sirrenberg et al., 1998; Luciano et al., 2001; Vial et al., 2002), and 13 ’twin Cx₉C’ proteins contribute to the stabilization and assembly of inner membrane proteins (Potting et al., 2010; Vögtle et al., 2012; Longen et al., 2009; Modjtahedi et al., 2016; Horn et al., 2010; Bode et al., 2015). But recently also proteins with different disulfide configurations were discovered (Okamoto et al., 2014; Wrobel et al., 2013; Varabyova et al., 2013; Hangen et al., 2015; Klöppel et al., 2011; Wrobel et al., 2016). The import of these proteins into mitochondria relies on the mitochondrial disulfide relay (also called MIA pathway) which employs two conserved, essential proteins, Erv1 and Mia40. Erv1 is an FAD-binding sulfhydryl oxidase which can ‘generate’ disulfide bonds de novo thereby transferring electrons either directly to molecular oxygen or to cytochrome c of the respiratory chain (Lee et al., 2000; Dabir et al., 2007; Ang and Lu, 2009; Tienson et al., 2009; Bien et al., 2010; Mesecke et al., 2005; Allen et al., 2005; Bihlmaier et al., 2007; Fass, 2008). The oxidoreductase Mia40 contains a redox-active cysteine-proline-cysteine (CPC) motif (Terziyska et al., 2009; Chacinska et al., 2004; Banci et al., 2009; 2010; Kawano et al., 2009). Erv1 maintains this motif in an oxidized conformation which then permits the transfer of disulfide bonds to Mia40 substrates during their translocation into the IMS (von der Malsburg et al., 2011). Mia40 substrates are often small proteins that are unstructured in the reduced form but very stable as soon as they are oxidized (Morgan et al., 2009; Curran et al., 2004; 2002a; Baker et al., 2012; Banci et al., 2009; 2012). It was proposed that oxidation traps the incoming polypeptides in the IMS so that oxidation-induced folding drives their net translocation into the IMS (‘folding trap model’) (Lutz et al., 2003; Koehler, 2004). Mechanistically, this would be very different from protein translocation into the periplasm and the ER where the translocation of proteins is driven in ATP-dependent reactions by SecA or BiP, respectively, prior to and independent of their oxidative folding (Matlack et al., 1999; Economou and Wickner, 1994; Taufik et al., 2013). Moreover, the formation of mixed disulfides of Mia40 with incoming polypeptides was suggested to serve as a crucial reaction in the translocation reaction (von der Malsburg et al., 2011; Longen et al., 2014; Bien et al., 2010) that is critical to avoid the mistargeting of reduced IMS proteins to the cytosol (Bragoszewski et al., 2015; Wrobel et al., 2015).

Structural analysis revealed the presence of a hydrophobic substrate-binding pocket on the surface of Mia40 (Figure 1A); like the CPC motif this substrate-binding region of Mia40 is essential for its function in the import and folding of IMS proteins and mutants were shown to be inviable (Banci et al., 2009, 2010; Kawano et al., 2009; Weckbecker et al., 2012). This region is essential for the binding and hence the oxidation of Mia40 substrates as well as for binding to Erv1. Mia40 substrates contain internal signals, known as MISS or ITS sequences, which specifically dock onto this binding region thereby selecting cysteine residues for interaction with the redox-active cysteine pair in Mia40 (Koch and Schmid, 2014b; Sideris and Tokatlidis, 2007; Sideris et al., 2009; Peleh et al., 2014; Milenkovic et al., 2009).

Figure 1

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In this study, we analyzed the relevance of both structural elements of Mia40 in more detail. To this end, we generated Mia40 mutants which lack either the redox-active cysteine pair (Mia40-SPS) or the substrate-binding pocket (Mia40-FE and Mia40-STOP). Interestingly, the Mia40-SPS mutant still mediates protein import of Mia40 substrates with high efficiency. It also allows the accumulation of Mia40 substrates in mitochondria, albeit at reduced levels. 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. Hence, protein oxidation in the IMS is not directly linked to translocation, and mechanistically is independent of the transport reaction, similar to the situation in the ER and the periplasm.

Mia40 is a highly conserved IMS protein which consists of two functionally and structurally distinct elements. Here we show that both the redox-active cysteine pair and the substrate-binding region of Mia40 are essential and mutants lacking individual elements cannot cross-complement each other. However, we found that cysteines in the CPC motif of Mia40 are dispensable for the import reaction of all Mia40 substrates we tested. This was unexpected because it was suggested that the mitochondrial disulfide relay operates as a folding trap (Figure 7E) (Lutz et al., 2003; Koehler, 2004). This model postulated that substrates would enter the IMS via the TOM pore in a reversible reaction. The reversible nature of the outer membrane translocation of Mia40 substrates is experimentally well documented (Lutz et al., 2003; Bragoszewski et al., 2015). According to the folding trap hypothesis, the directivity of the import reaction is driven by the introduction of disulfide bonds thereby locking proteins in a folded confirmation which traps them in the IMS (Mesecke et al., 2005; Bihlmaier et al., 2007). Based on the identification of the MISS/ITS signal as specific recognition sites in Mia40 substrates (Koch and Schmid, 2014b; Sideris and Tokatlidis, 2007; Sideris et al., 2009; Peleh et al., 2014; Milenkovic et al., 2009) and the observation of Mia40-linked import intermediates that span the TOM complex (von der Malsburg et al., 2011) it was suggested that Mia40 serves as an IMS-located receptor protein critical for outer membrane translocation of its substrates (Figure 7F). According to both models, disulfide bond formation by Mia40 is the critical feature for its role in protein translocation. However, our observation that the Mia40-SPS variant can efficiently drive the import reaction suggests that the affinity to the substrate-binding pocket provides the driving force for the translocation reaction rather than protein oxidation. Hence, we propose that Mia40 serves as a 'trans-site receptor' on the IMS site of the TOM complex that mediates protein translocation across the outer membrane (Figure 7G). At present it is not known whether the trapping function of Mia40 is restricted to disulfide-containing proteins or plays a broader, more general role in mitochondrial protein import. However, the recent identification of non-canonical substrates of Mia40 (Petrungaro et al., 2015; Okamoto et al., 2014; Wrobel et al., 2013; Hangen et al., 2015) might indeed suggest a much more general role of Mia40 in protein translocation across the outer membrane than previously expected.

The oxidoreductase activity of Mia40 is obviously important, but it apparently plays a role after translocation. This is consistent with a recent study which showed that mutants of small Tim proteins, in which all cysteine residues were replaced by serine residues, accumulated to wild type levels in the IMS as soon as the iAAA protease Yme1 was deleted (Baker et al., 2012).

Surprisingly, the presence of diamide can partially suppress the growth defect of Mia40-SPS cells suggesting that the substrate-specificity of the mitochondrial disulfide relay is not absolutely necessary. The observation of oxidized Tim10 in the Mia40-SPS mutant indicates that the CPC of Mia40 is not absolutely essential for protein oxidation in the IMS. Whether Erv1 can directly oxidize IMS proteins or whether other factors might serve as oxidoreductases here is not known, but it will be interesting to test the relevance of the recently identified thioredoxins and glutaredoxins in the IMS in this process (Vögtle et al., 2012; Kojer et al., 2015). However, it should be noted that, even in the presence of diamide, Mia40-SPS cells were very sick and protein oxidation in the IMS is obviously still an essential process.

Our observations indicate that Mia40 is rate-limiting for the import into mitochondria as overexpression of Mia40 leads to a considerable increase of the levels of many Mia40 substrates. Apparently, a fraction of the Mia40 substrates that are initially synthesized in the cytosol are degraded in the cytosol or the IMS, an assumption that is supported by pulse labeling experiments in whole cells (Wrobel et al., 2015; Longen et al., 2014; Kojer et al., 2015; Fischer et al., 2013). Increased levels of Mia40 might reduce the degradation either by accelerating the import or the folding reaction. In contrast, up- or down-regulation of the levels of Erv1 had no positive effect on the steady state levels of IMS proteins again supporting the idea that the trapping rather than the oxidase function of Mia40 is decisive (Bien et al., 2010; Mesecke et al., 2005). The strong increase in Mia40 substrates in Mia40-overexpressing cells suggests that more Mia40 leads to more import sites for Mia40 substrates. It is conceivable that mitochondria contain specific import sites for different types of preproteins which differ at the level of the 'trans-site receptors' in the IMS (Figure 7G). Thus, matrix-targeted proteins might specifically encounter TOM complexes that are associated to Tim50 (Lytovchenko et al., 2013; Marom et al., 2011) whereas IMS proteins might use TOM complexes that are in proximity to Mia40. This hypothesis is supported by studies on the Mic60 subunit of the MICOS complex which presumably tethers Mia40 to a subpopulation of the TOM complex (von der Malsburg et al., 2011; Herrmann, 2011; Varabyova et al., 2013). The existence of a discrete translocation route into the IMS was recently proposed for Mia40 substrates on the basis of in organello competition import experiments (Gornicka et al., 2014) and, even much earlier, for the most abundant IMS protein, cytochrome c (Wiedemann et al., 2003; Diekert et al., 2001; Mayer et al., 1995).

The bacterial periplasm, the ER and the mitochondrial IMS are the three compartments in which dedicated machineries mediate the oxidative folding of a broad range of proteins (Riemer et al., 2009). Members of the thioredoxin protein family mediate protein oxidation in the periplasm (DsbA) and the ER (PDI). Apparently, the thioredoxin-based oxidation system of the periplasm of the endosymbiont that served as the ancestor of mitochondria was replaced during evolution by the Mia40 system. This replacement during evolution was hardly driven to increase protein oxidation in the IMS since Mia40 is a poor catalyst: in its oxidizing power, it is weaker than DsbA, and in forming correct disulfides, it is slower than PDI (Koch and Schmid, 2014b; 2014a; Hudson and Thorpe, 2015; Tienson et al., 2009). However, Mia40 binds much stronger and for much longer interaction times to its substrates than DsbA or PDIs (Koch and Schmid, 2014b; Mesecke et al., 2005; Naoé et al., 2004; Chacinska et al., 2004). It therefore appears likely that the ability of Mia40 to trap incoming preproteins efficiently at the trans-site of the TOM complex made Mia40 the superior oxidoreductase to mediate the translocation and oxidation of IMS proteins.

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Author details

1. Valentina Peleh

   Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany

   Contribution

   VP, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Emmanuelle Cordat

   Department of Physiology, University of Alberta, Edmonton, Canada

   Contribution

   EC, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Johannes M Herrmann

   Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany

   Contribution

   JMH, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   hannes.herrmann@biologie.uni-kl.de

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-2081-4506

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