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

Abstract

The intermembrane space (IMS) is a very small mitochondrial sub-compartment with critical relevance for many cellular processes. IMS proteins fulfil important functions in transport of proteins, lipids, metabolites and metal ions, in signalling, in metabolism and in defining the mitochondrial ultrastructure. Our understanding of the IMS proteome has become increasingly refined although we still lack information on the identity and function of many of its proteins. One characteristic of many IMS proteins are conserved cysteines. Different post-translational modifications of these cysteine residues can have critical roles in protein function, localization and/or stability. The close localization to different ROS-producing enzyme systems, a dedicated machinery for oxidative protein folding, and a unique equipment with antioxidative systems, render the careful balancing of the redox and modification states of the cysteine residues, a major challenge in the IMS. In this review, we discuss different functions of human IMS proteins, the involvement of cysteine residues in these functions, the consequences of cysteine modifications and the consequences of cysteine mutations or defects in the machinery for disulfide bond formation in terms of human health. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Figure 1

The layout of the mitochondrial intermembrane space. Mitochondria contain four sub‐compartments: the matrix, the OMM and IMM and the IMS. The IMS is subdivided into a peripheral IMS and cristae. The peripheral IMS is in contact with the OMM and the IBM. The cristae are enclosed by invaginations of the IMM and the CM. CJ segregate cristae and peripheral IMS. Contact sites (CS) formed by parts of the MICOS complex and the sorting and assembly machinery (SAM) complex mediate contacts between OMM and IBM. The OMM contains few proteins that have a high mobility in the membrane. Conversely, IMM proteins are more restricted in their movement. The structure of the IMM leads to heterogeneous protein distribution, for example, different protein sets between IBM and CM.

Figure 2

Functions of the mitochondrial intermembrane space. (A) A list of IMS proteins was compiled from the IMS proteomes of human cells, the interactome of CHCHD4 (Vogtle et al., 2012; Hung et al., 2014; Petrungaro et al., 2015; Morgenstern et al., 2017) and the bioinformatics analyses of twin CX₉C‐like proteins (Cavallaro, 2010; Fischer et al., 2013) (Supporting Information Table S1). Some dually localized proteins were added manually to the list. Of the so far identified IMS proteins (about 150) approximately 20% are still of unknown function. According to published data or database entries, we assigned the remaining proteins to four different functional classes: Logistics hub, Metabolism, Morphology and Signalling. Proteins were assigned to the group ‘Logistics hub’ if they were annotated as being involved in transport of proteins/small molecules, protein degradation or assembly of protein complexes. Proteins of the ‘Metabolism’ group are integral subunits of respiratory chain complexes (e.g. ATP5I) or enzymes in metabolic pathways (e.g. CPOX). The ‘Morphology’ group contains proteins, which are involved in maintenance of mitochondrial structure (e.g. OPA1). Proteins were assigned to ‘Signalling’ if they were annotated as being involved in, for example, apoptotic, redox or calcium signalling. Assignment to multiple groups was undertaken where applicable (e.g. voltage‐dependent anion channels (VDAC1‐3) in ‘Logistics’, ‘Metabolism’ and ‘Signalling’). (B) Logistics hub: IMS proteins are involved in distributing mitochondrial proteins to their respective mitochondrial sub‐compartments. After entering through the TOM, proteins can become sorted into the OMM by the SAM complex. Chaperone complexes in the IMS support this process (small TIMM proteins). The translocase complex of the IMM, TIM23, either sorts proteins with mitochondrial targeting signals to the IMM or guides them to the matrix, while TIM22 handles members of the mitochondrial carrier family. OXA1 shuttles proteins into the IMM from the matrix side. Lastly, many soluble IMS proteins are folded by the mitochondrial disulfide relay. IMS proteins also contribute to folding, quality control and removal of proteins as well as their assembly into protein complexes such as the MCU or the respiratory chain. Additionally, the IMS plays an important role in transferring metabolites and lipids between cytosol and matrix and OMM and IMM respectively. PA, phosphatidic acid; CIC, mitochondrial citrate transporter; ANT, adenine nucleotide translocator; TIM, translocase of the inner membrane. (C) Metabolism: The cristae are functionally important for the formation of an electrochemical potential across the IMM. This electrochemical potential is utilized to generate ATP in the matrix (OXPHOS). The IMS also harbours enzymes that catalyse parts of metabolic pathways. For example, coproporphyrinogen oxidase (CPOX) catalyses the penultimate step in porphyrin biosynthesis. Nucleotide‐converting enzymes balance nucleotide pools and enable their efficient diffusion, membrane transport and enzyme function. They include adenylate kinase 2 and mitochondrial CK. In the IMM, membrane lipids are converted, for example, production of cardiolipin takes place. (D) Morphology: Proteins of the IMS and its adjacent membranes define mitochondrial morphology and dynamics. This not only includes formation of cristae junctions and contact sites by MICOS complex and SAM complex but also balancing membrane fission and fusion by DRP1 (also DNML1, dynamin‐1‐like protein) and MFN1/2 (mitofusin 1/2) respectively. (E) Signalling: The IMS is important for signalling processes. Regulators of mitochondrial calcium signalling such as MICU1/2 are resident in the IMS and modulate activity of the MCU. The IMS is an important production site for ROS and therefore is relevant in ROS (depicted as H₂O₂) release/signalling to the cytosol, for example, via VDACs in the OMM. Upon apoptotic stimuli and membrane remodelling IMS proteins, for example, cleaved AIF and cytochrome c can be released into the cytosol. For a detailed description, see text.

Figure 3

Conserved cysteines in mammalian IMS enzymes. (A) The list of IMS proteins (Supporting Information Table S1) was analysed for cysteine presence and their conservation. Many IMS proteins contain at least one conserved cysteine while only a few IMS proteins contain non‐conserved or no cysteines. Conservation was assessed in Homo sapiens (H.s.), Mus musculus (M.m.), Rattus norvegicus (R.n.), Danio rerio (D.r.) and Xenopus laevis (X.l.). (B) Examples for the function of conserved cysteine residues. (1) Reduced cysteines can act in metal ion transfer. Specifically, SCO1/2 can chelate copper and transfer it onto COX2, which assembles into complex IV of the respiratory chain. (2) Reduced cysteines can act in substrate binding sites. In CK, a reduced cysteine in its binding groove is involved in orientating creatine for phosphorylation. (3) Redox‐active cysteines are involved in oxidative folding while structural disulfides form a binding groove. In CHCHD4, the redox‐active cysteines C53 and C55 oxidize substrates during oxidative folding. The structural disulfides stabilize the hydrophobic binding groove necessary for substrate recognition. (4) Redox‐active cysteines are involved in the detoxification of H₂O₂. PRDX3/4 contain redox‐active cysteines, which are oxidized by H₂O₂, while H₂O₂ is reduced to H₂O. (5) Structural disulfides are necessary for complex assembly. CHCHD3/6 are oxidatively folded via the disulfide relay. The stable fold conferred by two disulfides per protein allow for stable insertion into the MICOS complex. (6) Structural disulfides lead to protein dimerization. MICU1/2 form a disulfide‐linked heterodimer, which allows for calcium‐dependent regulation of the MCU. For a detailed description, see text.

Figure 4

Cysteine modifications in the IMS. (A) Cysteine modifications. The reactive thiol group of cysteine residues can exist in the protonated state or as the deprotonated thiolate anion. This thiol group can be further modified as indicated. (B) Various thiol modifications are introduced into IMS proteins. (1) ROS‐generation in the IMS. Transfer of a single electron to O₂ leads to formation of superoxide anion (O₂°⁻). Superoxide generators of the IMS include complex III of the respiratory chain (CIII), the ALR, and GPDH. Superoxide is enzymically converted to H₂O₂ by SOD1. Dihydroorotate dehydrogenase (DHODH) can generate O₂°⁻ and H₂O₂. Likewise, MAO B and p66Shc have been reported to directly generate H₂O₂. (2) Cysteine modification by H₂O₂. H₂O₂ can result in the direct oxidation of target thiols (–SH) to sulfenic (–SOH), sulfinic (–SO₂H) and sulfonic (–SO₃H) acid depending on thiol reactivity and H₂O₂ availability. Most protein thiols exhibit low reactivity towards H₂O₂ and will thus be primarily metabolised by dedicated H₂O₂‐scavenging enzymes such as peroxiredoxins (PRDX). PRDX3/4 are located in the IMS and their oxidation by H₂O₂ can be reversed by thioredoxins (TRX), but might also be passed on to other proteins. H₂O₂ also mediates phospholipid (PL) peroxidation (PL–OOH). PL peroxidation can be removed by GSH peroxidase‐4 (GPX4). (3) The mitochondrial disulfide relay. During oxidative protein folding, reduced cysteine residues (–SH) are oxidized to disulfide bonds (–SS) by oxidized (ox.) CHCHD4. See Figure 5 for additional details. (4) S‐nitrosations of cysteine residues. Reduced GSH reacts with ·NO to S‐nitrosoglutathione (GSNO), inducing S‐nitrosations (–SNO). NO is also able to directly induce S‐nitrosations. (5) S‐glutathionylation of cysteine residues. Reduced glutaredoxin‐1 [GRX1 (–SH)] reacts with GSSG and becomes glutathionylated [GRX1 (–SSG)]. GRX1 subsequently mediates the S‐glutathionylation (–SSG). (6) S‐sulfhydration of cysteine residues. Disulfides and sulfenic acids (–SOH) can react with H₂S, resulting in S‐sulfhydration (–SSH) of cysteines. Thiols on the other hand can only be sulfhydrated by H₂S in the presence of superoxide anions. (C) Removal of thiol modifications. (1) Reduction of disulfide bonds by the thioredoxin system (TRX).Thioredoxin 1 (TRX1) is likely to mediate the reduction of disulfides and become oxidized itself. Thioredoxin reductase (TRXR) reduces TRX1 in an NADPH‐dependent manner. (2) Reduction of disulfide bonds and S‐glutathionylations by GRX1. GRX1 reduces disulfides and S‐glutathionylated thiols (–SSG) while oxidizing reduced GSH. GSH disulfide (GSSG) is reduced by GSH reductase (GLRX) in an NADPH‐dependent manner.

Figure 5

Enzyme‐catalysed disulfide formation in the IMS. (A) The mitochondrial disulfide relay. Disulfide relay substrates are synthesized on cytosolic ribosomes and are targeted to the TOM pore (step 1). During translocation, the substrates' mitochondrial IMS‐sorting signal (MISS, also ITS) binds the hydrophobic cleft of CHCHD4 and a mixed disulfide between the substrate and active site cysteines of CHCHD4 is formed (step 2). After releasing the oxidized and folded substrate (step 3), CHCHD4 is present in a reduced state. Electrons from reduced CHCHD4 are transferred to ALR (step 4). ALR shuffles electrons onto cytochrome c, which in turn transfers them to complex IV (step 5). The disulfide relay is supported by different auxiliary factors. RCHY/Hot13 complexes Zn²⁺ and thereby accelerates oxidation (step a). AIF facilitates CHCHD4 biogenesis and thus ensures the presence of this critical protein in the IMS (step b). The GSH redox buffer (E_GSH) and glutaredoxins proofread wrongly formed or trapped disulfides. Glutaredoxins are present only in limiting amounts to allow disulfide formation (step c). (B) CHCHD4 substrates fall into three different structural classes. Proteins that lack classical mitochondrial targeting sequences and instead contain conserved cysteine residues form classes 1 and 2. The cysteines can either be ordered in twin CX₃C of twin CX₉C motifs (class 1) or not (class 2). Proteins from both classes acquire disulfide bonds during import, and it appears that this also is the prerequisite for mitochondrial accumulation and retention in the IMS. CHCHD4 also oxidizes substrates that rely on N‐terminal mitochondrial targeting sequences (MTS) for import (class 3).

Figure 6

Cysteine‐linked defects in IMS enzymes associated with disease. (A) Substrates of the mitochondrial disulfide relay associated with disease phenotypes. Mutations in genes of diverse CHCHD4 substrates result in the indicated diseases. The CHCHD4 substrates are sorted according to their function as described in Figure 2 (also see Supporting Information Table S1). (B) Examples of CHCHD4 substrate mutations in which disulfide formation is affected. For TIMM8A (C66W) and NDUFB10 (C107S), cysteine mutations were reported to result in human disease. Specifically, these mutations affect disulfide formation, oxidative folding and mitochondrial import of TIMM8A and NDUFB10 that results in incomplete formation of the TIMM chaperone complex and complex I of the respiratory chain respectively. Disease‐associated mutations in COA5 (A53P) and COA6 (W59C/W66R) affect residues in proximity to cysteines which probably interferes with disulfide formation. For a detailed description, see text.