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The mitochondrial intermembrane space (IMS) houses a large spectrum of proteins with distinct and critical functions. Protein import into this mitochondrial sub-compartment is underpinned by an intriguing variety of pathways, many of which are still poorly understood. The constricted volume of the IMS and the topological segregation by the inner membrane cristae into a bulk area surrounded by the boundary inner membrane and the lumen within the cristae is an important factor that adds to the complexity of the protein import, folding and assembly processes. We discuss the main import pathways into the IMS, but also how IMS proteins are degraded or even retro-translocated to the cytosol in an integrated network of interactions that is necessary to maintain a healthy balance of IMS proteins under physiological and cellular stress conditions. We conclude this review by highlighting new and exciting perspectives in this area with a view to develop a better understanding of yet unknown, likely unconventional import pathways, how presequence-less proteins can be targeted and the basis for dual localisation in the IMS and the cytosol. Such knowledge is critical to understanding the dynamic changes of the IMS proteome in response to stress, and particularly important for maintaining optimal mitochondrial fitness.

Mitochondrial membrane proteins play an essential role in all major mitochondrial functions. The respiratory complexes of the inner membrane are key for the generation of energy. The carrier proteins for the influx/efflux of essential metabolites to/from the matrix. Many other inner membrane proteins play critical roles in the import and processing of nuclear encoded proteins (∼99% of all mitochondrial proteins). The outer membrane provides another lipidic barrier to nuclear-encoded protein translocation and is home to many proteins involved in the import process, maintenance of ionic balance, as well as the assembly of outer membrane components. While many aspects of the import and assembly pathways of mitochondrial membrane proteins have been elucidated, many open questions remain, especially surrounding the assembly of the respiratory complexes where certain highly hydrophobic subunits are encoded by the mitochondrial DNA and synthesised and inserted into the membrane from the matrix side. This review will examine the various assembly pathways for inner and outer mitochondrial membrane proteins while discussing the most recent structural and biochemical data examining the biogenesis process.

Synapses are a primary pathological target in neurodegenerative diseases. Identifying therapeutic targets at the synapse could delay progression of numerous conditions. The mitochondrial protein SFXN3 is a neuronally enriched protein expressed in synaptic terminals and regulated by key synaptic proteins, including α-synuclein. We first show that SFXN3 uses the carrier import pathway to insert into the inner mitochondrial membrane. Using high-resolution proteomics on Sfxn3-KO mice synapses, we then demonstrate that SFXN3 influences proteins and pathways associated with neurodegeneration and cell death (including CSPα and Caspase-3), as well as neurological conditions (including Parkinson's disease and Alzheimer's disease). Overexpression of SFXN3 orthologues in Drosophila models of Parkinson's disease significantly reduced dopaminergic neuron loss. In contrast, the loss of SFXN3 was insufficient to trigger neurodegeneration in mice, indicating an anti- rather than pro-neurodegeneration role for SFXN3. Taken together, these results suggest a potential role for SFXN3 in the regulation of neurodegeneration pathways.

Mitochondria have a central role in cellular metabolism; they are responsible for the biosynthesis of amino acids, lipids, iron–sulphur clusters and regulate apoptosis. About 99% of mitochondrial proteins are encoded by nuclear genes, so the biogenesis of mitochondria heavily depends on protein import pathways into the organelle. An intricate system of well-studied import machinery facilitates the import of mitochondrial proteins. In addition, folding of the newly synthesized proteins takes place in a busy environment. A system of folding helper proteins, molecular chaperones and co-chaperones, are present to maintain proper conformation and thus avoid protein aggregation and premature damage. The components of the import machinery are well characterised, but the targeting signals and how they are recognised and decoded remains in some cases unclear. Here we provide some detail on the types of targeting signals involved in the protein import process. Furthermore, we discuss the very elaborate chaperone systems of the intermembrane space that are needed to overcome the particular challenges for the folding process in this compartment. The mechanisms that sustain productive folding in the face of aggregation and damage in mitochondria are critical components of the stress response and play an important role in cell homeostasis.

Mitochondria are double-membrane organelles that contain their own genome, the mitochondrial DNA (mtDNA), and reminiscent of its endosymbiotic origin. Mitochondria are responsible for cellular respiration via the function of the electron oxidative phosphorylation system (OXPHOS), located in the mitochondrial inner membrane and composed of the four electron transport chain (ETC) enzymes (complexes I-IV), and the ATP synthase (complex V). Even though the mtDNA encodes essential OXPHOS components, the large majority of the structural subunits and additional biogenetical factors (more than seventy proteins) are encoded in the nucleus and translated in the cytoplasm. To incorporate these proteins and the rest of the mitochondrial proteome, mitochondria have evolved varied, and sophisticated import machineries that specifically target proteins to the different compartments defined by the two membranes. The intermembrane space (IMS) contains a high number of cysteine-rich proteins, which are mostly imported via the MIA40 oxidative folding system, dependent on the reduction, and oxidation of key Cys residues. Several of these proteins are structural components or assembly factors necessary for the correct maturation and function of the ETC complexes. Interestingly, many of these proteins are involved in the metalation of the active redox centers of complex IV, the terminal oxidase of the mitochondrial ETC. Due to their function in oxygen reduction, mitochondria are the main generators of reactive oxygen species (ROS), on both sides of the inner membrane, i.e., in the matrix and the IMS. ROS generation is important due to their role as signaling molecules, but an excessive production is detrimental due to unwanted oxidation reactions that impact on the function of different types of biomolecules contained in mitochondria. Therefore, the maintenance of the redox balance in the IMS is essential for mitochondrial function. In this review, we will discuss the role that redox regulation plays in the maintenance of IMS homeostasis as well as how mitochondrial ROS generation may be a key regulatory factor for ETC biogenesis, especially for complex IV.

International audience; Mitochondria are critical for several cellular functions as they control metabolism, cell physiology, and cell death. The mitochondrial proteome consists of around 1500 proteins, the vast majority of which (about 99% of them) are encoded by nuclear genes, with only 13 polypeptides in human cells encoded by mitochondrial DNA. Therefore, it is critical for all the mitochondrial proteins that are nuclear-encoded to be targeted precisely and sorted specifically to their site of action inside mitochondria. These processes of targeting and sorting are catalysed by protein translocases that operate in each one of the mitochondrial sub-compartments. The main protein import pathway for the intermembrane space (IMS) recognises proteins that are cysteine-rich, and it is the only import pathway that chemically modifies the imported precursors by introducing disulphide bonds to them. In this manner, the precursors are trapped in the IMS in a folded state. The key component of this pathway is Mia40 (called CHCHD4 in human cells), which itself contains cysteine motifs and is subject to redox regulation. In this review, we detail the basic components of the MIA pathway and the disulphide relay mechanism that underpins the electron transfer reaction along the oxidative folding mechanism. Then, we discuss the key protein modulators of this pathway and how they are interlinked to the small redox-active molecules that critically affect the redox state in the IMS. We present also evidence that the mitochondrial redox processes that are linked to iron–sulfur clusters biogenesis and calcium homeostasis coalesce in the IMS at the MIA machinery. The fact that the MIA machinery and several of its interactors and substrates are linked to a variety of common human diseases connected to mitochondrial dysfunction highlight the potential of redox processes in the IMS as a promising new target for developing new treatments for some of the most complex and devastating human diseases.

The mitochondrial intermembrane space (IMS) is the most constricted sub-mitochondrial compartment, housing only about 5% of the mitochondrial proteome, and yet is endowed with the largest variability of protein import mechanisms. In this review, we summarize our current knowledge of the major IMS import pathway based on the oxidative protein folding pathway and discuss the stunning variability of other IMS protein import pathways. As IMS-localized proteins only have to cross the outer mitochondrial membrane, they do not require energy sources like ATP hydrolysis in the mitochondrial matrix or the inner membrane electrochemical potential which are critical for import into the matrix or insertion into the inner membrane. We also explore several atypical IMS import pathways that are still not very well understood and are guided by poorly defined or completely unknown targeting peptides. Importantly, many of the IMS proteins are linked to several human diseases, and it is therefore crucial to understand how they reach their normal site of function in the IMS. In the final part of this review, we discuss current understanding of how such IMS protein underpin a large spectrum of human disorders.

International audience; In the mitochondria of healthy cells, Apoptosis-Inducing factor (AIF) is required for the optimal functioning of the respiratory chain machinery, mitochondrial integrity, cell survival, and proliferation. In all analysed species, it was revealed that the downregulation or depletion of AIF provokes mainly the post-transcriptional loss of respiratory chain Complex I protein subunits. Recent progress in the field has revealed that AIF fulfils its mitochondrial pro-survival function by interacting physically and functionally with CHCHD4, the evolutionarily-conserved human homolog of yeast Mia40. The redox-regulated CHCHD4/Mia40-dependent import machinery operates in the intermembrane space of the mitochondrion and controls the import of a set of nuclear-encoded cysteine-motif carrying protein substrates. In addition to their participation in the biogenesis of specific respiratory chain protein subunits, CHCHD4/Mia40 substrates are also implicated in the control of redox regulation, antioxidant response, translation, lipid homeostasis and mitochondrial ultrastructure and dynamics. Here, we discuss recent insights on the AIF/CHCHD4-dependent protein import pathway and review current data concerning the CHCHD4/Mia40 protein substrates in metazoan. Recent findings and the identification of disease-associated mutations in AIF or in specific CHCHD4/Mia40 substrates have highlighted these proteins as potential therapeutic targets in a variety of human disorders.

Porin is crucial for metabolite flux in mitochondria. In this issue of Molecular Cell, Sakaue et al. (2019) and Ellenrieder et al. (2019) describe an unexpected role for Porin in mitochondrial protein import by regulating the oligomeric state of the major protein import gate, the TOM complex, and the inner membrane insertion of metabolite carriers.

Mammalian glutamate dehydrogenase (GDH), a nuclear-encoded enzyme central to cellular metabolism, is among the most abundant mitochondrial proteins (constituting up to 10% of matrix proteins). To attain such high levels, GDH depends on very efficient mitochondrial targeting that, for human isoenzymes hGDH1 and hGDH2, is mediated by an unusually long cleavable presequence (N53). Here, we studied the mitochondrial transport of these proteins using isolated yeast mitochondria and human cell lines. We found that both hGDHs were very rapidly imported and processed in isolated mitochondria, with their presequences (N53) alone being capable of directing non-mitochondrial proteins into mitochondria. These presequences were predicted to form two α helices (α1: N 1–10; α2: N 16–32) separated by loops. Selective deletion of the α1 helix abolished the mitochondrial import of hGDHs. While the α1 helix alone had a very weak hGDH mitochondrial import capacity, it could direct efficiently non-mitochondrial proteins into mitochondria. In contrast, the α2 helix had no autonomous mitochondrial-targeting capacity. A peptide consisting of α1 and α2 helices without intervening sequences had GDH transport efficiency comparable with that of N53. Mutagenesis of the cleavage site blocked the intra-mitochondrial processing of hGDHs, but did not affect their mitochondrial import. Replacement of all three positively charged N-terminal residues (Arg3, Lys7 and Arg13) by Ala abolished import. We conclude that the synergistic interaction of helices α1 and α2 is crucial for the highly efficient import of hGDHs into mitochondria.

Evidence is accumulating that unrelated species have independently evolved the same way of importing proteins in their mitochondria.

The introduction of disulfide bonds in proteins of the mitochondrial intermembrane space (IMS) is fundamental for their folding and assembly. This oxidative folding process depends on the disulfide donor/import receptor Mia40 and the flavin adenine dinucleotide oxidase Erv1 and concerns proteins involved in mitochondrial biogenesis, respiratory complex assembly, and metal transfer.The recently determined structural basis of the interaction between Mia40 and some substrates provides a framework for the electron transfer process. A possible proofreading role for the cellular reductant glutathione has been proposed, while other studies suggest the association of Mia40 and Erv1 in dynamic multiprotein complexes in the IMS.The association of Mia40 with Erv1 and substrates in large multiprotein complexes is critical. Completion of substrate folding by additional disulfide bonds after initial binding to Mia40 remains unclear. Furthermore, a more general role for Mia40 in recognizing substrates targeted to other compartments, or even without specific cysteine motifs, remains an intriguing possibility.Dissecting a regulatory role of intramitochondrial protein complex organization and small redox-active molecules will be crucial for understanding oxidative folding in the IMS. This should have an impact on the physiology of human cells, as disease-linked mutations of key components of this process have been manifested, and their expression in stem cells appears crucial for development.

Mitochondria are organelles that play a central role in cellular metabolism, as they are responsible for processes such as iron/sulfur cluster biogenesis, respiration and apoptosis. Here, we describe briefly the various protein import pathways for sorting of mitochondrial proteins into the different subcompartments, with an emphasis on the targeting to the intermembrane space. The discovery of a dedicated redox-controlled pathway in the intermembrane space that links protein import to oxidative protein folding raises important questions on the redox regulation of this process. We discuss the salient features of redox regulation in the intermembrane space and how such mechanisms may be linked to the more general redox homeostasis balance that is crucial not only for normal cell physiology but also for cellular dysfunction.

The functional role of unstructured protein domains is an emerging field in the frame of intrinsically disordered proteins. The involvement of intrinsically disordered domains (IDDs) in protein targeting and biogenesis processes in mitochondria is so far not known. Here, we have characterized the structural/dynamic and functional properties of an IDD of the sulfhydryl oxidase ALR (augmenter of liver regeneration) located in the intermembrane space of mitochondria. At variance to the unfolded-to-folded structural transition of several intrinsically disordered proteins, neither substrate recognition events nor redox switch of its shuttle cysteine pair is linked to any such structural change. However, this unstructured domain performs a dual function in two cellular compartments: it acts (i) as a mitochondrial targeting signal in the cytosol and (ii) as a crucial recognition site in the disulfide relay system of intermembrane space. This domain provides an exciting new paradigm for IDDs ensuring two distinct functions that are linked to intracellular organelle targeting.

Members of the coiled-coil-helix-coiled-coil-helix (CHCH) domain-containing protein family that carry (CX9C) type motifs are imported into the mitochondrion with the help of the disulfide relay-dependent MIA import pathway. These evolutionarily conserved proteins are emerging as new cellular factors that control mitochondrial respiration, redox regulation, lipid homeostasis, and membrane ultrastructure and dynamics. We discuss recent insights on the activity of known (CX9C) motif-carrying proteins in mammals and review current data implicating the Mia40/CHCHD4 import machinery in the regulation of their mitochondrial import. Recent findings and the identification of disease-associated mutations in specific (CX9C) motif-carrying proteins have highlighted members of this family of proteins as potential therapeutic targets in a variety of human disorders.

The interaction of Mia40 with Erv1/ALR is central to the oxidative protein folding in the intermembrane space of mitochondria (IMS) as Erv1/ALR oxidizes reduced Mia40 to restore its functional state. Here we address the role of Mia40 in the import and maturation of Erv1/ALR. The C-terminal FAD-binding domain of Erv1/ALR has an essential role in the import process by creating a transient intermolecular disulfide bond with Mia40. The action of Mia40 is selective for the formation of both intra and intersubunit structural disulfide bonds of Erv1/ALR, but the complete maturation process requires additional binding of FAD. Both of these events must follow a specific sequential order to allow Erv1/ALR to reach the fully functional state, illustrating a new paradigm for protein maturation in the IMS.

Disulfide bond formation is a crucial step for oxidative folding and necessary for the acquisition of a protein's native conformation. Introduction of disulfide bonds is catalyzed in specialized subcellular compartments and requires the coordinated action of specific enzymes. The intermembrane space of mitochondria has recently been found to harbor a dedicated machinery that promotes the oxidative folding of substrate proteins by shuttling disulfide bonds. The newly identified oxidative pathway consists of the redox-regulated receptor Mia40 and the sulfhydryl oxidase Erv1. Proteins destined to the intermembrane space are trapped by a disulfide relay mechanism that involves an electron cascade from the incoming substrate to Mia40, then on to Erv1, and finally to molecular oxygen via cytochrome c. This thiol-disulfide exchange mechanism is essential for the import and for maintaining the structural stability of the incoming precursors. In this review we describe the mechanistic parameters that define the interaction and oxidation of the substrate proteins in light of the recent publications in the mitochondrial oxidative folding field.

A nine-residue intermembrane-targeting signal brings the active Cys of substrate proteins into contact with Mia40 oxidase for folding and import into mitochondria., Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step–specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys.

Mitochondria possess a dedicated-chaperone system in the intermembrane space, the small Tims that are ubiquitous in all eukaryotes from yeast to man. They escort membrane proteins to the outer or the inner membrane for proper insertion. These mitochondrial chaperones do not require external energy to perform their function and have structural similarities to other ATP-independent chaperones. Here, we discuss their structural properties and how these relate to their chaperoning function in the mitochondrial intermembrane space.

A number of nuclear encoded proteins are imported in to the intermembrane space of mitochondria where they adopt a coiled coil-helix-coiled coil-helix (CHCH) fold. Two disulfide bonds formed by twin CX3C or CX9C motifs stabilize this fold. Some of these proteins are also characterized at their N-termini by the presence of two additional cysteine residues which can perform oxidoreductase or metallochaperone functions or both. This fold represents the most ‘minimal’ oxidoreductase domain described so far.

The small Tims chaperone hydrophobic precursors across the mitochondrial intermembrane space. Tim9 and Tim10 form the soluble TIM10 complex that binds precursors exiting from the outer membrane. Tim12 functions downstream, as the only small Tim peripherally attached on the inner membrane. We show that Tim12 has an intrinsic affinity for inner mitochondrial membrane lipids, in contrast to the other small Tims. We find that the C-terminal end of Tim12 is essential in vivo. Its deletion crucially abolishes assembly of Tim12 in complexes with the other Tims. The N-terminal end contains targeting information and also mediates direct binding of Tim12 to the transmembrane segments of the carrier substrates. These results provide a molecular basis for the concept that the essential role of Tim12 relies on its unique assembly properties that allow this subunit to bridge the soluble and membrane-embedded translocases in the carrier import pathway.

Mitochondria were derived from intracellular bacteria and the mitochondrial intermembrane space is topologically equivalent to the bacterial periplasm. Both compartments contain ATP-independent chaperones involved in the transport of hydrophobic membrane proteins. The mitochondrial TIM (translocase of the mitochondrial inner membrane) 10 complex and the periplasmic chaperone SurA were examined in terms of evolutionary relation, structural similarity, substrate binding specificity and their function in transporting polypeptides for insertion into membranes. The two chaperones are evolutionarily unrelated; structurally, they are also distinct both in their characteristics, as determined by SAXS (small-angle X-ray scattering), and in pairwise structural comparison using the distance matrix alignment (DALILite server). Despite their structural differences, SurA and the TIM10 complex share a common binding specificity in Pepscan assays of substrate proteins. Comprehensive analysis of the binding on a total of 1407 immobilized 13-mer peptides revealed that the TIM10 complex, like SurA, does not bind hydrophobic peptides generally, but that both chaperones display selectivity for peptides rich in aromatic residues and with net positive charge. This common binding specificity was not sufficient for SurA to completely replace TIM10 in yeast cells in vivo. In yeast cells lacking TIM10, when SurA is targeted to the intermembrane space of mitochondria, it binds translocating substrate proteins, but fails to completely transfer the substrate to the translocase in the mitochondrial inner membrane. We suggest that SurA was incapable of presenting substrates effectively to the primitive TOM (translocase of the mitochondrial outer membrane) and TIM complexes in early mitochondria, and was replaced by the more effective small Tim chaperone.

Most of the mitochondrial inner-membrane proteins are generated without a presequence and their targeting depends on inadequately defined internal segments. Despite the numerous components of the import machinery identified by proteomics, the properties of hydrophobic import substrates remain poorly understood. Recent studies support several principles for these membrane proteins: first, they become organized into partially assembled forms within the translocon; second, they present noncontiguous targeting signals; and third, they induce conformational changes in translocase subunits, thereby mediating "assembly on demand" of the import machinery. It is possible that the energy needed for these proteins to pass across the outer membrane, to travel through the intermembrane space and to target the inner-membrane surface is provided by conformational changes involving import components that seem to have natively unfolded structures. Such structural malleability might render some of the translocase subunits more adept at driving the protein import process.

The yeast ADP/ATP carrier (AAC) is a mitochondrial protein that is targeted to the inner membrane via the TIM10 and TIM22 translocase complexes. AAC is devoid of a typical mitochondrial targeting signal and its targeting and insertion are thought to be guided by internal amino acid sequences. Here we show that AAC contains a cryptic matrix targeting signal that can target up to two thirds of the N-terminal part of the protein to the matrix. This event is coordinated by the TIM23 translocase and displays all the features of the matrix-targeting pathway. However, in the context of the whole protein, this signal is ‘masked’ and rendered non-functional as the polypeptide is targeted to the inner membrane via the TIM10 and TIM22 translocases. Our data suggest that after crossing the outer membrane the whole polypeptide chain of AAC is necessary to commit the precursor to the TIM22-mediated inner membrane insertion pathway.

Tim9 and Tim10 are essential components of the "small Tim" family of proteins that facilitate insertion of polytopic proteins at the inner mitochondrial membrane. The small Tims are themselves imported from the cytosol and are organized in specific translocation assemblies in the intermembrane space. Their conformational properties and how these influence the mechanism of assembly remain poorly understood. Moreover, the three-dimensional structure of the TIM10 complex is unknown. We have characterized the structural properties of these proteins in their free and assembled states using NMR, circular dichroism, and small angle x-ray scattering. We show that the free proteins are largely unfolded in their reduced assembly-incompetent state and molten globules in their oxidized assembly-competent state. Tim10 appears less structured than Tim9 in their respective free oxidized forms and undergoes a larger structural change than Tim9 upon complexation. The NMR data here demonstrates unequivocally that only the oxidized states of the Tim9 and Tim10 proteins are capable of forming a complex. Zinc binding stabilizes the reduced state against proteolysis without significantly affecting the secondary structure. Solution x-ray scattering was used to obtain a molecular envelope for the subunits individually and for their fully functional TIM10 complex. Ab initio shape reconstructions based on the scattering data has allowed us to obtain the first low resolution three-dimensional structure of the TIM10 complex. This is a novel structure that displays extensive surface hydrophobicity. The structure also provides an explanation for the escorting function of this non-ATP-powered chaperone particle.

The TIM10 chaperone facilitates the insertion of hydrophobic proteins at the mitochondrial inner membrane. Here we report the novel molecular mechanism of TIM10 assembly. This process crucially depends on oxidative folding in mitochondria and involves: (i) import of the subunits in a Cys-reduced and unfolded state; (ii) folding to an assembly-competent structure maintained by intramolecular disulfide bonding of their four conserved cysteines; and (iii) assembly of the oxidized zinc-devoid subunits to the functional complex. We show that intramolecular disulfide bonding occurs in vivo, whereas intermolecular disulfides observed in vitro are abortive intermediates in the assembly pathway. This novel mechanism of compartment-specific redox-regulated assembly is crucial for the formation of a functional TIM10 chaperone.

The TIM10 complex, composed of the homologous proteins Tim10 and Tim9, chaperones hydrophobic proteins inserted at the mitochondrial inner membrane. A salient feature of the TIM10 complex subunits is their conserved "twin CX3C" motif. Systematic mutational analysis of all cysteines of Tim10 showed that their underlying molecular defect is impaired folding (demonstrated by circular dichroism, aberrant homo-oligomer formation, and thiol trapping assays). As a result of defective folding, clear functional consequences were manifested in (i) complex formation with Tim9, (ii) chaperone activity, and (iii) import into tim9ts mitochondria lacking both endogenous Tim9 and Tim10. The organization of the four cysteines in intrachain disulfides was determined by trypsin digestion and mass spectrometry. The two distal CX3C motifs are juxtaposed in the folded structure and disulfide-bonded to each other rather than within each other, with an inner cysteine pair connecting Cys44 with Cys61 and an outer pair between Cys40 and Cys65. These cysteine pairs are not equally important for folding and assembly; mutations of the inner Cys are severely affected and form wrong, non-native disulfides, in contrast to mutations of the outer Cys that can still maintain the native inner disulfide pair and display weaker functional defects. Taken together these data reveal this specific intramolecular disulfide bonding as the crucial mechanism for Tim10 folding and show that the inner cysteine pair has a more prominent role in this process.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.

The TIM10 complex is localized in the mitochondrial intermembrane space and mediates insertion of hydrophobic proteins at the inner membrane. We have characterized TIM10 assembly and analyzed the structural properties of its subunits, Tim9 and Tim10. Both proteins are alpha-helical with a protease-resistant central domain, and each self-associates to form mainly dimers and trimers in solution. Tim9 and Tim10 bound to one another with submicromolar affinity in equimolar amounts and assembled in a stable, significantly extended complex that was indistinguishable from the native mitochondrial TIM10 complex. Importantly, the reconstituted TIM10 complex is functional because it bound to the physiological substrate ADP/ATP carrier and displayed chaperone activity in refolding the model substrate firefly luciferase. These data demonstrate that the individual subunits can exist as independent, dynamically self-associating proteins. Assembly into the thermodynamically stable hexameric complex is necessary for the TIM10 chaperone function.

Import of the ADP/ATP carrier (AAC) into mitochondria requires the soluble TIM10 complex to cross the intermembrane space. We report here that Tim9 and Tim10 purified from Escherichia coli can form a complex of the same size as the endogenous complex from yeast mitochondria. This shows that no other mitochondrial protein is required for the formation of the TIM10 complex. Co-expression of both proteins rendered Tim9 more soluble and allowed purification of the reconstituted complex in a single step. Urea/EDTA treatment of recombinant Tim10 allowed its import into tim10-ts mitochondria that lack endogenous Tim10 and cannot import AAC. In this way, we were able to (i) reconstitute the TIM10 complex in the intermembrane space and (ii) restore import of AAC to almost wild-type levels. The reconstituted TIM10 complex not only facilitated passage of AAC across the outer membrane but also ensured its accurate membrane insertion. We conclude that the TIM10 complex can be formed exclusively from Tim9 and Tim10 and that the reconstituted complex efficiently restores AAC import in a strain lacking the TIM10 complex.

The protein import pathway that targets proteins to the mitochondrial matrix has been extensively characterized in the past 15 years. Variations of this import pathway account for the sorting of proteins to other compartments as well, but the insertion of integral inner membrane proteins lacking a presequence is mediated by distinct translocation machinery. This consists of a complex of Tim9 and Tim10, two homologous, Zn2+-binding proteins that chaperone the passage of the hydrophobic precursor across the aqueous inter-membrane space. The precursor is then targeted to another, inner-membrane-bound, complex of at least five subunits that facilitates insertion. Biochemical and genetic experiments have identified the key components of this process; we are now starting to understand the molecular mechanism. This review highlights recent advances in this new membrane protein insertion pathway.

The mitochondrial inner membrane of most organisms studied so far contains about a dozen proteins made by the mitochondrial genetic system and on the order of 10 proteins imported from the cytoplasm. This review briefly describes the two major pathways by which proteins made inside or outside the mitochondria are inserted into the inner membrane. These pathways were discovered only during the past few years; one of them involves a novel family of chaperone-like proteins in the mitochondrial intermembrane space. Import of proteins from the cytoplasm into mitochondria occurs by distinct routes that are dictated by the properties and final destination of each protein within the mitochondria (1–3). However, most of the different routes described until recently have been variants of the general “matrix pathway.” In this pathway, a protein carrying a positively charged amphiphilic helix at its N terminus is bound by cytosolic chaperones, delivered to an array of import receptors on the mitochondrial surface, and then transported across two distinct, hetero-oligomeric protein translocation channels, the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. During transport, the two complexes are transiently linked by the translocating precursor chain (4, 5). Transport across the TOM complex appears to be driven by binding of the basic N-terminal “presequence” to a series of acidic receptor domains of increasing avidity (“acid chain hypothesis”) (6, 7); insertion of the presequence into the TIM23 complex is driven electrophoretically by the potential across the inner membrane; and complete transport of the precursor into the matrix is driven by an ATP-powered import motor attached to the inner mouth of the TIM23 complex. Proteins destined for compartments other than the matrix can diverge from the general matrix pathway at different points (8–10). Some proteins destined for the outer membrane can arrest in the TOM complex and then escape into the outer membrane (11); and some intermembrane space proteins can arrest in the TIM23 complex and then be proteolytically released into the intermembrane space (8). A few years ago it seemed likely that a variation of this general matrix pathway would also sort proteins to the inner membrane. According to this view, a protein destined for the inner membrane would arrest in the TIM23 complex and escape “sidewise” into the phospholipid bilayer of the inner membrane. So far, however, no inner membrane protein has been shown to follow such a pathway. Arrest in the TIM23 complex has only been found for some proteins destined for the intermembrane space.

Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane. We now identify Tim9p, another essential component of this import pathway. Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and Tim10p co-purify in successive chromatographic fractionations and co-immunoprecipitated with each other. Tim9p can be cross-linked to a partly translocated carrier protein. A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p. The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p. A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants. Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.

In order to reach the inner membrane of the mitochondrion, multispanning carrier proteins must cross the aqueous intermembrane space. Two essential proteins of that space, Tim10p and Tim12p, were shown to mediate import of multispanning carriers into the inner membrane. Both proteins formed a complex with the inner membrane protein Tim22p. Tim10p readily dissociated from the complex and was required to transport carrier precursors across the outer membrane; Tim12p was firmly bound to Tim22p and mediated the insertion of carriers into the inner membrane. Neither protein was required for protein import into the other mitochondrial compartments. Both proteins may function as intermembrane space chaperones for the highly insoluble carrier proteins.

Mitochondria biogenesis is a fundamental process for the organization and normal function of all cells. Since the majority of mitochondrial proteins are synthesized in the cytosol, protein import is the major mechanism for mitochondria biogenesis. We describe the different pathways that ensure correct targeting and intra mitochondrial sorting of mitochondrial proteins. The import process of several proteins of the mitochondrial intermembrane space relies on the Mitochondrial Import and Assembly 40 and Essential for respiration and vegetative growth 1 (Erv1) proteins that together constitute the oxidative folding machinery of the mitochondrial intermembrane space. Recent work has implicated the FAD-oxidase protein Erv1 (ad its human homolog Augmenter of Liver Regeneration) as an anti-apoptotic factor in mammalian cells (including neuronal cells) that undergo Reactive Oxygen Species-triggered apoptosis. The different roles of this protein as a key factor in mitochondria biogenesis, iron-sulfur cluster biogenesis and in neuronal protection against apoptosis are discussed.

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.

Erv1 and Mia40 constitute the two important components of the disulfide relay system that mediates oxidative protein folding in the mitochondrial intermembrane space. Mia40 is the import receptor that recognizes the substrates introducing disulfide bonds while it is reduced. A key function of Erv1 is to recycle Mia40 to its active oxidative state. Our aims here were to dissect the domain of Erv1 that mediates the protein-protein interaction with Mia40 and to investigate the interactions between the shuttle domain of Erv1 and its catalytic core and their relevance for the interaction with Mia40. We purified these domains separately as well as cysteine mutants in the shuttle and the active core domains. The noncovalent interaction of Mia40 with Erv1 was measured by isothermal titration calorimetry, whereas their covalent mixed disulfide intermediate was analyzed in reconstitution experiments in vitro and in organello. We established that the N-terminal shuttle domain of Erv1 is necessary and sufficient for interaction to occur. Furthermore, we provide direct evidence for the intramolecular electron transfer from the shuttle cysteine pair of Erv1 to the core domain. Finally, we reconstituted the system by adding in trans the N- and C- terminal domains of Erv1 together with its substrate Mia40.

The MIA40 pathway is a novel import pathway in mitochondria specific for cysteine-rich proteins of the intermembrane space (IMS). The newly synthesised precursors are trapped in the IMS by a disulfide relay mechanism that involves introduction of disulfides from the sulfhydryl oxidase Erv1 to the redox-regulated import receptor Mia40 and then on to the substrate. This thiol-disulfide exchange mechanism is essential for the import and oxidative folding of the incoming cysteine-rich substrate proteins. In this chapter we will describe the experimental methods that have been developed in order to study and characterise disulfide-trapped intermediates in yeast mitochondria.

The small Tims are chaperones that facilitate insertion of hydrophobic precursors into the inner mitochondrial membrane. We purified Tim12 and found it forms dimers that bind to Tim9. In this interaction, Tim12 undergoes structural changes that may be important for transport of its substrates in the mitochondrial carrier import pathway.

Tim9 and Tim10 belong to the small Tim family of mitochondrial ATP-independent chaperones. They are organised in a specific hetero-oligomeric complex (TIM10) that escorts polytopic proteins into the mitochondrial inner membrane. The contributions of the individual subunits to the assembly and function of the TIM10 complex remain poorly understood. Here, we show that substrate recognition and assembly of the complex are mediated by distinct domains of the subunits. These are unrelated to the characteristic “twin CX3C” motif that is present in all small Tims and ensures proper folding of the unassembled subunits. Specifically, we show that substrate recognition is achieved by the Tim10 subunit, whilst Tim9 serves a more structural role. The N-terminal domain of Tim10 is a substrate sensor whilst its C-terminal part is essential for complex formation. By contrast, both N and C-terminal domains of Tim9 are involved in the stability of the complex.

The ADP/ATP carrier (AAC) that facilitates the translocation of ATP made in mitochondria is inserted at the inner mitochondrial membrane by the TIM10-TIM22 protein import system. Here we addressed the state of the AAC precursor during insertion (stage IV of import) and identified residues of the carrier important for dimerization. By a combination of (i) import of a mix of His-tagged and untagged versions of AAC either 35S-labeled or unlabeled, (ii) import of a tandem covalent dimer AAC into wild-type mitochondria, and (iii) import of monomeric AAC into mitochondria expressing only the tandem covalent dimer AAC, we found that the stage IV intermediate is a monomer, and this stage is probably the rate-limiting step of insertion in the membrane. Subsequent dimerization occurs extremely rapidly (within less than a minute). The incoming monomer dimerizes with monomeric endogenous AAC suggesting that the AAC dimer is very dynamic. Conserved Cys residues were found not to affect insertion significantly, but they are crucial for the dimerization process to obtain a functional carrier.

This is a series of papers (three reviews and five regular papers) focused on Mitochondria from authors contributing to two FEBS courses on Mitochondria held in 2012. These papers focus on: (i) mitochondrial biogenesis (regulation by phosphorylation, import of SOD1 and Mia40, mitophagy and tRNA variants) or (ii) mitochondrial pathology (ischaemia, obesity and Parkinson's disease).

Fragments of the 25OkDa SI subunit of the Clostridium thermocellum cellulosome were obtained by protease-induced or spontaneous degradation. All detectable fragments, down to a mass of about 30 kDa, retained the ability to bind to 125I-labelled endoglucanase CelD, one of the catalytic subunits of the cellulosome. Several fragments were able to bind both to cellulose and to CElD. However, some fragments that could still bind to CelD did not have the ability to bind to cellulose. Therefore, S1, a putative scaffolding protein of the cellulosome, is likely to carry two separate types of domains, one of which binds to cellulose, while the other type binds to the various catalytic subunits of the complex.

The formation of cytoplasmic inclusion bodies by Escherichia coli overproducing Clostridium thermocellum endoglucanase D (EGD) was investigated. EGD was found in inclusion bodies as a 68 kDa form, whereas the size of the cytoplasmic form was 65 kDa. Upon solubilization with urea followed by dialysis, the 68 kDa form was converted to the 65 kDa species. Proteolysis occurred within the COOH-terminal, reiterated region of the 68 kDa form, which is conserved among most C. thermocellum endoglucanase, but is not required for catalytic activity. The specific activity of the enzyme embedded in inclusion bodies was close to that of the purified protein. Thus, inclusion body formation does not involve denaturation of the catalytic domain of EGD, but more likely, the participation of the reiterated, conserved region in intermolecular interactions.

The function of the non-catalytic, duplicated segment found in C. thermocellum cellulases was investigated. Rabbit antibodies reacting with the duplicated segment of endoglucanase CelD cross-reacted with a variety of cellulosome components ranging between 50 and 100 kDa. 125I-labeled forms of CelD and of xylanase XynZ carrying the duplicated segment bound to a set of cellulosome proteins ranging between 66 and 250 kDa, particularly to the 250 kDa SL (or S1) subunit. 125I-labeled forms of CelD and XynZ devoid of the duplicated segment failed to bind to any cellulosome protein. The duplicated segment appears thus to serve to anchor the various cellulosome subunits to the complex by binding to SL, which may be a scaffolding element of the cellulosome.

Mitochondrial NADH-cytochrome b5 reductase (Mcr1p) is encoded by a single nuclear gene and imported into two different submitochondrial compartments: the outer membrane and the intermembrane space. We now show that the amino-terminal 47 amino acids suffice to target the Mcr1 protein to both destinations. The first 12 residues of this sequence function as a weak matrix-targeting signal; the remaining residues are mostly hydrophobic and serve as an intramitochondrial sorting signal for the outer membrane and the intermembrane space. A double point mutation within the hydrophobic region of the targeting sequence virtually abolishes the ability of the precursor to be inserted into the outer membrane but increases the efficiency of transport into the intermembrane space. Import of Mcr1p into the intermembrane space requires an electrochemical potential across the inner membrane, as well as ATP in the matrix, and is strongly impaired in mitochondria lacking Tom7p or Tim11p, two components of the translocation machineries in the outer and inner mitochondrial membranes, respectively. These results indicate that intramitochondrial sorting of the Mcr1 protein is mediated by specific interactions between the bipartite targeting sequence and components of both mitochondrial translocation systems.