Mia40, a novel factor for protein import into the intermembrane space of mitochondria is able to bind metal ions

Many proteins located in the intermembrane space (IMS) of mitochondria are characterized by a low molecular mass, contain highly conserved cysteine residues and coordinate metal ions. Studies on one of these proteins, Tim13, revealed that net translocation across the outer membrane is driven by metal-dependent folding in the IMS . We have identified an essential component, Mia40/Tim40/Ykl195w, with a highly conserved domain in the IMS that is able to bind zinc and copper ions. In cells lacking Mia40, the endogenous levels of Tim13 and other metal-binding IMS proteins are strongly reduced due to the impaired import of these proteins. Furthermore, Mia40 directly interacts with newly imported Tim13 protein. We conclude that Mia40 is the first essential component of a specific translocation pathway of metal-binding IMS proteins.     

A disulfide relay system in the intermembrane space of mitochondria that mediates protein import

We describe here a pathway for the import of proteins into the intermembrane space (IMS) of mitochondria. Substrates of this pathway are proteins with conserved cysteine motifs, which are critical for import. After passage through the TOM channel, these proteins are covalently trapped by Mia40 via disulfide bridges. Mia40 contains cysteine residues, which are oxidized by the sulfhydryl oxidase Erv1. Depletion of Erv1 or conditions reducing Mia40 prevent protein import. We propose that Erv1 and Mia40 function as a disulfide relay system that catalyzes the import of proteins into the IMS by an oxidative folding mechanism. The existence of a disulfide exchange system in the IMS is unexpected in view of the free exchange of metabolites between IMS and cytosol via porin channels. We suggest that this process reflects the evolutionary origin of the IMS from the periplasmic space of the prokaryotic ancestors of mitochondria.     

The essential mitochondrial protein Erv1 cooperates with Mia40 in biogenesis of intermembrane space proteins

The proteins of the mitochondrial intermembrane space (IMS) are encoded by nuclear genes and synthesized on cytosolic ribosomes. While some IMS proteins are imported by the classical presequence pathway that involves the membrane potential deltapsi across the inner mitochondrial membrane and proteolytic processing to release the mature protein to the IMS, the import of numerous small IMS proteins is independent of a deltapsi and does not include proteolytic processing. The biogenesis of small IMS proteins requires an essential mitochondrial IMS import and assembly protein, termed Mia40. Here, we show that Erv1, a further essential IMS protein that has been reported to function as a sulfhydryl oxidase and participate in biogenesis of Fe/S proteins, is also required for the biogenesis of small IMS proteins. We generated a temperature-sensitive yeast mutant of Erv1 and observed a strong reduction of the levels of small IMS proteins upon shift of the cells to non-permissive temperature. Isolated erv1-2 mitochondria were selectively impaired in import of small IMS proteins while protein import pathways to other mitochondrial subcompartments were not affected. Small IMS precursor proteins remained associated with Mia40 in erv1-2 mitochondria and were not assembled into mature oligomeric complexes. Moreover, Erv1 associated with Mia40 in a reductant-sensitive manner. We conclude that two essential proteins, Mia40 and Erv1, cooperate in the assembly pathway of small proteins of the mitochondrial IMS.     

A disulfide relay system in mitochondria

In this issue of Cell, show that there is a disulfide relay system in the intermembrane space (IMS) of mitochondria that is comprised of the proteins Mia40 and Erv1. This disulfide relay system promotes the import and oxidative folding of proteins. Oxidized Mia40 traps newly imported proteins through mixed disulfide bridges. Subsequent isomerization of these disulfide bridges allows the imported protein to be folded in the IMS. The reduced Mia40 generated is then reoxidized by the sulfhydryl oxidase Erv1, promoting the next round of disulfide exchange. The new work clarifies the molecular function of Mia40 and reveals Mia40 to be the first physiological substrate for the FAD-linked Erv1.     

Targeting and maturation of Erv1/ALR in the mitochondrial intermembrane space

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.     

The Erv1-Mia40 disulfide relay system in the intermembrane space of mitochondria

The compartment between the outer and the inner membranes of mitochondria, the intermembrane space (IMS), harbours a variety of proteins that contain disulfide bonds. Many of these proteins possess a conserved twin Cx(3)C motif or twin Cx(9)C motif. Recently, a disulfide relay system in the IMS has been identified which consists of two essential components, the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40/Tim40. The disulfide relay system drives the import of these cysteine-rich proteins into the IMS of mitochondria by an oxidative folding mechanism. In order to enable Mia40 to perform the oxidation of substrate proteins, the sulfhydryl oxidase Erv1 mediates the oxidation of Mia40 in a disulfide transfer reaction. To recycle Erv1 into its oxidized form, electrons are transferred to cytochrome c connecting the disulfide relay system to the electron transport chain of mitochondria. Despite the lack of homology of the components, the disulfide relay system in the IMS resembles the oxidation system in the periplasm of bacteria presumably reflecting the evolutionary origin of the IMS from the bacterial periplasm.     

The Disulfide Relay System of Mitochondria Is Required for the Biogenesis of Mitochondrial Ccs1 and Sod1

Cells protect themselves against oxygen stress and reactive oxygen species. An important enzyme in this process is superoxide dismutase, Sod1, which converts superoxide radicals into water and hydrogen peroxide. The biogenesis of functional Sod1 is dependent on its copper chaperone, Ccs1, which introduces a disulfide bond and a copper ion into Sod1. Ccs1 and Sod1 are present in the cytosol but are also found in the mitochondrial intermembrane space (IMS), the compartment between the outer and the inner membrane of mitochondria. Ccs1 mediates mitochondrial localization of Sod1.Here, we report on the biogenesis of the fractions of Ccs1 and Sod1 present in mitochondria of Saccharomyces cerevisiae. The IMS of mitochondria harbors a disulfide relay system consisting of the import receptor Mia40 and the thiol oxidase Erv1, which drives the import of substrates with conserved cysteine residues arranged in typical twin Cx₃C and twin Cx₉C motifs. We show that depletion of Mia40 results in decreased levels of Ccs1 and Sod1. On the other hand, overexpression of Mia40 increased the mitochondrial fraction of both proteins. In addition, the import rates of Ccs1 were enhanced by increased levels of Mia40 and reduced upon depletion of Mia40. Mia40 forms mixed disulfides with Ccs1, suggesting a role of Mia40 for the generation of disulfide bonds in Ccs1. We suggest that the disulfide relay system transfers disulfide bonds via Mia40 to Ccs1, which then shuttles disulfide bonds to Sod1. In conclusion, the disulfide relay system is crucial for the import of Ccs1, thereby affecting the transport of Sod1, and it can control the distribution of Ccs1 and Sod1 between the IMS of mitochondria and the cytosol.     

A Highlights from MBoC Selection: Kinetic control by limiting glutaredoxin amounts enables thiol oxidation in the reducing mitochondrial intermembrane space

The mitochondrial intermembrane space (IMS) harbors an oxidizing machinery that drives import and folding of small cysteine-containing proteins without targeting signals. The main component of this pathway is the oxidoreductase Mia40, which introduces disulfides into its substrates. We recently showed that the IMS glutathione pool is maintained as reducing as that of the cytosol. It thus remained unclear how equilibration of protein disulfides with the IMS glutathione pool is prevented in order to allow oxidation-driven protein import. Here we demonstrate the presence of glutaredoxins in the IMS and show that limiting amounts of these glutaredoxins provide a kinetic barrier to prevent the thermodynamically feasible reduction of Mia40 substrates by the IMS glutathione pool. Moreover, they allow Mia40 to exist in a predominantly oxidized state. Consequently, overexpression of glutaredoxin 2 in the IMS results in a more reduced Mia40 redox state and a delay in oxidative folding and mitochondrial import of different Mia40 substrates. Our findings thus indicate that carefully balanced glutaredoxin amounts in the IMS ensure efficient oxidative folding in the reducing environment of this compartment.     

Biogenesis of the essential Tim9-Tim10 chaperone complex of mitochondria: site-specific recognition of cysteine residues by the intermembrane space receptor Mia40

The mitochondrial intermembrane space (IMS) contains an essential machinery for protein import and assembly (MIA). Biogenesis of IMS proteins involves a disulfide relay between precursor proteins, the cysteine-rich IMS protein Mia40 and the sulfhydryl oxidase Erv1. How precursor proteins are specifically directed to the IMS has remained unknown. Here we systematically analyzed the role of cysteine residues in the biogenesis of the essential IMS chaperone complex Tim9-Tim10. Although each of the four cysteines of Tim9, as well as of Tim10, is required for assembly of the chaperone complex, only the most amino-terminal cysteine residue of each precursor is critical for translocation across the outer membrane and interaction with Mia40. Mia40 selectively recognizes cysteine-containing IMS proteins in a site-specific manner in organello and in vitro. Our results indicate that Mia40 acts as a trans receptor in the biogenesis of mitochondrial IMS proteins.     

Erv1 mediates the Mia40-dependent protein import pathway and provides a functional link to the respiratory chain by shuttling electrons to cytochrome c

Unlike matrix-targeted or inner membrane proteins, those that are targeted to the mitochondrial intermembrane space (IMS) do not require ATP or the inner membrane electrochemical potential. Their import is mediated primarily by the essential IMS protein Mia40/Tim40. Here, we show that the mitochondrial flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidase Erv1 (essential for respiration and vegetative growth 1) plays a central role in the biogenesis of small, cysteine proteins of the IMS that are import substrates for Mia40. In a temperature-sensitive strain of Erv1, steady-state levels of small translocases of the inner membrane (Tims) are specifically affected when cells are grown at the non-permissive temperature. Furthermore, mitochondria isolated from the erv1-ts show a specific import and assembly defect for the small Tims but not in any other protein import pathway. Erv1 does not directly oxidise the small Tims, as thiol trapping assays show that the small Tims can still be oxidised in erv1-ts cells grown at the non-permissive temperature and in isolated mitochondria from this strain. Moreover, addition of pure Erv1 into erv1-ts mitochondria lacking the endogenous protein restores import and assembly of the small Tims only to an extent, arguing for a cascade of interactions with Erv1 rather than for a direct interaction of Erv1 with the small Tims. Cytochrome c (cyt c) is the in vivo oxidase for Erv1, as yeast cells mutated in cyt c cannot grow under anaerobic conditions. Therefore, Erv1 functionally links the Mia40-dependent import pathway to the Mia40-independent cyt c import pathway transferring electrons from the incoming precursors to cyt c as an acceptor. In this context, the protein import process is linked to the respiratory chain via the communication of Erv1 with cyt c.     

Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins

Mitochondria import nuclear-encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.     

The sulfhydryl oxidase Erv1 is a substrate of the Mia40-dependent protein translocation pathway

The thiol oxidase Erv1 and the redox-regulated receptor Mia40/Tim40 are components of a disulfide relay system which mediates import of proteins into the intermembrane space (IMS) of mitochondria. Here we report that Erv1 requires Mia40 for its import into mitochondria. After passage across the translocase of the mitochondrial outer membrane Erv1 interacts via disulfide bonds with Mia40. Erv1 does not contain twin “CX₃C” or twin “CX₉C” motifs which are crucial for import of typical substrates of this pathway and it does not need two “CX₂C” motifs for import into mitochondria. Thus, Erv1 represents an unusual type of substrate of the Mia40-dependent import pathway.     

Mitochondrial disulfide relay: redox-regulated protein import into the intermembrane space

99% of all mitochondrial proteins are synthesized in the cytosol, from where they are imported into mitochondria. In contrast to matrix proteins, many proteins of the intermembrane space (IMS) lack presequences and are imported in an oxidation-driven reaction by the mitochondrial disulfide relay. Incoming polypeptides are recognized and oxidized by the IMS-located receptor Mia40. Reoxidation of Mia40 is facilitated by the sulfhydryl oxidase Erv1 and the respiratory chain. Although structurally unrelated, the mitochondrial disulfide relay functionally resembles the Dsb (disufide bond) system of the bacterial periplasm, the compartment from which the IMS was derived 2 billion years ago.     

Distinctive biochemistry in the trypanosome mitochondrial intermembrane space suggests a model for stepwise evolution of the MIA pathway for import of cysteine-rich proteins

Mia40-dependent disulphide bond exchange is used by animals, yeast, and probably plants for import of small, cysteine-rich proteins into the mitochondrial intermembrane space (IMS). During import, electrons are transferred from the imported substrate to Mia40 then, via the sulphydryl oxidase Erv1, into the respiratory chain. Curiously, however, there are protozoa which contain substrates for Mia40-dependent import, but lack Mia40. There are also organisms where Erv1 is present in the absence of respiratory chain components. In accommodating these and other relevant observations pertaining to mitochondrial cell biology, we hypothesise that the ancestral IMS import pathway for disulphide-bonded proteins required only Erv1 (but not Mia40) and identify parasites in which O(2) is the likely physiological oxidant for Erv1.     

Atp23 biogenesis reveals a chaperone‐like folding activity of Mia40 in the IMS of mitochondria

Mia40 is a recently identified oxidoreductase in the intermembrane space (IMS) of mitochondria that mediates protein import in an oxidation‐dependent reaction. Substrates of Mia40 that were identified so far are of simple structure and receive one or two disulphide bonds. Here we identified the protease Atp23 as a novel substrate of Mia40. Atp23 contains ten cysteine residues which are oxidized during several rounds of interaction with Mia40. In contrast to other Mia40 substrates, oxidation of Atp23 is not essential for its import; an Atp23 variant in which all ten cysteine residues were replaced by serine residues still accumulates in mitochondria in a Mia40‐dependent manner. In vitro Mia40 can mediate the folding of wild‐type Atp23 and prevents its aggregation. In these reactions, the hydrophobic substrate‐binding pocket of Mia40 was found to be essential for its chaperone‐like activity. Thus, Mia40 plays a much broader role in import and folding of polypeptides than previously expected and can serve as folding factor for proteins with complex disulphide patterns as well as for cysteine‐free polypeptides.     

The zinc-binding protein Hot13 promotes oxidation of the mitochondrial import receptor Mia40

A disulphide relay system mediates the import of cysteine-containing proteins into the intermembrane space of mitochondria. This system consists of two essential proteins, Mia40 and Erv1, which bind to newly imported proteins by disulphide transfer. A third component, Hot13, was proposed to be important in the biogenesis of cysteine-rich proteins of the intermembrane space, but the molecular function of Hot13 remained unclear. Here, we show that Hot13, a conserved zinc-binding protein, interacts functionally and physically with the import receptor Mia40. It improves the Erv1-dependent oxidation of Mia40 both in vivo and in vitro. As a consequence, in mutants lacking Hot13, the import of substrates of Mia40 is impaired, particularly in the presence of zinc ions. In mitochondria as well as in vitro, Hot13 can be functionally replaced by zinc-binding chelators. We propose that Hot13 maintains Mia40 in a zinc-free state, thereby facilitating its efficient oxidation by Erv1.     

Genome-wide analysis of eukaryotic twin CX9C proteins

Twin CX(9)C proteins are eukaryotic proteins that derive their name from their characteristic motif, consisting of two pairs of cysteines that form two disulfide bonds stabilizing a coiled coil-helix-coiled coil-helix (CHCH) fold. The best characterized of these proteins are Cox17, a copper chaperone acting in cytochrome c oxidase biogenesis, and Mia40, the central component of a system for protein import into the mitochondrial inter-membrane space (IMS). However, the range of possible functions for these proteins is unclear. Here, we performed a systematic search of twin CX(9)C proteins in eukaryotic organisms, and classified them into groups of putative homologues, by combining bioinformatics methods with literature analysis. Our results suggest that the functions of most twin CX(9)C proteins vary around the common theme of playing a scaffolding role, which can tie their observed roles in mitochondrial structure and function. This study will enhance the present annotation of eukaryotic proteomes, and will provide a rational basis for future experimental work aimed at a deeper understanding of this remarkable class of proteins.     

Oxidative folding of small Tims is mediated by site-specific docking onto Mia40 in the mitochondrial intermembrane space

Oxidative folding in the mitochondrial intermembrane space (IMS) is crucial for the import of certain cysteine-rich IMS proteins. The essential proteins Mia40 and Erv1 are key components for this mechanism functioning as a disulphide protein cascade that is functionally linked to the respiratory chain by shuttling electrons onto CytC. The subunits of the chaperone complex Tim9-Tim10 require Mia40 for their biogenesis. Previously, it was shown that the four cysteines of Tim10 are crucial for folding and assembly, that they are connected intramolecularly into an inner and an outer disulphide bridge, and that the inner disulphide has a more prominent role in these processes. Here we show that interaction with Mia40 is a site-specific event: (i) the N-terminal first cysteine of the precursor is crucial for docking onto Mia40 via a mixed disulphide; (ii) release is triggered by disulphide pairing of the C-terminal cysteine onto the N-terminal one; and (iii) formation of the inner disulphide between the second and third cysteines apparently precedes the release reaction and is critical for assembly with Tim9. The Tim10-Mia40 interaction is independent of divalent cations, any other mitochondrial proteins or membranes, and is shown to occur efficiently in organello and in vitro.     

Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the inner membrane of mitochondria

The mitochondrial intermembrane space assembly (MIA) pathway is generally considered to be dedicated to the redox-dependent import and biogenesis of proteins localized to the intermembrane space of mitochondria. The oxidoreductase Mia40 is a central component of the pathway responsible for the transfer of disulfide bonds to intermembrane space precursor proteins, causing their oxidative folding. Here we present the first evidence that the function of Mia40 is not restricted to the transport and oxidative folding of intermembrane space proteins. We identify Tim22, a multispanning membrane protein and core component of the TIM22 translocase of inner membrane, as a protein with cysteine residues undergoing oxidation during Tim22 biogenesis. We show that Mia40 is involved in the biogenesis and complex assembly of Tim22. Tim22 forms a disulfide-bonded intermediate with Mia40 upon import into mitochondria. Of interest, Mia40 binds the Tim22 precursor also via noncovalent interactions. We propose that Mia40 not only is responsible for disulfide bond formation, but also assists the Tim22 protein in its integration into the inner membrane of mitochondria.     

Targeting and Import Mechanism of Coiled-coil Helix Coiled-coil Helix Domain-containing Protein 3 (ChChd3) into the Mitochondrial Intermembrane Space

Coiled-coil helix coiled-coil helix domain-containing protein 3 (ChChd3) is a mitochondrial inner membrane (IM) protein facing toward the intermembrane space (IMS). In the IMS, ChChd3 complexes with multiple proteins at the crista junctions and contact sites and plays a key role in maintaining crista integrity. ChChd3 is myristoylated at the N terminus and has a CHCH domain with twin CX₉C motifs at its C terminus. The CHCH domain proteins are traditionally imported and trapped in the IMS by using a disulfide relay system mediated by Mia40 and Erv1. In this study, we systematically analyzed the role of the myristoylation and the CHCH domain in the import and mitochondrial localization of ChChd3. Based on our results, we predict that myristoylation promotes binding of ChChd3 to the outer membrane and that the CHCH domain translocates the protein across the outer membrane. By analysis of the CHCH domain cysteine mutants, we further show that they have distinct roles in binding to Mia40 in the IMS and proper folding of the protein. The transient disulfide-bonded intermediate with Mia40 is formed preferentially between the second cysteine in helix 1, Cys¹⁹³, and the active site cysteine in Mia40, Cys⁵⁵. Although each of the four cysteines is essential for folding of the protein and binding to mitofilin and Sam50, they are not involved in import. Together our results indicate that both the myristoylation and the CHCH domain are essential for the import and mitochondrial localization of ChChd3. Once imported, ChChd3 binds to Mia40 for further folding and assembly into macromolecular complexes.