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.     

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 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 Ccs1 contains a structural disulfide bond crucial for the import of this unconventional substrate by the disulfide relay system

The copper chaperone for superoxide dismutase 1 (Ccs1) provides an important cellular function against oxidative stress. Ccs1 is present in the cytosol and in the intermembrane space (IMS) of mitochondria. Its import into the IMS depends on the Mia40/Erv1 disulfide relay system, although Ccs1 is, in contrast to typical substrates, a multidomain protein and lacks twin Cx(n)C motifs. We report on the molecular mechanism of the mitochondrial import of Saccharomyces cerevisiae Ccs1 as the first member of a novel class of unconventional substrates of the disulfide relay system. We show that the mitochondrial form of Ccs1 contains a stable disulfide bond between cysteine residues C27 and C64. In the absence of these cysteines, the levels of Ccs1 and Sod1 in mitochondria are strongly reduced. Furthermore, C64 of Ccs1 is required for formation of a Ccs1 disulfide intermediate with Mia40. We conclude that the Mia40/Erv1 disulfide relay system introduces a structural disulfide bond in Ccs1 between the cysteine residues C27 and C64, thereby promoting mitochondrial import of this unconventional substrate. Thus the disulfide relay system is able to form, in addition to double disulfide bonds in twin Cx(n)C motifs, single structural disulfide bonds in complex protein domains.     

The disulfide relay system of mitochondria is connected to the respiratory chain

All proteins of the intermembrane space of mitochondria are encoded by nuclear genes and synthesized in the cytosol. Many of these proteins lack presequences but are imported into mitochondria in an oxidation-driven process that relies on the activity of Mia40 and Erv1. Both factors form a disulfide relay system in which Mia40 functions as a receptor that transiently interacts with incoming polypeptides via disulfide bonds. Erv1 is a sulfhydryl oxidase that oxidizes and activates Mia40, but it has remained unclear how Erv1 itself is oxidized. Here, we show that Erv1 passes its electrons on to molecular oxygen via interaction with cytochrome c and cytochrome c oxidase. This connection to the respiratory chain increases the efficient oxidation of the relay system in mitochondria and prevents the formation of toxic hydrogen peroxide. Thus, analogous to the system in the bacterial periplasm, the disulfide relay in the intermembrane space is connected to the electron transport chain of the inner membrane.     

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.     

Functional characterization of Mia40p, the central component of the disulfide relay system of the mitochondrial intermembrane space

Mia40p and Erv1p are components of a translocation pathway for the import of cysteine-rich proteins into the intermembrane space of mitochondria. We have characterized the redox behavior of Mia40p and reconstituted the disulfide transfer system of Mia40p by using recombinant functional C-terminal fragment of Mia40p, Mia40C, and Erv1p. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC motif. The second and the third bonds belong to the twin CX(9)C motif and bridge the cysteine residues of two CX(9)C segments. In contrast to the stabilizing disulfide bonds of the twin CX(9)C motif, the first disulfide bond was easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond as well as fully reduced Mia40C were oxidized by Erv1p in vitro. In the course of this reaction, mixed disulfides of Mia40C and Erv1p were formed. Reoxidation of fully reduced Mia40C required the presence of the first two cysteine residues in Mia40C. However, efficient reoxidation of a Mia40C variant containing only the cysteine residues of the twin CX(9)C motif was observed when in addition to Erv1p low amounts of wild type Mia40C were present. In the reconstituted system the thiol oxidase Erv1p was sufficient to transfer disulfide bonds to Mia40C, which then could oxidize the variant of Mia40C. In summary, we reconstituted a disulfide relay system consisting of Mia40C and Erv1p.     

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.     

Cell polarization during monopolar cytokinesis

The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.     

Role of twin cys-xaa9-cys motif cysteines in mitochondrial import of the cytochrome C oxidase biogenesis factor cmc1

The Mia40 import pathway facilitates the import and oxidative folding of cysteine-rich protein substrates into the mitochondrial intermembrane space. Here we describe the in vitro and in organello oxidative folding of Cmc1, a twin CX(9)C-containing substrate, which contains an unpaired cysteine. In vitro, Cmc1 can be oxidized by the import receptor Mia40 alone when in excess or at a lower rate by only the sulfhydryl oxidase Erv1. However, physiological and efficient Cmc1 oxidation requires Erv1 and Mia40. Cmc1 forms a stable intermediate with Mia40 and is released from this interaction in the presence of Erv1. The three proteins are shown to form a ternary complex in mitochondria. Our results suggest that this mechanism facilitates efficient formation of multiple disulfides and prevents the formation of non-native disulfide bonds.     

Thiol oxidation in bacteria, mitochondria and chloroplasts: common principles but three unrelated machineries?

The intermembrane space of mitochondria and the thylakoid lumen of chloroplasts are evolutionary descendents of the periplasmic space of bacteria. Presumably due to their common ancestry, the active oxidation of cysteinyl thiols is used in these three compartments in order to stabilize protein folding or to regulate protein function. In contrast, compartments of the eukaryotic cell which developed from the bacterial cytosol maintain cysteine residues largely reduced. Whereas the oxidizing machinery of bacteria is well characterized, that of mitochondria was only recently discovered and that of thylakoids still awaits to be identified. In mitochondria, protein oxidation is mediated by the sulfhydryl oxidase Erv1 which is highly conserved among eukaryotes. Erv1 oxidizes its substrate protein Mia40 which serves as an import receptor for proteins destined for the intermembrane space. This review summarizes the current knowledge on the mitochondrial disulfide relay system and compares its features to those of the periplasm and the thylakoid lumen. Although the sulfhydryl oxidases in the intermembrane space, Erv1, and the bacterial periplasm, DsbA-DsbB, share key structural features their primary sequence is not related and the evolutionary origin of Erv1 is unclear. On the basis of phylogenetic analyses of Erv1 sequences we propose that the mitochondrial oxidation machinery originated from a lateral gene transfer from flavobacteria-like prokaryotes early in eukaryotic evolution.     

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.     

In vivo evidence for cooperation of Mia40 and Erv1 in the oxidation of mitochondrial proteins

The intermembrane space of mitochondria accommodates the essential mitochondrial intermembrane space assembly (MIA) machinery that catalyzes oxidative folding of proteins. The disulfide bond formation pathway is based on a relay of reactions involving disulfide transfer from the sulfhydryl oxidase Erv1 to Mia40 and from Mia40 to substrate proteins. However, the substrates of the MIA typically contain two disulfide bonds. It was unclear what the mechanisms are that ensure that proteins are released from Mia40 in a fully oxidized form. In this work, we dissect the stage of the oxidative folding relay, in which Mia40 binds to its substrate. We identify dynamics of the Mia40–substrate intermediate complex. Our experiments performed in a native environment, both in organello and in vivo, show that Erv1 directly participates in Mia40–substrate complex dynamics by forming a ternary complex. Thus Mia40 in cooperation with Erv1 promotes the formation of two disulfide bonds in the substrate protein, ensuring the efficiency of oxidative folding in the intermembrane space 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.     

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.     

Novel mitochondrial intermembrane space proteins as substrates of the MIA import pathway

Mitochondria consist of four compartments, the outer membrane, intermembrane space (IMS), inner membrane and the matrix. Most mitochondrial proteins are synthesized as precursors in the cytosol and have to be imported into these compartments. While the protein import machineries of the outer membrane, inner membrane and matrix have been investigated in detail, a specific mitochondrial machinery for import and assembly of IMS proteins, termed MIA, was identified only recently. To date, only a very small number of substrate proteins of the MIA pathway have been identified. The substrates contain characteristic cysteine motifs, either a twin Cx(3)C or a twin Cx(9)C motif. The largest MIA substrates known possess a molecular mass of 11 kDa, implying that this new import pathway has a very small size limit. Here, we have compiled a list of Saccharomyces cerevisiae proteins with a twin Cx(9)C motif and identified three IMS proteins that were previously localized to incorrect cellular compartments by tagging approaches. Mdm35, Mic14 (YDR031w) and Mic17 (YMR002w) require the two essential subunits, Mia40 and Erv1, of the MIA machinery for their localization in the mitochondrial IMS. With a molecular mass of 14 kDa and 17 kDa, respectively, Mic14 and Mic17 are larger than the known MIA substrates. Remarkably, the precursor of Erv1 itself is imported via the MIA pathway. As Erv1 has a molecular mass of 22 kDa and a twin Cx(2)C motif, this study demonstrates that the MIA pathway can transport substrates that are twice as large as the substrates known to date and is not limited to proteins with twin Cx(3)C or Cx(9)C motifs. However, tagging of MIA substrates can interfere with their subcellular localization, indicating that the proper localization of mitochondrial IMS proteins requires the characterization of the authentic untagged proteins.     

Reconstitution of the Mia40-Erv1 Oxidative Folding Pathway for the Small Tim Proteins

Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, Mia40 served as a direct oxidant of Tim13, and Erv1 was required to reoxidize Mia40. During oxidation, four electrons were transferred from Tim13 with the insertion of two disulfide bonds in succession. The extent of Tim13 oxidation was directly dependent on Mia40 concentration and independent of Erv1 concentration. Characterization of the midpoint potentials showed that electrons flowed from Tim13 with a more negative midpoint potential of −310 mV via Mia40 with an intermediate midpoint potential of −290 mV to the C130-C133 pair of Erv1 with a positive midpoint potential of −150 mV. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Last, mutating C133 of the catalytic C130-C133 pair or C30 of the shuttle C30-C33 pair in Erv1 abolished oxidation of Tim13, whereas mutating the cysteines in the redox-active CPC motif, but not the structural disulfide linkages of the CX₉C motif of Mia40, prevented Tim13 oxidation. Thus, we demonstrate that Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation.     

Precursor oxidation by Mia40 and Erv1 promotes vectorial transport of proteins into the mitochondrial intermembrane space

The mitochondrial intermembrane space contains chaperone complexes that guide hydrophobic precursor proteins through this aqueous compartment. The chaperones consist of hetero-oligomeric complexes of small Tim proteins with conserved cysteine residues. The precursors of small Tim proteins are synthesized in the cytosol. Import of the precursors requires the essential intermembrane space proteins Mia40 and Erv1 that were proposed to form a relay for disulfide formation in the precursor proteins. However, experimental evidence for a role of Mia40 and Erv1 in the oxidation of intermembrane space precursors has been lacking. We have established a system to directly monitor the oxidation of precursors during import into mitochondria and dissected distinct steps of the import process. Reduced precursors bind to Mia40 during translocation into mitochondria. Both Mia40 and Erv1 are required for formation of oxidized monomers of the precursors that subsequently assemble into oligomeric complexes. Whereas the reduced precursors can diffuse back into the cytosol, the oxidized precursors are retained in the intermembrane space. Thus, oxidation driven by Mia40 and Erv1 determines vectorial transport of the precursors into the mitochondrial intermembrane space.     

Structure of Yeast Sulfhydryl Oxidase Erv1 Reveals Electron Transfer of the Disulfide Relay System in the Mitochondrial Intermembrane Space

The disulfide relay system in the mitochondrial intermembrane space drives the import of proteins with twin CX₉C or twin CX₃C motifs by an oxidative folding mechanism. This process requires disulfide bond transfer from oxidized Mia40 to a substrate protein. Reduced Mia40 is reoxidized/regenerated by the FAD-linked sulfhydryl oxidase Erv1 (EC 1.8.3.2). Full-length Erv1 consists of a flexible N-terminal shuttle domain (NTD) and a conserved C-terminal core domain (CTD). Here, we present crystal structures at 2.0 Å resolution of the CTD and at 3.0 Å resolution of a C30S/C133S double mutant of full-length Erv1 (Erv1FL). Similar to previous homologous structures, the CTD exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The structure of Erv1FL enabled us to identify, for the first time, the three-dimensional structure of the Erv1NTD, which is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the NTD to the CTD of another subunit. Comparative structural analysis revealed that the four-helix bundle of the CTD forms a wide platform for the electron donor NTD. Moreover, computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia40, the upstream electron donor. These findings provide structural insights into electron transfer from Mia40 via the shuttle domain of one subunit of Erv1 to the CTD of another Erv1 subunit.     

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.