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.     

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.     

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.     

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.     

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.     

Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways.     

Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol

The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient samples and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but causalities remain unclear. We used Saccharomyces cerevisiae to analyze how mitochondrial processes regulate the behavior of aggregation‐prone polyQ protein derived from human huntingtin. Expression of Q97‐GFP rapidly led to insoluble cytosolic aggregates and cell death. Although aggregation impaired mitochondrial respiration only slightly, it considerably interfered with the import of mitochondrial precursor proteins. Mutants in the import component Mia40 were hypersensitive to Q97‐GFP, whereas Mia40 overexpression strongly suppressed the formation of toxic Q97‐GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the post‐translational import of mitochondrial precursor proteins into mitochondria competes with aggregation‐prone cytosolic proteins for chaperones and proteasome capacity. Mia40 regulates this competition as it has a rate‐limiting role in mitochondrial protein import. Therefore, Mia40 is a dynamic regulator in mitochondrial biogenesis that can be exploited to stabilize cytosolic proteostasis.     

Mia40 Protein Serves as an Electron Sink in the Mia40-Erv1 Import Pathway

A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 is the oxidoreductase that inserts two disulfide bonds into the substrate simultaneously. However, Mia40 has one redox-active cysteine pair, resulting in ambiguity about how Mia40 accepts numerous electrons during substrate oxidation. In this study, we have addressed the oxidation of Tim13 in vitro and in organello. Reductants such as glutathione and ascorbate inhibited both the oxidation of the substrate Tim13 in vitro and the import of Tim13 and Cmc1 into isolated mitochondria. In addition, a ternary complex consisting of Erv1, Mia40, and substrate, linked by disulfide bonds, was not detected in vitro. Instead, Mia40 accepted six electrons from substrates, and this fully reduced Mia40 was sensitive to protease, indicative of conformational changes in the structure. Mia40 in mitochondria from the erv1–101 mutant was also trapped in a completely reduced state, demonstrating that Mia40 can accept up to six electrons as substrates are imported. Therefore, these studies support that Mia40 functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates.     

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.     

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.     

Mia40-dependent oxidation of cysteines in domain I of Ccs1 controls its distribution between mitochondria and the cytosol

Superoxide dismutase 1 (Sod1) is an important antioxidative enzyme that converts superoxide anions to hydrogen peroxide and water. Active Sod1 is a homodimer containing one zinc ion, one copper ion, and one disulfide bond per subunit. Maturation of Sod1 depends on its copper chaperone (Ccs1). Sod1 and Ccs1 are dually localized proteins that reside in the cytosol and in the intermembrane space of mitochondria. The import of Ccs1 into mitochondria depends on the mitochondrial disulfide relay system. However, the exact mechanism of this import process has been unclear. In this study we detail the import and folding pathway of Ccs1 and characterize its interaction with the oxidoreductase of the mitochondrial disulfide relay Mia40. We identify cysteines at positions 27 and 64 in domain I of Ccs1 as critical for mitochondrial import and interaction with Mia40. On interaction with Mia40, these cysteines form a structural disulfide bond that stabilizes the overall fold of domain I. Although the cysteines are essential for the accumulation of functional Ccs1 in mitochondria, they are dispensable for the enzymatic activity of cytosolic Ccs1. We propose a model in which the Mia40-mediated oxidative folding of domain I controls the cellular distribution of Ccs1 and, consequently, active Sod1.     

In vivo expression and mitochondrial targeting of yeast apoiso-1-cytochrome c fusion proteins.

To define the import pathway for apoiso-1-cytochrome c in vivo, the coding region for bacterial chloramphenicol acetyltransferase (CAT) or yeast copper metallothionein (CuMT) was fused to the carboxy terminus of the apoiso-1-cytochrome c (iso-1) coding region. When the resulting iso-1/CAT and iso-1/CuMT fusion proteins were individually expressed in Saccharomyces cerevisiae, they were specifically targeted to the mitochondria and protected from trypsin digestion. Although iso-1/CAT was accessible to heme modification, it remained membrane associated because of the folded conformation of the CAT domain. A small deletion disrupting CAT structure resulted in the translocation of the resulting fusion protein, iso-1/CAT delta, to the intermembrane space, where it functioned efficiently in respiratory electron transfer. Similarly, iso-1/CuMT was heme modified and nearly identical to iso-1 in its ability to support respiratory growth, indicating that the CuMT domain was compatible with translocation to the IMS. Inclusion of copper in the growth medium, which converts the loosely structured apo-CuMT to a tightly folded holo-CuMT, inhibited both heme attachment and respiratory growth without affecting mitochondrial targeting. Thus, by altering the folded conformation of the reporter moiety of these fusion proteins, it was possible to differentiate between those molecules arrested at the mitochondrial targeting step of the cytochrome c import pathway and those translocated to the intermembrane space. By replacing the heme-binding cysteine residues with serines, this system was used to demonstrate that the import requirement for heme attachment operated at the level of membrane translocation and not on mitochondrial targeting in vivo.     

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.     

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.     

An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins

Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR (‘augmenter of liver regeneration’), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process.     

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.