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.     

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX₉C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX₉C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.     

Mia40 Combines Thiol Oxidase and Disulfide Isomerase Activity to Efficiently Catalyze Oxidative Folding in Mitochondria

Mia40 (a mitochondrial import and assembly protein) catalyzes disulfide bond formation in proteins in the mitochondrial intermembrane space. By using Cox17 (a mitochondrial copper-binding protein) as a natural substrate, we discovered that, in the presence of Mia40, the formation of native disulfides is strongly favored. The catalytic mechanism of Mia40 involves a functional interplay between the chaperone site and the catalytic disulfide. Mia40 forms a specific native disulfide in Cox17 much more rapidly than other disulfides, in particular, non-native ones, which originates from the recently described high affinity for hydrophobic regions near target cysteines and the long lifetime of the mixed disulfide. In addition to its thiol oxidase function, Mia40 is active also as a disulfide reductase and isomerase. We found that species with inadvertently formed incorrect disulfides are rebound by Mia40 and reshuffled, revealing a proofreading mechanism that is steered by the conformational folding of the substrate protein.     

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.     

Oxidative Protein Folding in the Secretory Pathway and Redox Signaling Across Compartments and Cells

The endoplasmic reticulum (ER) is central for many essential cellular activities, such as folding, assembly and quality control of secretory and membrane proteins, disulfide bond formation, glycosylation, lipid biosynthesis, Ca²⁺ storage and signaling. In addition, this multifunctional organelle integrates many adaptive and/or maladaptive signaling cues reporting on metabolism, proteostasis, Ca²⁺ and redox homeostasis. We are beginning to understand how these functions and pathways are integrated with one another to regulate homeostasis at cell, tissue and organism levels. The mechanisms underlying the introduction of the proper set of disulfide bonds into secretory proteins (oxidative folding) are strictly related to redox homeostasis, ER stress sensing and signaling and provide a good example of the integration systems operative in the early secretory compartment.     

Biogenesis of Tim Proteins of the Mitochondrial Carrier Import Pathway: Differential Targeting Mechanisms and Crossing Over with the Main Import Pathway

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23-Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22-Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23-Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.     

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.     

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.     

Cotranslational Folding of Proteins

We suppose that folding of proteins occurs cotranslationally by the following scheme. The polypeptide chains enter the folding sites from protein translocation complexes (ribosome, translocation machinery incorporated in membranes) directionally with the N-terminus and gradually. The chain starts to fold as soon as its N-terminal residue enters the folding site from the translocation complex. The folding process accompanies the translocation of the chain to its folding site and is completed after the C-terminal residue leaves the translocation complex. Proteins fold in sequential stages, by translocation of their polypeptide into folding compartments. At each stage a particular conformation of the N-terminal part of the chain that has emerged from the translocation complex is formed. The formation of both the particular conformations of the N-terminal chain segment at each folding stage and the final native protein conformation at the last stage occurs in a time that does not exceed the duration of the fastest elongation cycle on the ribosome.     

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.     

Functional Relationship between Protein Disulfide Isomerase Family Members during the Oxidative Folding of Human Secretory Proteins

To examine the relationship between protein disulfide isomerase family members within the mammalian endoplasmic reticulum, PDI, ERp57, ERp72, and P5 were depleted with high efficiency in human hepatoma cells, either singly or in combination. The impact was assessed on the oxidative folding of several well-characterized secretory proteins. We show that PDI plays a predominant role in oxidative folding because its depletion delayed disulfide formation in all secretory proteins tested. However, the phenotype was surprisingly modest suggesting that other family members are able to compensate for PDI depletion, albeit with reduced efficacy. ERp57 also exhibited broad specificity, overlapping with that of PDI, but with preference for glycosylated substrates. Depletion of both PDI and ERp57 revealed that some substrates require both enzymes for optimal folding and, furthermore, led to generalized protein misfolding, impaired export from the ER, and degradation. In contrast, depletion of ERp72 or P5, either alone or in combination with PDI or ERp57 had minimal impact, revealing a narrow substrate specificity for ERp72 and no detectable role for P5 in oxidative protein folding.     

Chaperonin-Affected Folding of Globular Proteins

We studied the effect of GroEL on the kinetic refolding of-lactalbumin by stopped-flow fluorescence techniques. We usedwild-type GroEL and its ATPase-defficient mutant D398A, and studied thebinding constants between GroEL and the molten globule foldingintermediate at various concentrations of ADP and ATP. The results arecompared with titration of GroEL with the nucleotides, ADP, ATP-analogs(ATP-S and AMP-PNP) and ATP, which have shown that bothADP and the ATP analogs are bound to GroEL in a non-cooperativemanner but that ATP shows a cooperative effect. Similarly, the bindingconstant between GroEL and the folding intermediate decreased in acooperative manner with an increase in ATP concentration although itshowed non-cooperative decrease with respect to ADP concentration. Itis shown that the allosteric control of GroEL by the nucleotides isresponsible for the above behavior of GroEL and that the observeddifference between the ATP- and ADP-induced transitions of GroEL isbrought about by a small difference in an allosteric parameter (the ratio ofthe nucleotide affinities of GroEL in the high-affinity and the low-affinitystates), i.e., 4.1 for ATP and 2.6 for ADP.     

Atomistic Picture for the Folding Pathway of a Hybrid-1 Type Human Telomeric DNA G-quadruplex

In this work we studied the folding process of the hybrid-1 type human telomeric DNA G-quadruplex with solvent and ions explicitly modeled. Enabled by the powerful bias-exchange metadynamics and large-scale conventional molecular dynamic simulations, the free energy landscape of this G-DNA was obtained for the first time and four folding intermediates were identified, including a triplex and a basically formed quadruplex. The simulations also provided atomistic pictures for the structures and cation binding patterns of the intermediates. The results showed that the structure formation and cation binding are cooperative and mutually supporting each other. The syn/anti reorientation dynamics of the intermediates was also investigated. It was found that the nucleotides usually take correct syn/anti configurations when they form native and stable hydrogen bonds with the others, while fluctuating between two configurations when they do not. Misfolded intermediates with wrong syn/anti configurations were observed in the early intermediates but not in the later ones. Based on the simulations, we also discussed the roles of the non-native interactions. Besides, the formation process of the parallel conformation in the first two G-repeats and the associated reversal loop were studied. Based on the above results, we proposed a folding pathway for the hybrid-1 type G-quadruplex with atomistic details, which is new and more complete compared with previous ones. The knowledge gained for this type of G-DNA may provide a general insight for the folding of the other G-quadruplexes.     

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.     

Desolvation Barrier Effects Are a Likely Contributor to the Remarkable Diversity in the Folding Rates of Small Proteins

The variation in folding rate among single-domain natural proteins is tremendous, but common models with explicit representations of the protein chain are either demonstrably insufficient or unclear as to their capability for rationalizing the experimental diversity in folding rates. In view of the critical role of water exclusion in cooperative folding, we apply native-centric, coarse-grained chain modeling with elementary desolvation barriers to investigate solvation effects on folding rates. For a set of 13 proteins, folding rates simulated with desolvation barriers cover ∼ 4.6 orders of magnitude, spanning a range essentially identical to that observed experimentally. In contrast, folding rates simulated without desolvation barriers cover only ∼ 2.2 orders of magnitude. Following a Hammond-like trend, the folding transition-state ensemble (TSE) of a protein model with desolvation barriers generally has a higher average number of native contacts and is structurally more specific, that is, less diffused, than the TSE of the corresponding model without desolvation barriers. Folding is generally significantly slower in models with desolvation barriers because of their higher overall macroscopic folding barriers as well as slower conformational diffusion speeds in the TSE that are ≈ 1/50 times those in models without desolvation barriers. Nonetheless, the average root-mean-square deviation between the TSE and the native conformation is often similar in the two modeling approaches, a finding suggestive of a more robust structural requirement for the folding rate-limiting step. The increased folding rate diversity in models with desolvation barriers originates from the tendency of these microscopic barriers to cause more heightening of the overall macroscopic folding free-energy barriers for proteins with more nonlocal native contacts than those with fewer such contacts. Thus, the enhancement of folding cooperativity by solvation effects is seen as positively correlated with a protein's native topological complexity.     

Biochemical Reconstitution of the Mimiviral Base Excision Repair Pathway

Viruses are believed to be the obligate intracellular parasites that only carry genes essential for infecting and hijacking the host cell machinery. However, a recently discovered group of viruses belonging to the phylum nucleocytovirocota, also known as the nucleo-cytoplasmic large DNA viruses (NCLDVs), possess a number of genes that code for proteins predicted to be involved in metabolism, and DNA replication, and repair. In the present study, first, using proteomics of viral particles, we show that several proteins required for the completion of the DNA base excision repair (BER) pathway are packaged within the virions of Mimivirus as well as related viruses while they are absent from the virions of Marseillevirus and Kurlavirus that are NCLDVs with smaller genomes. We have thoroughly characterized three putative base excision repair enzymes from Mimivirus, a prototype NCLDV and successfully reconstituted the BER pathway using the purified recombinant proteins. The mimiviral uracil-DNA glycosylase (mvUDG) excises uracil from both ssDNA and dsDNA, a novel finding contrary to earlier studies. The putative AP-endonuclease (mvAPE) specifically cleaves at the abasic site created by the glycosylase while also exhibiting the 3′-5′ exonuclease activity. The Mimivirus polymerase X protein (mvPolX) can bind to gapped DNA substrates and perform single nucleotide gap-filling followed by downstream strand displacement. Furthermore, we show that when reconstituted in vitro, mvUDG, mvAPE, and mvPolX function cohesively to repair a uracil-containing DNA predominantly by long patch BER and together, may participate in the BER pathway during the early phase of Mimivirus life-cycle.     

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.     

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.     

Oxidative folding competes with mitochondrial import of the small Tim proteins

All small Tim proteins of the mitochondrial intermembrane space contain two conserved CX(3)C motifs, which form two intramolecular disulfide bonds essential for function, but only the cysteine-reduced, but not oxidized, proteins can be imported into mitochondria. We have shown that Tim10 can be oxidized by glutathione under cytosolic concentrations. However, it was unknown whether oxidative folding of other small Tims can occur under similar conditions and whether oxidative folding competes kinetically with mitochondrial import. In the present study, the effect of glutathione on the cysteine-redox state of Tim9 was investigated, and the standard redox potential of Tim9 was determined to be approx. -0.31 V at pH 7.4 and 25 degrees C with both the wild-type and Tim9F43W mutant proteins, using reverse-phase HPLC and fluorescence approaches. The results show that reduced Tim9 can be oxidized by glutathione under cytosolic concentrations. Next, we studied the rate of mitochondrial import and oxidative folding of Tim9 under identical conditions. The rate of import was approx. 3-fold slower than that of oxidative folding of Tim9, resulting in approx. 20% of the precursor protein being imported into an excess amount of mitochondria. A similar correlation between import and oxidative folding was obtained for Tim10. Therefore we conclude that oxidative folding and mitochondrial import are kinetically competitive processes. The efficiency of mitochondrial import of the small Tim proteins is controlled, at least partially in vitro, by the rate of oxidative folding, suggesting that a cofactor is required to stabilize the cysteine residues of the precursors from oxidation in vivo.