Kostas Tokatlidis

MIA40 is an oxidoreductase that catalyzes oxidative protein folding in mitochondria

MIA40 has a key role in oxidative protein folding in the mitochondrial intermembrane space. We present the solution structure of human MIA40 and its mechanism as a catalyst of oxidative folding. MIA40 has a 66-residue folded domain made of an α-helical hairpin core stabilized by two structural disulfides and a rigid N-terminal lid, with a characteristic CPC motif that can donate its disulfide bond to substrates. The CPC active site is solvent-accessible and sits adjacent to a hydrophobic cleft. Its second cysteine (Cys55) is essential in vivo and is crucial for mixed disulfide formation with the substrate. The hydrophobic cleft functions as a substrate binding domain, and mutations of this domain are lethal in vivo and abrogate binding in vitro. MIA40 represents a thioredoxin-unrelated, minimal oxidoreductase, with a facile CPC redox active site that ensures its catalytic function in oxidative folding in mitochondria.

Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins

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

Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components

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

A novel intermembrane space–targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding

A nine-residue intermembrane-targeting signal brings the active Cys of substrate proteins into contact with Mia40 oxidase for folding and import into mitochondria.

High activity of inclusion bodies formed in Escherichia coli overproducing Clostridium thermocellum endoglucanase D

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

Molecular chaperone function of Mia40 triggers consecutive induced folding steps of the substrate in mitochondrial protein import

Several proteins of the mitochondrial intermembrane space are targeted by internal targeting signals. A class of such proteins with α-helical hairpin structure bridged by two intramolecular disulfides is trapped by a Mia40-dependent oxidative process. Here, we describe the oxidative folding mechanism underpinning this process by an exhaustive structural characterization of the protein in all stages and as a complex with Mia40. Two consecutive induced folding steps are at the basis of the protein-trapping process. In the first one, Mia40 functions as a molecular chaperone assisting α-helical folding of the internal targeting signal of the substrate. Subsequently, in a Mia40-independent manner, folding of the second substrate helix is induced by the folded targeting signal functioning as a folding scaffold. The Mia40-induced folding pathway provides a proof of principle for the general concept that internal targeting signals may operate as a folding nucleus upon compartment-specific activation.

Juxtaposition of the Two Distal CX3C Motifs via Intrachain Disulfide Bonding Is Essential for the Folding of Tim10

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

Oxidative Protein Folding in the Mitochondrial Intermembrane Space

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

Molecular recognition and substrate mimicry drive the electron-transfer process between MIA40 and ALR

Oxidative protein folding in the mitochondrial intermembrane space requires the transfer of a disulfide bond from MIA40 to the substrate. During this process MIA40 is reduced and regenerated to a functional state through the interaction with the flavin-dependent sulfhydryl oxidase ALR. Here we present the mechanistic basis of ALR–MIA40 interaction at atomic resolution by biochemical and structural analyses of the mitochondrial ALR isoform and its covalent mixed disulfide intermediate with MIA40. This ALR isoform contains a folded FAD-binding domain at the C-terminus and an unstructured, flexible N-terminal domain, weakly and transiently interacting one with the other. A specific region of the N-terminal domain guides the interaction with the MIA40 substrate binding cleft (mimicking the interaction of the substrate itself), without being involved in the import of ALR. The hydrophobicity-driven binding of this region ensures precise protein–protein recognition needed for an efficient electron transfer 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.