Functional TIM10 Chaperone Assembly Is Redox-regulated in Vivo

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Tim9 and Tim10 are essential components of the "small Tim" family of proteins that facilitate insertion of polytopic proteins at the inner mitochondrial membrane. The small Tims are themselves imported from the cytosol and are organized in specific translocation assemblies in the intermembrane space. Their conformational properties and how these influence the mechanism of assembly remain poorly understood. Moreover, the three-dimensional structure of the TIM10 complex is unknown. We have characterized the structural properties of these proteins in their free and assembled states using NMR, circular dichroism, and small angle x-ray scattering. We show that the free proteins are largely unfolded in their reduced assembly-incompetent state and molten globules in their oxidized assembly-competent state. Tim10 appears less structured than Tim9 in their respective free oxidized forms and undergoes a larger structural change than Tim9 upon complexation. The NMR data here demonstrates unequivocally that only the oxidized states of the Tim9 and Tim10 proteins are capable of forming a complex. Zinc binding stabilizes the reduced state against proteolysis without significantly affecting the secondary structure. Solution x-ray scattering was used to obtain a molecular envelope for the subunits individually and for their fully functional TIM10 complex. Ab initio shape reconstructions based on the scattering data has allowed us to obtain the first low resolution three-dimensional structure of the TIM10 complex. This is a novel structure that displays extensive surface hydrophobicity. The structure also provides an explanation for the escorting function of this non-ATP-powered chaperone particle.A universal event for the successful construction and function of all of the cells is that proteins are targeted to their correct location both spatially and temporally. The extent of these targeting events is reflected by the fact that more than one-third of the proteome of the cell are translocated across or inserted into a membrane. Mitochondria represent a major site for extensive protein translocation. Virtually all of the mitochondrial proteins (approximately one thousand different polypeptides) are imported from the cytosol and are sorted within the organelle. This event is orchestrated by distinct translocation machineries in the outer (translocase of the outer membrane, TOM) 1 and inner (translocase of the inner membrane, TIM) membranes and within the intermembrane space. An intriguing new class of translocase proteins is the small Tim family, which specifically mediates the import of presequencedevoid polytopic proteins.There are two main pathways for proteins destined to mitochondria. The matrix pathway is used mainly by precursors that contain a cleavable presequence (1), whereas precursors devoid of a presequence divert through the distinct carrier pathway (2-5). The small Tims are organized in two distinct 70-kDa complexes in the intermembrane space, the TIM10 complex that is made of Tim9 and Tim10, which are encoded by e...

mentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

The Structural Basis of the TIM10 Chaperone Assembly

Golovanov

Alcock

et al. 2004

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“…Although not directly shown so far, this suggests a transfer of disulfide bonds from Mia40p to the interacting IMS proteins. The generation of intramolecular disulfide bonds has been proposed to trigger folding of substrates and thereby trapping of imported substrates in the IMS (26,27). Two redox forms of Mia40p have been detected in mitochondria, an oxidized and a reduced form.…”

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confidence: 94%

Functional Characterization of Mia40p, the Central Component of the Disulfide Relay System of the Mitochondrial Intermembrane Space

Grumbt

Stroobant

Terziyska

et al. 2007

116

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.

“…AMS Assays-At various time points, protein aliquots were removed from reaction solutions and added to nonreducing gel sample buffer containing excess amount of AMS (10 mM) for 30 min in the dark at room temperature as described before (26). AMS interacts with free thiols of reduced proteins covalently, but not disulfide bonds.…”

Section: Methodsmentioning

confidence: 99%

Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

Ang

2009

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Erv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX 16 C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX 16 C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur.Disulfide bonds play very important roles in the structure and function of many proteins by stabilizing protein folding and/or acting as thiol/disulfide redox switches. The process of disulfide formation is catalyzed by dedicated enzymes in vivo (1-4). Erv1p is a FAD-dependent sulfhydryl oxidase located in the Saccharomyces cerevisiae mitochondrial intermembrane space (4 -6). It is an essential component of the redox regulated Mia40/Erv1 import and assembly pathway used by many of the cysteine-containing intermembrane space proteins, such as members of the "small Tim" and Cox17 families (7-10). Upon import of a Cys-reduced substrate, Mia40 interacts with the substrate via intermolecular disulfide bond and shuttles a disulfide to its substrate. Although oxidized Mia40 promotes disulfide bond formation in the substrates, Erv1p functions in catalyzing reoxidation of the reduced Mia40 and/or release of the substrate (11-13).The common features for the FAD-dependent sulfhydryl oxidases are that the enzymes can catalyze the electron transfer from substrate molecules (e.g. protein thiols) through the noncovalent bound FAD cofactor to molecular oxygen or oxidized cytochrome c (14). The sulfhydryl oxidases can be divided into three groups: Ero1 enzymes, multidomain quiesin sulfhydryl oxidases, and single domain Erv (essential for respiration and vegetative growth)/ALR proteins. The yeast Ero1p and the mammalian homologues (Ero1␣ and Ero1␤) are large flavoenzymes present in the ER with...