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.     

Mitochondrial biogenesis, switching the sorting pathway of the intermembrane space receptor Mia40

Mitochondrial precursor proteins are directed into the intermembrane space via two different routes, the presequence pathway and the redox-dependent MIA pathway. The pathways were assumed to be independent and transport different proteins. We report that the intermembrane space receptor Mia40 can switch between both pathways. In fungi, Mia40 is synthesized as large protein with an N-terminal presequence, whereas in metazoans and plants, Mia40 consists only of the conserved C-terminal domain. Human MIA40 and the C-terminal domain of yeast Mia40 (termed Mia40(core)) rescued the viability of Mia40-deficient yeast independently of the presence of a presequence. Purified Mia40(core) was imported into mitochondria via the MIA pathway. With cells expressing both full-length Mia40 and Mia40(core), we demonstrate that yeast Mia40 contains dual targeting information, directing the large precursor onto the presequence pathway and the smaller Mia40(core) onto the MIA pathway, raising interesting implications for the evolution of mitochondrial protein sorting.     

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.     

Allosteric and electrostatic protein-protein interactions regulate the assembly of the heterohexameric Tim9-Tim10 complex

Protein-protein interactions are crucial processes in virtually all cellular events. The heterohexameric Tim9-Tim10 complex of the mitochondrial intermembrane space plays an important role during import of mitochondrial membrane proteins. It consists of three molecules of each subunit arranged alternately in a ring-shaped structure. While the individual protein Tim9 forms a homodimer, Tim10 is a monomer. Further to our previous investigation on the complex formation pathway, in this study, the assembly mechanism of Tim9-Tim10 was investigated using a stopped-flow technique coupled with mutagenesis. We show that while the initial velocity of the assembly depends on Tim9 concentration linearly, it presents a sigmoid curve on Tim10. In addition, the overall rate of assembly depends on the pH level in a bell-shaped profile, and two pK_a values that are in good agreement with the respective isoelectric points of Tim9 and Tim10 were determined. Using a Tim10F70W mutant, we were able to show that there was clear salt concentration dependence in the rate of assembly at the early stages. Taken together, the results of pH and salt concentration dependence indicate that electrostatic interactions are important and provide an initial driving force for the complex formation. Thus, this study not only demonstrates that allosteric and electrostatic interactions are two key regulators for the assembly of the Tim9-Tim10 complex but also has important implications for our understanding of how proteins interact with their partners.     

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.     

Identification of Tim40 that mediates protein sorting to the mitochondrial intermembrane space

Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria, and sorted to one of the four mitochondrial subcompartments. Here we identified a new inner membrane protein, Tim40, that mediates sorting of small Tim proteins to the intermembrane space. Tim40 is essential for yeast cell growth, and its function in vivo requires six conserved Cys residues but not anchoring of the protein to the inner membrane by its N-terminal hydrophobic segment. Depletion of Tim40 impairs the import of small Tim proteins into mitochondria both in vivo and in vitro. In wild-type mitochondria, Tim40 forms a translocation intermediate with small Tim proteins prior to their assembly in the intermembrane space in vitro. These results suggest the essential role of Tim40 in sorting/assembly of small Tim proteins.     

Mia40, a novel factor for protein import into the intermembrane space of mitochondria is able to bind metal ions

Many proteins located in the intermembrane space (IMS) of mitochondria are characterized by a low molecular mass, contain highly conserved cysteine residues and coordinate metal ions. Studies on one of these proteins, Tim13, revealed that net translocation across the outer membrane is driven by metal-dependent folding in the IMS . We have identified an essential component, Mia40/Tim40/Ykl195w, with a highly conserved domain in the IMS that is able to bind zinc and copper ions. In cells lacking Mia40, the endogenous levels of Tim13 and other metal-binding IMS proteins are strongly reduced due to the impaired import of these proteins. Furthermore, Mia40 directly interacts with newly imported Tim13 protein. We conclude that Mia40 is the first essential component of a specific translocation pathway of metal-binding IMS proteins.     

To Mia or not to Mia: stepwise evolution of the mitochondrial intermembrane space disulfide relay

The disulfide relay system found in the intermembrane space (IMS) of mitochondria is an essential pathway for the import and oxidative folding of IMS proteins. Erv1, an essential member of this pathway, has been previously found to be ubiquitously present in mitochondria-containing eukaryotes. However, the other essential protein, Mia40, was found to be absent or not required in some organisms, raising questions about how the disulfide relay functions in these organisms. A recent study published in BMC Biology demonstrates for the first time that some Erv1 proteins can function in oxidative folding independently of a Mia40 protein, providing for the first time strong evidence that the IMS disulfide relay evolved in a stepwise manner.See research article: 10.1186/s12915-017-0445-8     

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 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.     

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.     

Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.     

Architecture and assembly dynamics of the essential mitochondrial chaperone complex TIM9·10·12

1. Download : Download high-res image (232KB)
2. Download : Download full-size image

     

Biogenesis of Tom40, core component of the TOM complex of mitochondria.

Tom40 is an essential component of the preprotein translocase of the mitochondrial outer membrane (TOM complex) in which it constitutes the core element of the protein conducting pore. We have investigated the biogenesis of Tom40. Tom40 is inserted into the outer membrane by the TOM complex. Initially, Tom40 is bound as a monomer at the mitochondrial surface. The import receptor Tom20 is involved in this initial step; it stimulates both binding and efficient insertion of the Tom40 precursor. This step is followed by the formation of a further intermediate at which the Tom40 precursor is partially inserted into the outer membrane. Finally, Tom40 is integrated into preexisting TOM complexes. Efficient import appears to require the Tom40 precursor to be in a partially folded conformation. Neither the NH(2) nor the COOH termini are necessary to target Tom40 to the outer membrane. However, the NH(2)-terminal segment is required for Tom40 to become assembled into the TOM complex. A model for the biogenesis of Tom40 is presented.     

The ionic strength of the intermembrane space of intact mitochondria is not affected by the pH or volume of the intermembrane space.

Ionic strength affects the electron transport activity of cytochrome c through its electrostatic interactions with redox partners and membrane lipids. We previously reported (Cortese, J.D., Voglino, A.L. and Hackenbrock, C.R. (1991) J. Cell Biol. 113, 1331-1340) that the ionic strength (I) of the intermembrane space (IMS-I) in isolated, intact condensed mitochondria is similar to the external, bulk I, over a wide range of bulk I. We now consider the possible effects of IMS-pH and IMS-volume, both variable parameters of mitochondrial function in situ, on IMS-I. IMS-pH and IMS-I were measured with pH- and I-sensitive fluorescent probes (highly fluorescent FITC-dextran for IMS-pH and FITC-BSA for IMS-I). These probes were delivered into the IMS of intact mitochondria via probe encapsulation into asolectin vesicles, followed by low pH-induced fusion of the vesicles with the outer membranes of intact mitochondria. IMS-pH was found to be 0.4-0.5 units lower than bulk pH over the pH range 6.0-8.5 for mitochondria with a large IMS-volume separating the two mitochondrial membranes (condensed configuration), and 0-0.2 units lower for mitochondria with a small IMS-volume and membranes closely opposed (orthodox configuration). This small pH difference between IMS-pM and bulk pH did not influence the similarity between IMS-I and bulk I. When the IMS-volume was osmotically decreased, bringing the two mitochondrial membranes in close proximity as in the orthodox configuration, IMS-I followed the bulk I above 10 mM but did not respond to changes in bulk I below 10 mM. The lack of response of the IMS-I below 10 mM indicates that the close proximity of the two mitochondrial membranes excludes ions only at low, nonphysiological I. Since the similarity of IMS-I and bulk I is unaffected by either IMS-pH or IMS-volume above a bulk I of 10 mM, at cytosolic physiological I (i.e., 100-150 mM) cytochrome c can be expected to be a free, three-dimensional diffusant in the IMS irrespective of the pH or volume of the IMS.     

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.     

Temperature‐dependent study reveals that dynamics of hydrophobic residues plays an important functional role in the mitochondrial Tim9–Tim10 complex

Protein–protein interaction is a fundamental process in all major biological processes. The hexameric Tim9–Tim10 (translocase of inner membrane) complex of the mitochondrial intermembrane space plays an essential chaperone‐like role during import of mitochondrial membrane proteins. However, little is known about the functional mechanism of the complex because the interaction is weak and transient. This study investigates how electrostatic and hydrophobic interactions affect the conformation and function of the complex at physiological temperatures, using both experimental and computational methods. The results suggest that, first, different complex conformational states exist at equilibrium, and the major difference between these states is the degree of hydrophobic interactions. Second, the conformational change mimics the biological activity of the complex as measured by substrate binding at the same temperatures. Finally, molecular dynamics simulation and detailed energy decomposition analysis provided supporting evidence at the atomic level for the presence of an excited state of the complex, the formation of which is largely driven by the disruption of hydrophobic interactions. Taken together, this study indicates that the dynamics of the hydrophobic residues plays an important role in regulating the function of the Tim9–Tim10 complex. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Biogenesis of the protein import channel Tom40 of the mitochondrial outer membrane: intermembrane space components are involved in an early stage of the assembly pathway

Tom40 forms the central channel of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The precursor of Tom40 is encoded in the nucleus, synthesized in the cytosol, and imported into mitochondria via a multi-step assembly pathway that involves the mature TOM complex and the sorting and assembly machinery of the outer membrane (SAM complex). We report that opening of the mitochondrial intermembrane space by swelling blocks the assembly pathway of the beta-barrel protein Tom40. Mitochondria with defects in small Tim proteins of the intermembrane space are impaired in the Tom40 assembly pathway. Swelling as well as defects in the small Tim proteins inhibit an early stage of the Tom40 import pathway that is needed for formation of a Tom40-SAM intermediate. We propose that the biogenesis pathway of beta-barrel proteins of the outer mitochondrial membrane not only requires TOM and SAM components, but also involves components of the intermembrane space.