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The functional role of unstructured protein domains is an emerging field in the frame of intrinsically disordered proteins. The involvement of intrinsically disordered domains (IDDs) in protein targeting and biogenesis processes in mitochondria is so far not known. Here, we have characterized the structural/dynamic and functional properties of an IDD of the sulfhydryl oxidase ALR (augmenter of liver regeneration) located in the intermembrane space of mitochondria. At variance to the unfolded-to-folded structural transition of several intrinsically disordered proteins, neither substrate recognition events nor redox switch of its shuttle cysteine pair is linked to any such structural change. However, this unstructured domain performs a dual function in two cellular compartments: it acts (i) as a mitochondrial targeting signal in the cytosol and (ii) as a crucial recognition site in the disulfide relay system of intermembrane space. This domain provides an exciting new paradigm for IDDs ensuring two distinct functions that are linked to intracellular organelle targeting.

SummaryHuman anamorsin was implicated in cytosolic iron-sulfur (Fe/S) protein biogenesis. Here, the structural and metal-binding properties of anamorsin and its interaction with Mia40, a well-known oxidoreductase involved in protein trapping in the mitochondrial intermembrane space (IMS), were characterized. We show that (1), anamorsin contains two structurally independent domains connected by an unfolded linker; (2), the C-terminal domain binds a [2Fe-2S] cluster through a previously unknown cysteine binding motif in Fe/S proteins; (3), Mia40 specifically introduces two disulfide bonds in a twin CX2C motif of the C-terminal domain; (4), anamorsin and Mia40 interact through an intermolecular disulfide-bonded intermediate; and (5), anamorsin is imported into mitochondria. Hence, anamorsin is the first identified Fe/S protein imported into the IMS, raising the possibility that it plays a role in cytosolic Fe/S cluster biogenesis also once trapped in the IMS.

The small Tims chaperone hydrophobic precursors across the mitochondrial intermembrane space. Tim9 and Tim10 form the soluble TIM10 complex that binds precursors exiting from the outer membrane. Tim12 functions downstream, as the only small Tim peripherally attached on the inner membrane. We show that Tim12 has an intrinsic affinity for inner mitochondrial membrane lipids, in contrast to the other small Tims. We find that the C-terminal end of Tim12 is essential in vivo. Its deletion crucially abolishes assembly of Tim12 in complexes with the other Tims. The N-terminal end contains targeting information and also mediates direct binding of Tim12 to the transmembrane segments of the carrier substrates. These results provide a molecular basis for the concept that the essential role of Tim12 relies on its unique assembly properties that allow this subunit to bridge the soluble and membrane-embedded translocases in the carrier import pathway.

Most of the mitochondrial inner-membrane proteins are generated without a presequence and their targeting depends on inadequately defined internal segments. Despite the numerous components of the import machinery identified by proteomics, the properties of hydrophobic import substrates remain poorly understood. Recent studies support several principles for these membrane proteins: first, they become organized into partially assembled forms within the translocon; second, they present noncontiguous targeting signals; and third, they induce conformational changes in translocase subunits, thereby mediating "assembly on demand" of the import machinery. It is possible that the energy needed for these proteins to pass across the outer membrane, to travel through the intermembrane space and to target the inner-membrane surface is provided by conformational changes involving import components that seem to have natively unfolded structures. Such structural malleability might render some of the translocase subunits more adept at driving the protein import process.

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.

The TIM10 chaperone facilitates the insertion of hydrophobic proteins at the mitochondrial inner membrane. Here we report the novel molecular mechanism of TIM10 assembly. This process crucially depends on oxidative folding in mitochondria and involves: (i) import of the subunits in a Cys-reduced and unfolded state; (ii) folding to an assembly-competent structure maintained by intramolecular disulfide bonding of their four conserved cysteines; and (iii) assembly of the oxidized zinc-devoid subunits to the functional complex. We show that intramolecular disulfide bonding occurs in vivo, whereas intermolecular disulfides observed in vitro are abortive intermediates in the assembly pathway. This novel mechanism of compartment-specific redox-regulated assembly is crucial for the formation of a functional TIM10 chaperone.

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.

The TIM10 complex is localized in the mitochondrial intermembrane space and mediates insertion of hydrophobic proteins at the inner membrane. We have characterized TIM10 assembly and analyzed the structural properties of its subunits, Tim9 and Tim10. Both proteins are alpha-helical with a protease-resistant central domain, and each self-associates to form mainly dimers and trimers in solution. Tim9 and Tim10 bound to one another with submicromolar affinity in equimolar amounts and assembled in a stable, significantly extended complex that was indistinguishable from the native mitochondrial TIM10 complex. Importantly, the reconstituted TIM10 complex is functional because it bound to the physiological substrate ADP/ATP carrier and displayed chaperone activity in refolding the model substrate firefly luciferase. These data demonstrate that the individual subunits can exist as independent, dynamically self-associating proteins. Assembly into the thermodynamically stable hexameric complex is necessary for the TIM10 chaperone function.

The mitochondrial inner membrane of most organisms studied so far contains about a dozen proteins made by the mitochondrial genetic system and on the order of 10 proteins imported from the cytoplasm. This review briefly describes the two major pathways by which proteins made inside or outside the mitochondria are inserted into the inner membrane. These pathways were discovered only during the past few years; one of them involves a novel family of chaperone-like proteins in the mitochondrial intermembrane space. Import of proteins from the cytoplasm into mitochondria occurs by distinct routes that are dictated by the properties and final destination of each protein within the mitochondria (1–3). However, most of the different routes described until recently have been variants of the general “matrix pathway.” In this pathway, a protein carrying a positively charged amphiphilic helix at its N terminus is bound by cytosolic chaperones, delivered to an array of import receptors on the mitochondrial surface, and then transported across two distinct, hetero-oligomeric protein translocation channels, the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. During transport, the two complexes are transiently linked by the translocating precursor chain (4, 5). Transport across the TOM complex appears to be driven by binding of the basic N-terminal “presequence” to a series of acidic receptor domains of increasing avidity (“acid chain hypothesis”) (6, 7); insertion of the presequence into the TIM23 complex is driven electrophoretically by the potential across the inner membrane; and complete transport of the precursor into the matrix is driven by an ATP-powered import motor attached to the inner mouth of the TIM23 complex. Proteins destined for compartments other than the matrix can diverge from the general matrix pathway at different points (8–10). Some proteins destined for the outer membrane can arrest in the TOM complex and then escape into the outer membrane (11); and some intermembrane space proteins can arrest in the TIM23 complex and then be proteolytically released into the intermembrane space (8). A few years ago it seemed likely that a variation of this general matrix pathway would also sort proteins to the inner membrane. According to this view, a protein destined for the inner membrane would arrest in the TIM23 complex and escape “sidewise” into the phospholipid bilayer of the inner membrane. So far, however, no inner membrane protein has been shown to follow such a pathway. Arrest in the TIM23 complex has only been found for some proteins destined for the intermembrane space.

The yeast mitochondrial processing peptidase (MPP) and its subunits were purified in Escherichia coli under conditions for which the enzyme retains most of its processing activity in the absence of externally added divalent cation. The holoenzyme exhibited a K m value of 1.35 μM and a V max value of 0.25 μM/min and was inhibited by metal chelators in a time-dependent manner. Measurement of the metal content showed that both, MPP and β-MPP, contained 0.86 and 1.05 atoms of Zn 2+ per molecule, respectively. An enzymatically inactive MPP mutant carrying a mutation of the first histidine of the putative metal-ion binding HXXEH motif in β-MPP retained less than 0.2 atom of Zn 2+ per molecule. A metal-free enzyme (apoenzyme) was prepared from the holoenzyme and shown to be devoid of any processing activity. Incubation of the apoenzyme with 50 nM and 500 nM Zn 2+ restored 50% and 80% of the processing activity, respectively. However, no reactivation occurred at concentrations of Zn 2+ higher than 1 μM. Addition of 500 nM Mn 2+ or higher concentrations (up to 50 μM) reactivated only 50% of the processing activity. The holoenzyme was competitively inhibited by molar excess of Zn 2+ ( K i of 3.1 μM) but not by molar excess of Mn 2+ . Taken together, our data suggest that the authentic MPP is a Zn 2+ rather than a Mn 2+ metallopeptidase.

In this issue of Cell, Mesecke et al. (2005) show that there is a disulfide relay system in the intermembrane space (IMS) of mitochondria that is comprised of the proteins Mia40 and Erv1. This disulfide relay system promotes the import and oxidative folding of proteins. Oxidized Mia40 traps newly imported proteins through mixed disulfide bridges. Subsequent isomerization of these disulfide bridges allows the imported protein to be folded in the IMS. The reduced Mia40 generated is then reoxidized by the sulfhydryl oxidase Erv1, promoting the next round of disulfide exchange. The new work clarifies the molecular function of Mia40 and reveals Mia40 to be the first physiological substrate for the FAD-linked Erv1.