Assembly of the three small Tim proteins precedes docking to the mitochondrial carrier translocase

The mitochondrial intermembrane space contains a family of small Tim proteins that function as essential chaperones for protein import. The soluble Tim9-Tim10 complex transfers hydrophobic precursor proteins through the aqueous intermembrane space to the carrier translocase of the inner membrane (TIM22 complex). Tim12, a peripheral membrane subunit of the TIM22 complex, is thought to recruit a portion of Tim9-Tim10 to the inner membrane. It is not known, however, how Tim12 is assembled. We have identified a new intermediate in the biogenesis pathway of Tim12. A soluble form of Tim12 first assembles with Tim9 and Tim10 to form a Tim12-core complex. Tim12-core then docks onto the membrane-integrated subunits of the TIM22 complex to form the holo-translocase. Thus, the function of Tim12 in linking soluble and membrane-integrated subunits of the import machinery involves a sequential assembly mechanism of the translocase through a soluble intermediate complex of the three essential small Tim proteins.     

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.     

Tim9, a new component of the TIM22.54 translocase in mitochondria.

We have identified Tim9, a new component of the TIM22.54 import machinery, which mediates transport of proteins into the inner membrane of mitochondria. Tim9, an essential protein of Saccharomyces cerevisiae, shares sequence similarity with Tim10 and Tim12. Tim9 is located in the mitochondrial intermembrane space and is organized into two distinct hetero-oligomeric assemblies with Tim10 and Tim12. One complex contains Tim9 and Tim10. The other complex contains Tim9, Tim10 and Tim12 and is tightly associated with Tim22 in the inner membrane. The TIM9.10 complex is more abundant than the TIM9.10.12 complex and mediates partial translocation of mitochondrial carriers proteins across the outer membrane. The TIM9.10.12 complex assists further translocation into the inner membrane in association with TIM22.54.     

Tim18p, a new subunit of the TIM22 complex that mediates insertion of imported proteins into the yeast mitochondrial inner membrane

Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.     

Distinct domains of small Tims involved in subunit interaction and substrate recognition

Tim9 and Tim10 belong to the small Tim family of mitochondrial ATP-independent chaperones. They are organised in a specific hetero-oligomeric complex (TIM10) that escorts polytopic proteins into the mitochondrial inner membrane. The contributions of the individual subunits to the assembly and function of the TIM10 complex remain poorly understood. Here, we show that substrate recognition and assembly of the complex are mediated by distinct domains of the subunits. These are unrelated to the characteristic "twin CX3C" motif that is present in all small Tims and ensures proper folding of the unassembled subunits. Specifically, we show that substrate recognition is achieved by the Tim10 subunit, whilst Tim9 serves a more structural role. The N-terminal domain of Tim10 is a substrate sensor whilst its C-terminal part is essential for complex formation. By contrast, both N and C-terminal domains of Tim9 are involved in the stability of the complex.     

Tim23–Tim50 pair coordinates functions of translocators and motor proteins in mitochondrial protein import

Mitochondrial protein traffic requires coordinated operation of protein translocator complexes in the mitochondrial membrane. The TIM23 complex translocates and inserts proteins into the mitochondrial inner membrane. Here we analyze the intermembrane space (IMS) domains of Tim23 and Tim50, which are essential subunits of the TIM23 complex, in these functions. We find that interactions of Tim23 and Tim50 in the IMS facilitate transfer of precursor proteins from the TOM40 complex, a general protein translocator in the outer membrane, to the TIM23 complex. Tim23–Tim50 interactions also facilitate a late step of protein translocation across the inner membrane by promoting motor functions of mitochondrial Hsp70 in the matrix. Therefore, the Tim23–Tim50 pair coordinates the actions of the TOM40 and TIM23 complexes together with motor proteins for mitochondrial protein import.     

Mutation of conserved charged residues in mitochondrial TIM10 subunits precludes TIM10 complex assembly, but does not abolish growth of yeast cells

The Saccharomyces cerevisiae TIM10 complex (TIM10c) is an ATP-independent chaperone of the mitochondrial intermembrane space, involved in transport of polytopic membrane proteins. The complex is an alpha(3)beta(3) hexamer of Tim9 and Tim10 subunits. We have generated specific mutations in charged residues in the central core domain of each subunit delineated by the characteristic twin CX(3)C motif, and investigated the effect of these mutations on subunit folding, complex assembly and TIM10 function in vitro and in vivo. Any combination of mutations that included a specific glutamate residue, conserved in all known Tim9 and Tim10 sequences, abolished assembly of the TIM10 complex. In vivo complementation analyses using a MET3-TIM10 strain that is selectively inactivated for the expression of wild-type Tim10 showed that (i) an N-terminal deleted version of Tim10 that was previously shown to be defective in substrate binding is lethal under all conditions, but (ii) the charged residues mutant of Tim10 that is defective in assembly with Tim9 can restore growth in glucose, but not in non-fermentable carbon sources. These data suggest that formation of the hexamer is beneficial but not vital for TIM10 function, whilst the N-terminal substrate-binding region of Tim10 is essential in vivo.     

The J domain-related cochaperone Tim16 is a constituent of the mitochondrial TIM23 preprotein translocase

Mitochondria import the vast majority of their proteins from the cytosol. The mitochondrial import motor of the TIM23 translocase drives the translocation of precursor proteins across the outer and inner membrane in an ATP-dependent reaction. Tim44 at the inner face of the translocation pore recruits the chaperone mtHsp70, which binds the incoming precursor protein. This reaction is assisted by the cochaperones Tim14 and Mge1. We have identified a novel essential cochaperone, Tim16. It is related to J-domain proteins and forms a stable subcomplex with the J protein Tim14. Depletion of Tim16 has a marked effect on protein import into the mitochondrial matrix, impairs the interaction of Tim14 with the TIM23 complex and leads to severe structural changes of the import motor. In conclusion, Tim16 is a constituent of the TIM23 preprotein translocase, where it exerts crucial functions in the import motor.     

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.     

The Tim9p-Tim10p complex binds to the transmembrane domains of the ADP/ATP carrier

The soluble Tim9p-Tim10p (Tim, translocase of inner membrane) complex of the mitochondrial intermembrane space mediates the import of the carrier proteins and is a component of the TIM22 import system. The mechanism by which the Tim9p-Tim10p complex assembles and binds the carriers is not well understood, but previous studies have proposed that the conserved cysteine residues in the 'twin CX3C' motif coordinate zinc and potentially generate a zinc-finger-like structure that binds to the matrix loops of the carrier proteins. Here we have purified the native and recombinant Tim9p-Tim10p complex, and show that both complexes resemble each other and consist of three Tim9p and three Tim10p. Results from inductively coupled plasma--mass spectrometry studies failed to detect zinc in the Tim9p-Tim10p complex. Instead, the cysteine residues seemingly formed disulfide linkages. The Tim9p-Tim10p complex bound specifically to the transmembrane domains of the ADP/ATP carrier, but had no affinity for Tim23p, an inner membrane protein that is inserted via the TIM22 complex. The chaperone-like Tim9p-Tim10p complex thus may prevent aggregation of the unfolded carrier proteins in the aqueous intermembrane space.     

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.     

Structure and function of Tim14 and Tim16, the J and J-like components of the mitochondrial protein import motor

The import motor of the mitochondrial translocase of the inner membrane (TIM23) mediates the ATP-dependent translocation of preproteins into the mitochondrial matrix by cycles of binding to and release from mtHsp70. An essential step of this process is the stimulation of the ATPase activity of mtHsp70 performed by the J cochaperone Tim14. Tim14 forms a complex with the J-like protein Tim16. The crystal structure of this complex shows that the conserved domains of the two proteins have virtually identical folds but completely different surfaces enabling them to perform different functions. The Tim14-Tim16 dimer reveals a previously undescribed arrangement of J and J-like domains. Mutations that destroy the complex between Tim14 and Tim16 are lethal demonstrating that complex formation is an essential requirement for the viability of cells. We further demonstrate tight regulation of the cochaperone activity of Tim14 by Tim16. The first crystal structure of a J domain in complex with a regulatory protein provides new insights into the function of the mitochondrial TIM23 translocase and the Hsp70 chaperone system in general.     

Quaternary structure of the mitochondrial TIM23 complex reveals dynamic association between Tim23p and other subunits

Tim23p is an essential channel-forming component of the multisubunit TIM23 complex of the mitochondrial inner membrane that mediates protein import. Radiolabeled Tim23p monocysteine mutants were imported in vitro, incorporated into functional TIM23 complexes, and subjected to chemical cross-linking. Three regions of proximity between Tim23p and other subunits of the TIM23 complex were identified: Tim17p and the first transmembrane segment of Tim23p; Tim50p and the C-terminal end of the Tim23p hydrophilic region; and the entire hydrophilic domains of Tim23p molecules. These regions of proximity reversibly change in response to changes in membrane potential across the inner membrane and also when a translocating substrate is trapped in the TIM23 complex. These structural changes reveal that the macromolecular arrangement within the TIM23 complex is dynamic and varies with the physiological state of the mitochondrion.     

The structural basis of the TIM10 chaperone assembly

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 Tim8-Tim13 complex has multiple substrate binding sites and binds cooperatively to Tim23

The Tim8-Tim13 complex, located in the mitochondrial intermembrane space, functions in the TIM22 import pathway that mediates the import of the mitochondrial carriers Tim23, Tim22, and Tim17 into the mitochondrial inner membrane. The Tim8-Tim13 complex assembles as a hexamer and binds to the substrate Tim23 to chaperone the hydrophobic Tim23 across the aqueous intermembrane space. However, both structural features of the Tim8-Tim13 complex and the binding interaction to Tim23 remain poorly defined. The crystal structure of the yeast Tim8-Tim13 complex, reported here at 2.6 Å resolution, reveals that the architecture of the Tim8-Tim13 complex is similar to those of other chaperones such as Tim9-Tim10, prefoldin, and Skp, in which long helices extend from a central body like tentacles from a jellyfish. Surface plasmon resonance was applied to investigate interactions between the Tim8-Tim13 complex and Tim23. The Tim8-Tim13 complex contained approximately six binding sites and showed a complex binding interaction indicative of positive cooperativity rather than a simple bimolecular interaction. By combining results from the structural and binding studies, we provide a molecular model of the Tim8-Tim13 complex binding to Tim23. The regions where the tentacle helices attach to the body of the Tim8-Tim13 complex contain six hydrophobic pockets that likely interact with specific sequences of Tim23 and possibly other substrates. Smaller hydrophobic patches on the tentacles themselves likely interact nonspecifically with the substrate's transmembrane helices, shielding it from the aqueous intermembrane space. The central region of Tim23, which enters the intermembrane space first, may serve to nucleate the binding of the Tim8-Tim13 complex, thereby initiating the chaperoned translocation of Tim23 to the mitochondrial inner membrane.     

The essential function of the small Tim proteins in the TIM22 import pathway does not depend on formation of the soluble 70-kilodalton complex

The TIM22 protein import pathway of the yeast mitochondrion contains several components, including a family of five proteins (Tim8p, -9p, -10p, -12p, and -13p [Tim, for translocase of inner membrane]) that are located in the intermembrane space and are 25% identical. Tim9p and Tim10p have dual roles in mediating the import of inner membrane proteins. Like the Tim8p-Tim13p complex, the Tim9p-Tim10p complex functions as a putative chaperone to guide hydrophobic precursors across the intermembrane space. Like membrane-associated Tim12p, they are members of the Tim18p-Tim22p-Tim54p membrane complex that mediates precursor insertion into the membrane. To understand the role of this family in protein import, we have used a genetic approach to manipulate the complement of the small Tim proteins. A strain has been constructed that lacks the 70-kDa soluble Tim8p-Tim13p and Tim9p-Tim10p complexes in the intermembrane space. Instead, a functional version of Tim9p (Tim9(S67C)p), identified as a second-site suppressor of a conditional tim10 mutant, maintains viability. Characterization of this strain revealed that Tim9(S67C)p and Tim10p were tightly associated with the inner membrane, the soluble 70-kDa Tim8p-Tim13p and Tim9p-Tim10p complexes were not detectable, and the rate of protein import into isolated mitochondria proceeded at a slower rate. An arrested translocation intermediate bound to Tim9(S67C)p was located in the intermembrane space, associated with the inner membrane. We suggest that the 70-kDa complexes facilitate import, similar to the outer membrane receptors of the TOM (hetero-oligomeric translocase of the outer membrane) complex, and the essential role of Tim9p and Tim10p may be to mediate protein insertion in the inner membrane with the TIM22 complex.     

Mitochondrial Tim9 protects Tim10 from degradation by the protease Yme1

Translocase of IM (inner membrane; Tim)9 and Tim10 are essential homologue proteins of the mitochondrial intermembrane space (IMS) and form a stable hexameric Tim9–Tim10 complex there. Redox-switch of the four conserved cysteine residues plays a key role during the biogenesis of these proteins and, in turn, the Tim proteins play a vital chaperone-like role during import of mitochondrial membrane proteins. However, the functional mechanism of the small Tim chaperones is far from solved and it is unclear whether the individual proteins play specific roles or the complex functions as a single unit. In the present study, we examined the requirement and role for the individual disulfide bonds of Tim9 on cell viability, complex formation and stability using yeast genetic, biochemical and biophysical methods. Loss of the Tim9 inner disulfide bond led to a temperature-sensitive phenotype and degradation of both Tim9 and Tim10. The growth phenotype could be suppressed by deletion of the mitochondrial i-AAA (ATPases associated with diverse cellular activities) protease Yme1, and this correlates strongly with stabilization of the Tim10 protein regardless of Tim9 levels. Formation of both disulfide bonds is not essential for Tim9 function, but it can facilitate the formation and improve the stability of the hexameric Tim9–Tim10 complex. Furthermore, our results suggest that the primary function of Tim9 is to protect Tim10 from degradation by Yme1 via assembly into the Tim9–Tim10 complex. We propose that Tim10, rather than the hexameric Tim9–Tim10 complex, is the functional form of these proteins.     

The role of Tim9p in the assembly of the TIM22 import complexes

Tim9p is located in the soluble 70-kDa Tim9p–Tim10p complex and the 300-kDa membrane complex in the mitochondrial TIM22 protein import system, which mediates the import of inner membrane proteins. From a collection of temperature-sensitive mutants, we have analyzed two in detail. tim9–3 contained two mutations and tim9–19 contained one mutation, all located near the 'twin CX₃C' motif that is conserved in the small Tim proteins. As a result, the import components in the tim9–3 mutant mitochondria were severely reduced and assembled into complexes of aberrant sizes. Protein import was severely reduced and Tim9p and Tim10p binding to in vitro imported ADP/ATP carrier was impaired. In the tim9–19 mutant mitochondria, the 300-kDa membrane complex was assembled, although the soluble 70-kDa Tim9p–Tim10p complex was not detectable. Protein import was decreased only two-fold. When coexpressed in Escherichia coli , tim9–19 and TIM10 proteins failed to assemble into a 70-kDa complex. Our findings suggest that residues near the 'twin CX₃C' motif are important for the assembly of Tim9p in both the Tim9p–Tim10p complex and the 300-kDa membrane complex.     

The role of the TIM8-13 complex in the import of Tim23 into mitochondria

Tim8 and Tim13 are non-essential, conserved proteins of the mitochondrial intermembrane space, which are organized in a hetero-oligomeric complex. They are structurally related to Tim9 and Tim10, essential components of the import machinery for mitochondrial carrier proteins. Here we show that the TIM8-13 complex interacts with translocation intermediates of Tim23, which are partially translocated across the outer membrane but not with fully imported or assembled Tim23. The TIM8-13 complex binds to the N-terminal or intermediate domain of Tim23. It traps the incoming precursor in the intermembrane space thereby preventing retrograde translocation. The TIM8-13 complex is strictly required for import of Tim23 under conditions when a low membrane potential exists in the mitochondria. The human homologue of Tim8 is encoded by the DDP1 (deafness/dystonia peptide 1) gene, which is associated with the Mohr-Tranebjaerg syndrome (MTS), a progressive neurodegenerative disorder leading to deafness. It is demonstrated that import of human Tim23 is dependent on a high membrane potential. A mechanism to explain the pathology of MTS is discussed.     

Tim14, a novel key component of the import motor of the TIM23 protein translocase of mitochondria

The TIM23 translocase mediates the deltaPsi- and ATP-dependent import of proteins into mitochondria. We identified Tim14 as a novel component of the TIM23 translocase. Tim14 is an integral protein of the inner membrane with a typical J-domain exposed to the matrix space. TIM14 genes are present in the genomes of virtually all eukaryotes. In yeast, Tim14 is essential for viability. Mitochondria from cells depleted of Tim14 are deficient in the import of proteins mediated by the TIM23 complex. In particular, import of proteins that require the action of mtHsp70 is affected. Tim14 interacts with Tim44 and mtHsp70 in an ATP-dependent manner. A mutation in the HPD motif of the J-domain of Tim14 is lethal. Thus, Tim14 is a constituent of the mitochondrial import motor. We propose a model in which Tim14 is required for the activation of mtHsp70 and enables this chaperone to act in a rapid and regulated manner in the Tim44-mediated trapping of unfolded preproteins entering the matrix.