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.     

Association of the Tim14.Tim16 subcomplex with the TIM23 translocase is crucial for function of the mitochondrial protein import motor

Tim14 and Tim16 are essential components of the import motor of the mitochondrial TIM23 preprotein translocase. Tim14 contains a J domain in the matrix space that is anchored in the inner membrane by a transmembrane segment. Tim16 is a J-related protein with a moderately hydrophobic segment at its N terminus. The J and J-like domains function in the regulation of the ATPase activity of the Hsp70 chaperone of the import motor. We report here on the role of the hydrophobic segments of Tim16 and Tim14 in the TIM23 translocase. Yeast cells lacking the hydrophobic N-terminal segment in either Tim16 or Tim14 are viable but show growth defects and decreased import rates of matrix-targeted preproteins into mitochondria. The interaction of the Tim14.Tim16 complex with the core complex of the TIM23 translocase is destabilized in these cells. In particular, the N-terminal domain of Tim16 is crucial for the interaction of the Tim14.Tim16 complex with the TIM23 preprotein translocase. Deletion of hydrophobic segments in both, Tim16 and Tim14, is lethal. We conclude that import into the matrix space of mitochondria requires association of the co-chaperones Tim16 and Tim14 with the TIM23 preprotein translocase.     

The Tim9p/10p and Tim8p/13p complexes bind to specific sites on Tim23p during mitochondrial protein import

The import of polytopic membrane proteins into the mitochondrial inner membrane (IM) is facilitated by Tim9p/Tim10p and Tim8p/Tim13p protein complexes in the intermembrane space (IMS). These complexes are proposed to act as chaperones by transporting the hydrophobic IM proteins through the aqueous IMS and preventing their aggregation. To examine the nature of this interaction, Tim23p molecules containing a single photoreactive cross-linking probe were imported into mitochondria in the absence of an IM potential where they associated with small Tim complexes in the IMS. On photolysis and immunoprecipitation, a probe located at a particular Tim23p site (27 different locations were examined) was found to react covalently with, in most cases, only one of the small Tim proteins. Tim8p, Tim9p, Tim10p, and Tim13p were therefore positioned adjacent to specific sites in the Tim23p substrate before its integration into the IM. This specificity of binding to Tim23p strongly suggests that small Tim proteins do not function solely as general chaperones by minimizing the exposure of nonpolar Tim23p surfaces to the aqueous medium, but may also align a folded Tim23p substrate in the proper orientation for delivery and integration into the IM at the TIM22 translocon.     

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.     

A novel import route for an N-anchor mitochondrial outer membrane protein aided by the TIM23 complex

The membrane topology of Om45 in the yeast mitochondrial outer membrane (OM) is under debate. Here, we confirm that Om45 is anchored to the OM from the intermembrane space (IMS) by its N-terminal hydrophobic segment. We show that import of Om45 requires the presequence receptors, Tom20 and Tom22, and the import channel of Tom40. Unlike any of the known OM proteins, Om45 import requires the TIM23 complex in the inner membrane, a translocator for presequence-containing proteins, and the membrane potential (ΔΨ). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex.     

Tim50 is a subunit of the TIM23 complex that links protein translocation across the outer and inner mitochondrial membranes

Based on the results of site-specific photocrosslinking of translocation intermediates, we have identified Tim50, a component of the yeast TIM23 import machinery, which mediates translocation of presequence-containing proteins across the mitochondrial inner membrane. Tim50 is anchored to the inner mitochondrial membrane, exposing the C-terminal domain to the intermembrane space. Tim50 interacts with the N-terminal intermembrane space domain of Tim23. Functional defects of Tim50 either by depletion of the protein or addition of anti-Tim50 antibodies block the protein translocation across the inner membrane. A translocation intermediate accumulated at the TOM complex is crosslinked to Tim50. We suggest that Tim50, in cooperation with Tim23, facilitates transfer of the translocating protein from the TOM complex to the TIM23 complex     

Role of the import motor in insertion of transmembrane segments by the mitochondrial TIM23 complex

The TIM23 complex mediates translocation of proteins across, and their lateral insertion into, the mitochondrial inner membrane. Translocation of proteins requires both the membrane-embedded core of the complex and its ATP-dependent import motor. Insertion of some proteins, however, occurs in the absence of ATP, questioning the need for the import motor during lateral insertion. We show here that the import motor associates with laterally inserted proteins even when its ATPase activity is not required. Furthermore, our results suggest a role for the import motor in lateral insertion. Thus, the import motor is involved in ATP-dependent translocation and ATP-independent lateral insertion.     

Pam17 and Tim44 act sequentially in protein import into the mitochondrial matrix

Import of proteins into the matrix is driven by the Tim23 presequence translocase-associated import motor PAM. The core component of PAM is the mitochondrial chaperone mtHsp70, which ensures efficient translocation of proteins across the inner membrane through interactions with the J-protein complex Pam16–Pam18 (Tim16–Tim14) and its cochaperone Tim44. The recently identified non-essential Pam17 is a further member of PAM. Genetic and biochemical analyses reveal synthetic interactions between PAM17 and TIM44. Pam17 is involved in an early stage of protein translocation whereas Tim44 assists in a later step of transport, suggesting that both proteins can cooperate in a complementary manner in protein import.     

Two intermembrane space TIM complexes interact with different domains of Tim23p during its import into mitochondria

Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p-Tim13p and Tim9p-Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p-Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH(2)-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p-Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p-Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.     

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.     

The many faces of the mitochondrial TIM23 complex

The TIM23 complex in the inner membrane of mitochondria mediates import of essentially all matrix proteins and a large number of inner membrane proteins. Here we present an overview on the latest insights into the structure and function of this remarkable molecular machine.     

Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins.

We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi. A second import signal is located in the C-terminal membrane-integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C-terminal half and its import requires DeltaPsi. The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.     

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.     

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 import motor of the yeast mitochondrial TIM23 preprotein translocase contains two different J proteins, Tim14 and Mdj2

The import motor of the mitochondrial (mt)TIM23 complex drives translocation of presequence-containing preproteins across the mitochondrial inner membrane in an ATP-dependent manner. Tim44 is the central component of the motor. It recruits mtHsp70, which binds the incoming preproteins. The J protein Tim14 stimulates the ATPase activity of mtHsp70 and thereby enables efficient binding of mtHsp70 to preproteins. Tim16 is a J-like protein that forms a stable subcomplex with Tim14 and recruits it to the translocase. All subunits of the TIM23 translocase but one are essential for yeast cell viability. Yeast cells contain a close homologue of Tim14, Mdj2. In contrast to Tim14, its deletion leads to no obvious growth defect. In the present study we analyzed Mdj2 and compared it with Tim14. Mdj2 forms a complex with Tim16 and is recruited to the TIM23 translocase. It stimulates the ATPase activity of mtHsp70 to the same extent that Tim14 does. Mdj2 is expressed at a lower level compared with Tim14, and its complex with Tim16 is less stable. However, overexpressed Mdj2 fully restores the growth of cells lacking Tim14. We conclude that Mdj2 is a functional J protein and a component of the mitochondrial import motor.     

Protein import into and across the mitochondrial inner membrane: role of the TIM23 and TIM22 translocons

Import of nuclear-encoded mitochondrial proteins requires the action of at least two different import machines, called translocons, in the mitochondrial inner membrane (IM). The TIM23 complex mediates the translocation of proteins into the mitochondria matrix, whereas the TIM22 complex is required for the insertion of polytopic proteins into the IM. While the two translocons are distinct and composed of separate subunits, the essential reactions in each complex are carried out by homologous proteins. In addition, the core components of both the TIM23 and TIM22 translocons have been shown to form aqueous pores in the mitochondrial IM. In this review, we summarize what is known about import of proteins across the mitochondrial IM.     

Direct interaction of mitochondrial targeting presequences with purified components of the TIM23 protein complex

Precursor proteins that are imported from the cytosol into the matrix of mitochondria carry positively charged amphipathic presequences and cross the inner membrane with the help of vital components of the TIM23 complex. It is currently unclear which subunits of the TIM23 complex recognize and directly bind to presequences. Here we analyzed the binding of presequence peptides to purified components of the TIM23 complex. The interaction of three different presequences with purified soluble domains of yeast Tim50 (Tim50IMS), Tim23 (Tim23IMS), and full-length Tim44 was examined. Using chemical cross-linking and surface plasmon resonance we demonstrate, for the first time, the ability of purified Tim50IMS and Tim44 to interact directly with the yeast Hsp60 presequence. We also analyzed their interaction with presequences derived from precursors of yeast mitochondrial 70-kDa heat shock protein (mHsp70) and of bovine cytochrome P450SCC. Moreover, we characterized the nature of the interactions and determined their KDs. On the basis of our results, we suggest a mechanism of translocation where stronger interactions of the presequences on the trans side of the channel support the import of precursor proteins through TIM23 into the matrix.     

Conserved N-terminal negative charges in the Tim17 subunit of the TIM23 translocase play a critical role in the import of preproteins into mitochondria

The TIM23 complex of the mitochondrial inner membrane mediates the import of preproteins that contain positively charged targeting signals. This translocase consists of the two phylogenetically related membrane-embedded subunits Tim17 and Tim23 to which four largely hydrophilic subunits, Tim50, Tim44, Tim16, and Tim14, are attached. Whereas in vitro reconstitution experiments have suggested a pore-forming capacity of recombinant Tim23, virtually nothing is known about the properties and function of Tim17. We employed a combined genetic and biochemical approach to address the function of Tim17 in preprotein translocation. Tim17 exposes an N-terminal hydrophilic stretch into the intermembrane space. Truncation of the first 11 amino acid residues of this stretch did not affect the stability or integrity of TIM23 subunits but strongly impaired the import of preproteins. Moreover, expression of the truncated Tim17 variant led to a dominant negative effect on the mitochondrial membrane potential. By an alanine-scanning approach we identified two conserved negative charges in the N terminus of Tim17 as critical for Tim17 function. The replacement of these positions by positively charged residues results in a strong growth defect, which can be cured by reverting two conserved positive charges into aspartate residues between transmembrane domains two and three of Tim17. On the basis of these observations we propose that charged residues in Tim17 are critical for the preprotein-induced gating of the TIM23 translocase.     

Solution structure of the voltage-gated Tim23 channel in complex with a mitochondrial presequence peptide

Dear Editor, Most mitochondrial proteins are synthesized in the cytosol and transported into various mitochondrial subcompartments in a process that is mediated...