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.     

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.     

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.     

Tim50, a novel component of the TIM23 preprotein translocase of mitochondria

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. We isolated the TIM23 complex of Neurospora crassa. Besides Tim23 and Tim17, it contained a novel component, referred to as Tim50. Tim50 spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the intermembrane space. Tim50 is essential for viability of yeast. Mitochondria from cells depleted of Tim50 displayed strongly reduced import kinetics of preproteins using the TIM23 complex. Tim50 could be cross-linked to preproteins that were halted at the level of the translocase of the outer membrane (TOM complex) or spanning both TOM and TIM23 complexes. We suggest that Tim50 plays a crucial role in the transfer of preproteins from the TOM complex to the TIM23 complex through the intermembrane space.     

Modular structure of the TIM23 preprotein translocase of mitochondria

The TIM23 complex mediates import into mitochondria of nuclear encoded preproteins with a matrix-targeting signal. It is composed of the integral membrane proteins Tim17 and Tim23 and the peripheral membrane protein Tim44, which recruits mitochondrial Hsp70 to the sites of protein import. We have analyzed the functions of these constituents using a combined genetic and biochemical approach. Depletion of either Tim17 or Tim23 led to loss of import competence of mitochondria and to a reduction in the number of preprotein-conducting channels. Upon depletion of Tim44, mitochondria also lost their ability to import proteins but maintained normal numbers of import channels. In the absence of Tim44 precursor protein was specifically recognized. The presequence was translocated in a Delta psi-dependent manner across the inner membrane and cleaved by matrix-processing peptidase. However, the preprotein did not move further into the matrix but rather underwent retrograde sliding out of the TIM23 complex. Thus, the TIM23 complex is composed of functionally independent modules. Tim17 and Tim23 are necessary for initiating translocation, whereas Tim44 and mitochondrial Hsp70 are indispensable for complete transport of preproteins and for unfolding of folded domains of preproteins.     

A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23.

Proteins imported into the mitochondrial matrix are synthesized in the cytosol with an N-terminal presequence and are translocated through hetero-oligomeric translocase complexes of the outer and inner mitochondrial membranes. The channel across the inner membrane is formed by the presequence translocase, which consists of roughly six distinct subunits; however, it is not known which subunits actually form the channel. Here we report that purified Tim23 forms a hydrophilic, approximately 13-24 A wide channel characteristic of the mitochondrial presequence translocase. The Tim23 channel is cation selective and activated by a membrane potential and presequences. The channel is formed by the C-terminal domain of Tim23 alone, whereas the N-terminal domain is required for selectivity and a high-affinity presequence interaction. Thus, Tim23 forms a voltage-sensitive high-conductance channel with specificity for mitochondrial presequences.     

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.     

The mitochondrial TIM22 preprotein translocase is highly conserved throughout the eukaryotic kingdom

The Mohr-Tranebjaerg syndrome (MTS), a neurodegenerative syndrome characterized by progressive sensorineural hearing loss, dystonia, mental retardation and blindness, is a mitochondrial disease caused by mutations in the deafness/dystonia peptide 1 (DDP1) gene. DDP1 shows similarity to the yeast proteins Tim9, Tim10 and Tim12, components of the mitochondrial import machinery for carrier proteins. Here, we show that DDP1 belongs to a large family of evolutionarily conserved proteins. We report the identification, chromosomal localization and expressional analysis of six human family members which represent further candidate genes for neurodegenerative diseases.     

The structural characteristics of human preprotein translocase of the inner mitochondrial membrane Tim23: implications for its physiological activities

The preprotein translocase of the inner mitochondrial membrane (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23 forms a pore for preprotein transportation in TIM23 complex, which spans the inner membrane with transmembrane segments and exposes a hydrophilic domain in the intermembrane space. In this study, we expressed and purified the intermembrane space (IMS) domain of human Tim23 (Tim23(IMS)). The far-UV CD spectra of Tim23(IMS) in native and denatured states revealed that the protein has a limited secondary structure and a not well-defined tertiary packing. Its Stokes radius was larger than both its expected size as a folded globular protein and the size determined by size exclusion chromatography. A large increase in 8-anilino-1-naphthalene-sulfonate (ANS) fluorescence (>50-fold) was observed, indicating that hydrophobic clusters are exposed at its surface. And GlobPlot/DisEMBL program predicted that the protein is in a loose folding state. We therefore conclude that, the non-bound hydrophilic domain of the human Tim23 is in a molten globule configuration with marginal stability. Furthermore, size exclusion chromatography and sedimentation equilibrium analysis showed that Tim23(IMS) exists as a dimer. And the results, showed by ANS binding and fluorescence quenching, indicated that a pH-dependent conformational change of Tim23(IMS) occurs, and at pH 4 and 3, it forms a compact structure.     

Tim17p regulates the twin pore structure and voltage gating of the mitochondrial protein import complex TIM23

The TIM23 complex mediates import of preproteins into mitochondria, but little is known of the mechanistic properties of this translocase. Here patch clamping reconstituted inner membranes allowed for first time insights into the structure and function of the preprotein translocase. Our findings indicate that the TIM23 channel has "twin pores" (two equal sized pores that cooperatively gate) thereby strikingly resembling TOM, the translocase of the outer membrane. Tim17p and Tim23p are homologues, but their functions differ. Tim23p acts as receptor for preproteins and may largely constitute the preprotein-conducting passageway. Conversely depletion of Tim17p induces a collapse of the twin pores into a single pore, whereas N terminus deletion or C terminus truncation results in variable sized pores that cooperatively gate. Further analysis of Tim17p mutants indicates that the N terminus is vital for both voltage sensing and protein sorting. These results suggest that although Tim23p is the main structural unit of the pore Tim17p is required for twin pore structure and provides the voltage gate for the TIM23 channel.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Cytosolic thioredoxin system facilitates the import of mitochondrial small Tim proteins

Thiol‐disulphide redox regulation has a key role during the biogenesis of mitochondrial intermembrane space (IMS) proteins. Only the Cys‐reduced form of precursor proteins can be imported into mitochondria, which is followed by disulphide bond formation in the mitochondrial IMS. In contrast to the wealth of knowledge on the oxidation process inside mitochondria, little is known about how precursors are maintained in an import‐competent form in the cytosol. Here we provide the first evidence that the cytosolic thioredoxin system is required to maintain the IMS small Tim proteins in reduced forms and facilitate their mitochondrial import during respiratory growth.     

A cooperative action of the ATP-dependent import motor complex and the inner membrane potential drives mitochondrial preprotein import

The import of mitochondrial preproteins requires an electric potential across the inner membrane and the hydrolysis of ATP in the matrix. We assessed the contributions of the two energy sources to the translocation driving force responsible for movement of the polypeptide chain through the translocation channel and the unfolding of preprotein domains. The import-driving activity was directly analyzed by the determination of the protease resistances of saturating amounts of membrane-spanning translocation intermediates. The ability to generate a strong translocation-driving force was solely dependent on the activity of the ATP-dependent import motor complex in the matrix. For a sustained import-driving activity on the preprotein in transit, an unstructured N-terminal segment of more than 70 to 80 amino acid residues was required. The electric potential of the inner membrane was required to maintain the import-driving activity at a high level. The electrophoretic force of the potential exhibited only a limited capacity to unfold preprotein domains. We conclude that the membrane potential increases the probability of a dynamic interaction of the preprotein with the import motor. Polypeptide translocation and unfolding are mainly driven by the inward-directed translocation activity based on the functional cooperation of the import motor components.