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.     

Different import pathways through the mitochondrial intermembrane space for inner membrane proteins.

Earlier work on the protein import system of yeast mitochondria has identified two soluble 70 kDa protein complexes in the intermembrane space. One complex contains the essential proteins Tim9p and Tim10p and mediates transport of cytosolically-made metabolite carrier proteins from the outer to the inner membrane. The other complex contains the non-essential proteins Tim8p and Tim13p as well as loosely associated Tim9p; its function was unclear, but it interacted structurally or functionally with the Tim9p-Tim10p complex. We now show that the two 70 kDa complexes each mediate the import of a different subset of integral inner membrane proteins and that they can transfer these proteins to one of three different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as yet uncharacterized insertion site. Yeast mitochondria thus use multiple pathways for escorting hydrophobic inner membrane proteins across the aqueous intermembrane space.     

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.     

The role of Hot13p and redox chemistry in the mitochondrial TIM22 import pathway

The small Tim proteins in the mitochondrial intermembrane space participate in the TIM22 import pathway for assembly of the inner membrane. Assembly of the small TIM complexes requires the conserved "twin CX3C" motif that forms juxtapositional intramolecular disulfide bonds. Here we identify a new intermembrane space protein, Hot13p, as the first component of a pathway that mediates assembly of the small TIM complexes. The small Tim proteins require Hot13p for assembly into a 70-kDa complex in the intermembrane space. Once assembled the small TIM complexes escort hydrophobic inner membrane proteins en route to the TIM22 complex. The mechanism by which the small Tim proteins bind and release substrate is not understood, and we investigated the affect of oxidant/reductant treatment on the TIM22 import pathway. With in organello import studies, oxidizing agents arrest the ADP/ATP carrier (AAC) bound to the Tim9p-Tim10p complex in the intermembrane space; this productive intermediate can be chased into the inner membrane upon subsequent treatment with reductant. Moreover, AAC import is markedly decreased by oxidant treatment in Deltahot13 mitochondria and improved when Hot13p is overexpressed, suggesting Hot13p may function to remodel the small TIM complexes during import. Together these results suggest that the small TIM complexes have a specialized assembly pathway in the intermembrane space and that the local redox state of the TIM complexes may mediate translocation of inner membrane proteins.     

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.     

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.     

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.     

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.     

Protein translocation into mitochondria: the role of TIM complexes

Import of nuclear-encoded mitochondrial preproteins is mediated by a general translocase in the outer membrane, the TOM complex, and by two distinct translocases in the mitochondrial inner membrane, the TIM23 complex and the TIM22 complex. Both TIM complexes cooperate with the TOM complex but facilitate import of different classes of precursor proteins. Precursors with an N-terminal presequence are imported via the TIM23 complex, whereas mitochondrial carrier proteins require the TIM22 complex for insertion into the inner membrane. This review discusses recent advances in understanding the structure and function of the translocases of the inner membrane and the possible role of Tim proteins in the development of the Mohr-Tranebjaerg syndrome, a mitochondrial disorder leading to neurodegeneration.     

Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex

Import of the ADP/ATP carrier (AAC) into mitochondria requires the soluble TIM10 complex to cross the intermembrane space. We report here that Tim9 and Tim10 purified from Escherichia coli can form a complex of the same size as the endogenous complex from yeast mitochondria. This shows that no other mitochondrial protein is required for the formation of the TIM10 complex. Co-expression of both proteins rendered Tim9 more soluble and allowed purification of the reconstituted complex in a single step. Urea/EDTA treatment of recombinant Tim10 allowed its import into tim10-ts mitochondria that lack endogenous Tim10 and cannot import AAC. In this way, we were able to (i) reconstitute the TIM10 complex in the intermembrane space and (ii) restore import of AAC to almost wild-type levels. The reconstituted TIM10 complex not only facilitated passage of AAC across the outer membrane but also ensured its accurate membrane insertion. We conclude that the TIM10 complex can be formed exclusively from Tim9 and Tim10 and that the reconstituted complex efficiently restores AAC import in a strain lacking the TIM10 complex.     

The assembly pathway of the mitochondrial carrier translocase involves four preprotein translocases

The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multisubunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22, and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new posttranslocational manner: in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.     

Genetic and structural characterization of the human mitochondrial inner membrane translocase.

Translocation of nuclear-encoded mitochondrial preproteins is mediated by translocases in the outer and inner membranes. In the yeast Saccharomyces cerevisiae, translocation of preproteins into the matrix requires the membrane proteins Tim23, Tim17 and Tim44, which drive translocation in cooperation with mtHsp70 and its co-chaperone Mge1p. We have cloned and functionally analyzed the human homologues of Tim17, Tim23 and Tim44. In contrast to yeast, two TIM17 genes were found to be expressed in humans. TIM44, TIM23 and TIM17a genes were mapped to chromosomes 19p13.2-p13.3, 10q11. 21-q11.23 and 1q32. The TIM17b gene mapped to Xp11.23, near the fusion point where an autosomal region was proposed to have been added to the "ancient" part of the X chromosome about 80-130 MY ago. The primary sequences of the two proteins, hTim17a and hTim17b, are essentially identical, significant differences being restricted to their C termini. They are ubiquitously expressed in fetal and adult tissues, and both show expression levels comparable to that of hTim23. Biochemical characterization of the human Tim components revealed that hTim44 is localized in the matrix and, in contrast to yeast, only loosely associated with the inner membrane. hTim23 is organized into two distinct complexes in the inner membrane, one containing hTim17a and one containing hTim17b. Both TIM complexes display a native molecular mass of 110 kDa. We suggest that the structural organization of TIM23.17 preprotein translocases is conserved from low to high eukaryotes.     

The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel

Mitochondrial proteins with N-terminal targeting signals are transported across the inner membrane via the presequence translocase, which consists of membrane-integrated channel proteins and the matrix Hsp70 import motor. It has not been known how preproteins are directed to the import channel. We have identified the essential protein Tim50, which exposes its major domain to the intermembrane space. Tim50 interacts with preproteins in transit and directs them to the channel protein Tim23. Inactivation of Tim50 strongly inhibits the import of preproteins with a classical matrix-targeting signal, while preproteins carrying an additional inner membrane-sorting signal do not strictly depend on Tim50. Thus, Tim50 is crucial for guiding the precursors of matrix proteins to their insertion site in the inner membrane.     

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.     

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.     

The mitochondrial import machinery for preproteins.

Most mitochondrial proteins are transported from the cytosol into the organelle. Due to the division of mitochondria into an outer and inner membrane, an intermembrane space and a matrix, an elaborated system for recognition and transport of preproteins has evolved. The translocase of the outer mitochondrial membrane (TOM) and the translocases of the inner mitochondrial membrane (TIM) mediate these processes. Receptor proteins on the cytosolic face of mitochondria recognize the cargo proteins and transfer them to the general import pore (GIP) of the outer membrane. Following the passage of preproteins through the outer membrane they are transported with the aid of the TIM23 complex into either the matrix, inner membrane, or intermembrane space. Some preprotein families utilize the TIM22 complex for their insertion into the inner membrane. The identification of protein components, which are involved in these transport processes, as well as significant insights into the molecular function of some of them, has been achieved in recent years. Moreover, we are now approaching a new era in which elaborated techniques have already allowed and will enable us to gather information about the TOM and TIM complexes on an ultrastructural level.     

The essential function of Tim12 in vivo is ensured by the assembly interactions of its C-terminal domain

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.     

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 Tim54p–Tim22p Complex Mediates Insertion of Proteins into the Mitochondrial Inner Membrane

We have identified a new protein, Tim54p, located in the yeast mitochondrial inner membrane. Tim54p is an essential import component, required for the insertion of at least two polytopic proteins into the inner membrane, but not for the translocation of precursors into the matrix. Several observations suggest that Tim54p and Tim22p are part of a protein complex in the inner membrane distinct from the previously characterized Tim23p-Tim17p complex. First, multiple copies of the TIM22 gene, but not TIM23 or TIM17, suppress the growth defect of a tim54-1 temperature-sensitive mutant. Second, Tim22p can be coprecipitated with Tim54p from detergent-solubilized mitochondria, but Tim54p and Tim22p do not interact with either Tim23p or Tim17p. Finally, the tim54-1 mutation destabilizes the Tim22 protein, but not Tim23p or Tim17p. Our results support the idea that the mitochondrial inner membrane carries two independent import complexes: one required for the translocation of proteins across the inner membrane (Tim23p–Tim17p), and the other required for the insertion of proteins into the inner membrane (Tim54p–Tim22p).