Reconstitution of the protein insertion machinery of the mitochondrial inner membrane.

We have reconstituted the protein insertion machinery of the yeast mitochondrial inner membrane into proteoliposomes. The reconstituted proteoliposomes have a distinct morphology and protein composition and correctly insert the ADP/ATP carrier (AAC) and Tim23p, two multi-spanning integral proteins of the mitochondrial inner membrane. The reconstituted system requires a membrane potential, but not Tim44p or mhsp70, both of which are required for the ATP-driven translocation of proteins into the matrix. The protein insertion machinery can thus operate independently of the energy-transducing Tim44p-mhsp70 complex.     

Sequential action of two hsp70 complexes during protein import into mitochondria.

The mitochondrial chaperone mhsp70 mediates protein transport across the inner membrane and protein folding in the matrix. These two reactions are effected by two different mhsp70 complexes. The ADP conformation of mhsp70 favors formation of a complex on the inner membrane; this 'import complex' contains mhsp70, its membrane anchor Tim44 and the nucleotide exchange factor mGrpE. The ATP conformation of mhsp70 favors formation of a complex in the matrix; this 'folding complex' contains mhsp70, the mitochondrial DnaJ homolog Mdj1 and mGrpE. A precursor protein entering the matrix interacts first with the import complex and then with the folding complex. A chaperone can thus function as part of two different complexes within the same organelle.     

The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import.

Import of preproteins into the mitochondrial matrix is driven by the ATP-dependent interaction of mt-Hsp70 with the peripheral inner membrane import protein Tim44 and the preprotein in transit. We show that Mge1p, a co-chaperone of mt-Hsp70, plays a key role in the ATP-dependent import reaction cycle in yeast. Our data suggest a cycle in which the mt-Hsp70-Tim44 complex forms with ATP: Mge1p promotes assembly of the complex in the presence of ATP. Hydrolysis of ATP by mt-Hsp70 occurs in complex with Tim44. Mge1p is then required for the dissociation of the ADP form of mt-Hsp70 from Tim44 after release of inorganic phosphate but before release of ADP. ATP hydrolysis and complex dissociation are accompanied by tight binding of mt-Hsp70 to the preprotein in transit. Subsequently, the release of mt-Hsp70 from the polypeptide chain is triggered by Mge1p which promotes release of ADP from mt-Hsp70. Rebinding of ATP to mt-Hsp70 completes the reaction cycle.     

The mitochondrial protein import motor

Mitochondrial proteins are synthesized as precursor proteins in the cytosol and are posttranslationally imported into the organelle. A complex system of translocation machineries recognizes and transports the precursor polypeptide across the mitochondrial membranes. Energy for the translocation process is mainly supplied by the mitochondrial membrane potential (deltapsi) and the hydrolysis of ATP. Mitochondrial Hsp70 (mtHsp70) has been identified as the major ATPase driving the membrane transport of the precursor polypeptides into the mitochondrial matrix. Together with the partner proteins Tim44 and Mge1, mtHsp70 forms an import motor complex interacting with the incoming preproteins at the inner face of the inner membrane. This import motor complex drives the movement of the polypeptides in the translocation channel and the unfolding of carboxy-terminal parts of the preproteins on the outside of the outer membrane. Two models of the molecular mechanism of mtHsp70 during polypeptide translocation are discussed. In the 'trapping' model, precursor movement is generated by Brownian movement of the polypeptide chain in the translocation pore. This random movement is made vectorial by the interaction with mtHsp70 in the matrix. The detailed characterization of conditional mutants of the import motor complex provides the basis for an extended model. In this 'pulling' model, the attachment of mtHsp70 at the inner membrane via Tim44 and a conformational change induced by ATP results in the generation of an inward-directed force on the bound precursor polypeptide. This active role of the import motor complex is necessary for the translocation of proteins containing tightly folded domains. We suggest that both mechanisms complement each other to reach a high efficiency of preprotein import.     

Multiple interactions of components mediating preprotein translocation across the inner mitochondrial membrane.

The protein transport machinery of the inner mitochondrial membrane contains three essential Tim proteins. Tim17 and Tim23 are thought to build a preprotein translocation channel, while Tim44 transiently interacts with the matrix heat shock protein Hsp70 to form an ATP-driven import motor. For this report we characterized the biogenesis and interactions of Tim proteins. (i) Import of the precursor of Tim44 into the inner membrane requires mtHsp70, whereas import and inner membrane integration of the precursors of Tim17 and Tim23 are independent of functional mtHsp70. (ii) Tim17 efficiently associates with Tim23 and mtHsp70, but only weakly with Tim44. (iii) Depletion of Tim44 does not affect the co-precipitation of Tim17 with antibodies directed against mtHsp70. (iv) Tim23 associates with both Tim44 and Tim17, suggesting the presence of two Tim23 pools in the inner membrane, a Tim44-Tim23-containing sub-complex and a Tim23-Tim17-containing sub-complex. (v) The association of mtHsp70 with the Tim23-Tim17 sub-complex is ATP sensitive and can be distinguished from the mtHsp70-Tim44 interaction by the differential influence of an amino acid substitution in mtHsp70. (vi) Genetic evidence, suppression of the protein import defect of a tim17 yeast mutant by overexpression of mtHsp70 and synthetic lethality of conditional mutants in the genes of Tim17 and mtHsp70, supports a functional interaction of mtHsp70 with Tim17. We conclude that the protein transport machinery of the mitochondrial inner membrane consists of dynamically interacting sub-complexes, each of which transiently binds mtHsp70.     

The Pam18/Tim14-Pam16/Tim16 complex of the mitochondrial translocation motor: the formation of a stable complex from marginally stable proteins

The vast majority of mitochondrial proteins are imported from the cytosol. For matrix-localized proteins, the final step of translocation across the inner membrane is mediated by the mitochondrial translocation motor, of which mhsp70 is a key component. The ATP-dependent function of mhsp70 is regulated by a complex, composed of a J-protein (called Pam18 or Tim14) and a J-like protein (called Pam16 or Tim16), and the nucleotide exchange factor Mge1. In this study, we investigated the structural properties of a recombinant purified Pam18/Tim14-Pam16/Tim16 complex using cross-linking with the bifunctional reagent DSS and CD-spectroscopy. The results of the study show that both Pam18/Tim14 and Pam16/Tim16 are thermally unstable proteins that unfold at very low temperatures (T(m) values of 16.5 degrees C and 29 degrees C, respectively). Upon mixing the proteins in vitro, or when both proteins are co-overexpressed in bacteria, Pam18/Tim14 and Pam16/Tim16 form a heterodimer that is thermally more stable than the individual proteins (T(m) = 41 degrees C). Analysis of the properties of the complex in GdnHCl shows that dissociation of the heterodimer is the limiting step in achieving full denaturation.     

A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability.

Mitochondrial hsp70 (mhsp70) is located in the matrix and an essential component of the mitochondrial protein import system. To study the function of mhsp70 and to identify possible partner proteins we constructed a yeast strain in which all mhsp70 molecules carry a C-terminal hexa-histidine tag. The tagged mhsp70 appears to be functional in vivo. When an ATP depleted mitochondrial extract was incubated with a nickel-derivatized affinity resin, the resin bound not only mhsp70, but also a 23 kDa protein. This protein was dissociated from mhsp70 by ATP. ADP and GTP were much less effective in promoting dissociation whereas CTP and TTP were inactive. We cloned the gene encoding the 23 kDa protein. This gene, termed GRPE, encodes a 228 residue protein, whose sequence closely resembles that of the bacterial GrpE protein. Microsequencing the purified 23 kDa protein established it as the product of the yeast GRPE gene. Yeast GrpEp is made as a precursor that is cleaved upon import into isolated mitochondria. GrpEp is essential for viability. We suggest that this protein interacts with mhsp70 in a manner analogous to that of GrpE with DnaK of E.coli.     

Mitochondrial import of the ADP/ATP carrier: the essential TIM complex of the intermembrane space is required for precursor release from the TOM complex

The mitochondrial intermembrane space contains a protein complex essential for cell viability, the Tim9-Tim10 complex. This complex is required for the import of hydrophobic membrane proteins, such as the ADP/ATP carrier (AAC), into the inner membrane. Different views exist about the role played by the Tim9-Tim10 complex in translocation of the AAC precursor across the outer membrane. For this report we have generated a new tim10 yeast mutant that leads to a strong defect in AAC import into mitochondria. Thereby, for the first time, authentic AAC is stably arrested in the translocase complex of the outer membrane (TOM), as shown by antibody shift blue native electrophoresis. Surprisingly, AAC is still associated with the receptors Tom70 and Tom20 when the function of Tim10 is impaired. The nonessential Tim8-Tim13 complex of the intermembrane space is not involved in the transfer of AAC across the outer membrane. These results define a two-step mechanism for translocation of AAC across the outer membrane. The initial insertion of AAC into the import channel is independent of the function of Tim9-Tim10; however, completion of translocation across the outer membrane, including release from the TOM complex, requires a functional Tim9-Tim10 complex.     

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.     

Transport of the ADP/ATP carrier of mitochondria from the TOM complex to the TIM22.54 complex.

Members of the mitochondrial carrier family such as the ADP/ATP carrier (AAC) are composed of three structurally related modules. Here we show that each of the modules contains a mitochondrial import signal recognized by Tim10 and Tim12 in the intermembrane space. The first and the second module are translocated across the outer membrane independently of the membrane potential, DeltaDeltapsipsi, but they are not inserted into the inner membrane. The third module interacts tightly with the TOM complex and thereby prevents complete translocation of the precursor across the outer membrane. At this stage, binding of a TIM9.10 complex confers a topology to the translocation intermediate which reflects the modular structure of the AAC. The precursor is then transferred to the TIM9.10.12 complex, still interacting with the TOM complex. Release of the precursor from the TOM complex and insertion into the inner membrane by the TIM22.54 complex requires a DeltaDeltapsipsi-responsive signal in the third module.     

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.     

Residues of Tim44 involved in both association with the translocon of the inner mitochondrial membrane and regulation of mitochondrial Hsp70 tethering

Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by the action of the import motor, which is associated with the translocon on the matrix side of the membrane. It is well established that an essential peripheral membrane protein, Tim44, tethers mitochondrial Hsp70 (mtHsp70), the core of the import motor, to the translocon. This Tim44-mtHsp70 interaction, which can be recapitulated in vitro, is destabilized by binding of mtHsp70 to a substrate polypeptide. Here we report that the N-terminal 167-amino-acid segment of mature Tim44 is sufficient for both interaction with mtHsp70 and destabilization of a Tim44-mtHsp70 complex caused by client protein binding. Amino acid alterations within a 30-amino-acid segment affected both the release of mtHsp70 upon peptide binding and the interaction of Tim44 with the translocon. Our results support the idea that Tim44 plays multiple roles in mitochondrial protein import by recruiting Ssc1 and its J protein cochaperone to the translocon and coordinating their interactions to promote efficient protein translocation in vivo.     

Mitochondrial protein import: molecular basis of the ATP-dependent interaction of MtHsp70 with Tim44.

Protein translocation across the mitochondrial inner membrane is driven by cycles of binding and release of mitochondrial heat shock protein 70 (mtHsp70) in the matrix. The peripheral inner membrane protein, Tim44, recruits mtHsp70 in an ATP-dependent manner to the import sites. We show that DnaK, the closely related Hsp70 of Escherichia coli, when targeted to the matrix of yeast mitochondria, interacts in a specific manner with Tim44. The interaction is, however, not regulated by ATP, and DnaK cannot support protein translocation. We used truncated mtHsp70s and chimeric proteins consisting of segments of mtHsp70 and DnaK to analyze which portions of mtHsp70 bind and functionally interact with Tim44. We show that Tim44 interacts with the beta-stranded core of the peptide binding domain of mtHsp70 and of DnaK. The alpha-helices A and B of the peptide binding domain of mtHsp70 are required to transmit the nucleotide state of the ATPase domain to the peptide binding domain. Tim44, by interacting in this way with the peptide binding domain, is proposed to coordinate the binding of mtHsp70 to the incoming preprotein and the subsequent release of the mtHsp70-preprotein complex from the TIM23 complex, the translocase of the inner membrane.     

Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins.

The mitochondrial heat shock protein Hsp70 is essential for import of nuclear-encoded proteins, involved in both unfolding and membrane translocation of preproteins. mtHsp70 interacts reversibly with Tim44 of the mitochondrial inner membrane, yet the role of this interaction is unknown. We analysed this role by using two yeast mutants of mtHsp70 that differentially influenced its interaction with Tim44. One mutant mtHsp70 (Ssc1-2p) efficiently bound preproteins, but did not show a detectable complex formation with Tim44; the mitochondria imported loosely folded preproteins with wild-type kinetics, yet were impaired in unfolding of preproteins. The other mutant Hsp70 (Ssc1-3p') bound both Tim44 and preproteins, but the mitochondria did not import folded polypeptides and were impaired in import of unfolded preproteins; Ssc1-3p' was defective in its ATPase domain and did not undergo a nucleotide-dependent conformational change, resulting in permanent binding to Tim44. The following conclusions are suggested. (i) The import of loosely folded polypeptides (translocase function of mtHsp70) does not depend on formation of a detectable Hsp70-Tim44 complex. Two explanations are possible: a trapping mechanism by soluble mtHsp70, or a weak/very transient interaction of Ssc1-2p with Tim44 that leads to a weak force generation sufficient for import of loosely folded, but not folded, polypeptides. (ii) Import of folded preproteins (unfoldase function of mtHsp70) involves a reversible nucleotide-dependent interaction of mtHsp70 with Tim44, including a conformational change in mtHsp70. This is consistent with a model that the dynamic interaction of mtHsp70 with Tim44 generates a pulling force on preproteins which supports unfolding during translocation.     

The interplay between components of the mitochondrial protein translocation motor studied using purified components

The final step of protein translocation across the mitochondrial inner membrane is mediated by a translocation motor composed of 1) the matrix-localized, ATP-hydrolyzing, 70-kDa heat shock protein mHsp70; 2) its anchor to the import channel, Tim44; 3) the nucleotide exchange factor Mge1; and 4) a J-domain-containing complex of co-chaperones, Tim14/Pam18-Tim16/Pam16. Despite its essential role in the biogenesis of mitochondria, the mechanism by which the translocation motor functions is still largely unknown. The goal of this work was to carry out a structure-function analysis of the mitochondrial translocation motor utilizing purified components, with an emphasis on the formation of the Tim44-mHsp70 complex. To this end, we purified Tim44 and monitored its interaction with other components of the motor using cross-linking with bifunctional reagents. The effects of nucleotides, the J-domain-containing components, and the P5 peptide (CALLSAPRR, representing part of the mitochondrial targeting signal of aspartate aminotransferase) on the formation of the translocation motor were examined. Our results show that only the peptide and nucleotides, but not J-domain-containing proteins, affect the Tim44-mHsp70 interaction. Additionally, binding of Tim44 to mHsp70 prevents the formation of a complex between the latter and Tim14/Pam18-Tim16/Pam16. Thus, mutually exclusive interactions between various components of the motor with mHsp70 regulate its functional cycle. The results are discussed in light of known models for the function of the mitochondrial translocation motor.     

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.     

The J-related segment of tim44 is essential for cell viability: a mutant Tim44 remains in the mitochondrial import site, but inefficiently recruits mtHsp70 and impairs protein translocation.

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Delta18 in addition to the endogenous wild-type Tim44. Tim44Delta18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Delta18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Delta18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.     

Mitochondrial protein biogenesis: The essential functions of chaperones and their cofactors during preprotein translocation

Most mitochondrial proteins have to be imported from the cytosol through both mitochondrial membranes to their final localization. A dedicated translocation machinery is responsible for the specific recognition and the membrane transport of mitochondrial precursor proteins. Protein translocase complexes integrated into both mitochondrial membranes cooperate closely with receptor proteins at the surface and provide aqueous transport channels through the membranes. Energy for the membrane insertion is provided by the electric potential across the mitochondrial inner membrane. However, full translocation of the polypeptide chain requires ATP hydrolysis in the matrix. The responsible ATPase enzyme is a member of an ubiquitous family of molecular chaperones, the mitochondrial heat shock protein of 70 kDa (mtHsp70). A physical and functional interaction with a set of cofactors is indispensable for the translocation function of mtHsp70. By a specific and nucleotide-dependent binding to the inner membrane translocase component Tim44, the soluble chaperone mtHsp70 is anchored directly at the site of preprotein membrane insertion. The nucleotide exchange factor Mge1 enhances the ATPase activity of mtHsp70 and is required for the preprotein import reaction. Two novel proteins, Pam18 and Pam16, members of the inner membrane translocation channel, are required to couple the ATPase activity of mtHsp70 to the preprotein import reaction. We have collected experimental evidence indicating that mtHsp70 generates an inward directed translocation force on the polypeptide chain in transit by an ATP-regulated direct interaction with the precursor protein. The force generation results in the movement and active unfolding of the preprotein domains during the translocation process. Taken together, the chaperone mtHsp70 with its accessory proteine forms an import motor complex for mitochondrial preproteins that is driven by the hydrolysis of ATP.     

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.     

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.