Genetic analysis of complex interactions among components of the mitochondrial import motor and translocon in Saccharomyces cerevisiae

A highly conserved, Hsp70-based, import motor, which is associated with the translocase on the matrix side of the inner mitochondrial membrane, is critical for protein translocation into the matrix. Hsp70 is tethered to the translocon via interaction with Tim44. Pam18, the J-protein co-chaperone, and Pam16, a structurally related protein with which Pam18 forms a heterodimer, are also critical components of the motor. Their N termini are important for the heterodimer's translocon association, with Pam18's and Pam16's N termini interacting in the intermembrane space and the matrix, respectively. Here, using the model organism Saccharomyces cerevisiae, we report the identification of an N-terminal segment of Tim44, important for association of Pam16 with the translocon. We also report that higher amounts of Pam17, a nonessential motor component, are found associated with the translocon in both PAM16 and TIM44 mutants that affect their interaction with one another. These TIM44 and PAM16 mutations are also synthetically lethal with a deletion of PAM17. In contrast, a deletion of PAM17 has little, or no genetic interaction with a PAM18 mutation that affects translocon association of the Pam16:Pam18 heterodimer, suggesting a second role for the Pam16:Tim44 interaction. A similar pattern of genetic interactions and enhanced Pam17 translocon association was observed in the absence of the C terminus of Tim17, a core component of the translocon. We suggest the Pam16:Tim44 interaction may play two roles: (1) tethering the Pam16:Pam18 heterodimer to the translocon and (2) positioning the import motor for efficient engagement with the translocating polypeptide along with Tim17 and Pam17.     

Mitochondrial protein import motor: differential role of Tim44 in the recruitment of Pam17 and J-complex to the presequence translocase

The presequence translocase of the mitochondrial inner membrane (TIM23 complex) mediates the import of preproteins with amino-terminal presequences. To drive matrix translocation the TIM23 complex recruits the presequence translocase-associated motor (PAM) with the matrix heat shock protein 70 (mtHsp70) as central subunit. Activity and localization of mtHsp70 are regulated by four membrane-associated cochaperones: the adaptor protein Tim44, the stimulatory J-complex Pam18/Pam16, and Pam17. It has been proposed that Tim44 serves as molecular platform to localize mtHsp70 and the J-complex at the TIM23 complex, but it is unknown how Pam17 interacts with the translocase. We generated conditional tim44 yeast mutants and selected a mutant allele, which differentially affects the association of PAM modules with TIM23. In tim44-804 mitochondria, the interaction of the J-complex with the TIM23 complex is impaired, whereas unexpectedly the binding of Pam17 is increased. Pam17 interacts with the channel protein Tim23, revealing a new interaction site between TIM23 and PAM. Thus, the motor PAM is composed of functional modules that bind to different sites of the translocase. We suggest that Tim44 is not simply a scaffold for binding of motor subunits but plays a differential role in the recruitment of PAM modules to the inner membrane translocase.     

Pam17 is required for architecture and translocation activity of the mitochondrial protein import motor

Import of mitochondrial matrix proteins involves the general translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated motor (PAM) drives the completion of preprotein translocation into the matrix. Five subunits of PAM are known: the preprotein-binding matrix heat shock protein 70 (mtHsp70), the nucleotide exchange factor Mge1, Tim44 that directs mtHsp70 to the inner membrane, and the membrane-bound complex of Pam16-Pam18 that regulates the ATPase activity of mtHsp70. We have identified a sixth motor subunit. Pam17 (encoded by the open reading frame YKR065c) is anchored in the inner membrane and exposed to the matrix. Mitochondria lacking Pam17 are selectively impaired in the import of matrix proteins and the generation of an import-driving activity of PAM. Pam17 is required for formation of a stable complex between the cochaperones Pam16 and Pam18 and promotes the association of Pam16-Pam18 with the presequence translocase. Our findings suggest that Pam17 is required for the correct organization of the Pam16-Pam18 complex and thus contributes to regulation of mtHsp70 activity at the inner membrane translocation site.     

Pam16 has an essential role in the mitochondrial protein import motor

Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, the translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not for protein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with the presequence translocase and for formation of a mtHsp70-Tim44 complex. Thus, Pam16 is a newly identified type of motor subunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.     

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.     

Interaction of the J-protein heterodimer Pam18/Pam16 of the mitochondrial import motor with the translocon of the inner membrane

Import of proteins across the inner mitochondrial membrane through the Tim23:Tim17 translocase requires the function of an essential import motor having mitochondrial 70-kDa heat-shock protein (mtHsp70) at its core. The heterodimer composed of Pam18, the J-protein partner of mtHsp70, and the related protein Pam16 is a critical component of this motor. We report that three interactions contribute to association of the heterodimer with the translocon: the N terminus of Pam16 with the matrix side of the translocon, the inner membrane space domain of Pam18 (Pam18(IMS)) with Tim17, and the direct interaction of the J-domain of Pam18 with the J-like domain of Pam16. Pam16 plays a major role in translocon association, as alterations affecting the stability of the Pam18:Pam16 heterodimer dramatically affect association of Pam18, but not Pam16, with the translocon. Suppressors of the growth defects caused by alterations in the N terminus of Pam16 were isolated and found to be due to mutations in a short segment of TIM44, the gene encoding the peripheral membrane protein that tethers mtHsp70 to the translocon. These data suggest a model in which Tim44 serves as a scaffold for precise positioning of mtHsp70 and its cochaperone Pam18 at the translocon.     

Comparative Analysis of Putative Orthologues of Mitochondrial Import Motor Subunit: Pam18 and Pam16 in Plants

Pam18/Tim14 and Pam16/Tim16, highly conserved proteins among eukaryotes, are two essential subunits of protein import motors localized in the inner mitochondrial membrane. The heterodimer formed by Pam18 and Pam16 via their J-type domains serves a regulatory function in protein translocation. Here, we report that thirty-one Pam18 and twenty-six Pam16 putative orthologues in twelve plant species were identified and analyzed through bioinformatics strategy. Results data revealed that Pam18 and Pam16 were also highly conserved among plants including their J-type domains within the hydrophilic region. Key amino acid residues and an HPD motif of Pam18 were identical among the orthologues except OsPam18L5. N-myristoylation sites of Pam18 and casein kinase II phosphorylation sites of Pam 16 were more abundant, which might be important functional sites. Some Pam18 and Pam16 proteins contained a transmembrane region at the N-terminal region. Sub-cellular prediction results indicated that many orthologues localized at mitochondria. Gene expression analyses revealed that Pam18 and Pam16 in Arabidopsis might play roles in senescence and abiotic stress responses. Our detailed study provides a better understanding of Pam18 and Pam16 in plant kingdom.     

Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17

The presequence translocase of the inner mitochondrial membrane (TIM23 complex) operates at a central junction of protein import. It accepts preproteins from the outer membrane TOM complex and directs them to inner membrane insertion or, in cooperation with the presequence translocase-associated motor (PAM), to the matrix. Little is known of how the TIM23 complex coordinates these tasks. We have identified Tim21 (YGR033c) that interacts with the TOM complex. Tim21 is specific for a TIM23 form that cooperates with TOM and promotes inner membrane insertion. Protein translocation into the matrix requires a switch to a Tim21-free, PAM bound presequence translocase. Tim17 is crucial for the switch by performing two separable functions: promotion of inner membrane insertion and binding of Pam18 to form the functional TIM-PAM complex. Thus, the presequence translocase is not a static complex but switches between TOM tethering and PAM binding in a reaction cycle involving Tim21 and Tim17.     

The presequence translocase-associated protein import motor of mitochondria. Pam16 functions in an antagonistic manner to Pam18

Transport of preproteins into the mitochondrial matrix requires the presequence translocase of the inner membrane (TIM23 complex) and the presequence translocase-associated motor (PAM). The motor consists of five essential subunits, the mitochondrial heat shock protein 70 (mtHsp70) and four cochaperones, the nucleotide exchange-factor Mge1, the translocase-associated fulcrum Tim44, the J-protein Pam18, and Pam16. Pam16 forms a complex with Pam18 and displays similarity to J-proteins but lacks the canonical tripeptide motif His-Pro-Asp (HPD). We report that Pam16 does not function as a typical J-domain protein but, rather, antagonizes the function of Pam18. Pam16 specifically inhibits the Pam18-mediated stimulation of the ATPase activity of mtHsp70. The inclusion of the HPD motif in Pam16 does not confer the ability to stimulate mtHsp70 activity. Pam16-HPD fully substitutes for wild-type Pam16 in vitro and in vivo but is not able to replace Pam18. Pam16 represents a new type of cochaperone that controls the stimulatory effect of the J-protein Pam18 and regulates the interaction of mtHsp70 with precursor proteins during import into mitochondria.     

Ups1p and Ups2p antagonistically regulate cardiolipin metabolism in mitochondria

Cardiolipin, a unique phospholipid composed of four fatty acid chains, is located mainly in the mitochondrial inner membrane (IM). Cardiolipin is required for the integrity of several protein complexes in the IM, including the TIM23 translocase, a dynamic complex which mediates protein import into the mitochondria through interactions with the import motor presequence translocase–associated motor (PAM). In this study, we report that two homologous intermembrane space proteins, Ups1p and Ups2p, control cardiolipin metabolism and affect the assembly state of TIM23 and its association with PAM in an opposing manner. In ups1Δ mitochondria, cardiolipin levels were decreased, and the TIM23 translocase showed altered conformation and decreased association with PAM, leading to defects in mitochondrial protein import. Strikingly, loss of Ups2p restored normal cardiolipin levels and rescued TIM23 defects in ups1Δ mitochondria. Furthermore, we observed synthetic growth defects in ups mutants in combination with loss of Pam17p, which controls the integrity of PAM. Our findings provide a novel molecular mechanism for the regulation of cardiolipin metabolism.     

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 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).     

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.     

Distinct Forms of Mitochondrial TOM-TIM Supercomplexes Define Signal-Dependent States of Preprotein Sorting

Mitochondrial import of cleavable preproteins occurs at translocation contact sites, where the translocase of the outer membrane (TOM) associates with the presequence translocase of the inner membrane (TIM23) in a supercomplex. Different views exist on the mechanism of how TIM23 mediates preprotein sorting to either the matrix or inner membrane. On the one hand, two TIM23 forms were proposed, a matrix transport form containing the presequence translocase-associated motor (PAM; TIM23-PAM) and a sorting form containing Tim21 (TIM23^SORT). On the other hand, it was reported that TIM23 and PAM are permanently associated in a single-entity translocase. We have accumulated distinct transport intermediates of preproteins to analyze the translocases in their active, preprotein-carrying state. We identified two different forms of active TOM-TIM23 supercomplexes, TOM-TIM23^SORT and TOM-TIM23-PAM. These two supercomplexes do not represent separate pathways but are in dynamic exchange during preprotein translocation and sorting. Depending on the signals of the preproteins, switches between the different forms of supercomplex and TIM23 are required for the completion of preprotein import.The majority of mitochondrial proteins are nuclear encoded and posttranslationally transported into the organelle. A major class of mitochondrial proteins possess cleavable targeting signals at their amino termini, so-called presequences (5, 9, 12, 19, 30, 32). These α-helical segments are positively charged and direct the proteins across the outer and inner mitochondrial membranes toward the matrix space, where the presequences are proteolytically removed. However, a number of proteins of the inner mitochondrial membrane, among them subunits of the respiratory chain complexes, also utilize presequences as targeting signals. In addition to the presequence, they contain a hydrophobic sorting signal, which arrests precursor translocation across the inner membrane and mediates the lateral release of the polypeptide into the lipid phase (16, 30). In some cases, the membrane-inserted precursors undergo a second processing event by the inner membrane protease that cleaves behind the sorting signal and therefore leads to the release of the protein into the intermembrane space (25, 30, 31). Thus, a large variety of proteins destined for three different intramitochondrial compartments use presequences as the primary signal for transport.Cleavable preproteins initially enter mitochondria via the TOM complex and are translocated into or across the inner membrane by the TIM23 complex. The TIM23 complex consists of four integral membrane proteins, Tim23, Tim17, Tim50, and Tim21. Tim23 forms the protein-conducting channel of the translocase and is tightly associated with Tim17 (8, 26, 43). Tim50 acts as a regulator for the Tim23 channel and is involved in early steps of precursor transfer from the outer to the inner membranes (23, 29, 41). Tim21 transiently interacts with the TOM complex via binding to the intermembrane space domain of Tom22. This interaction promotes the release of presequences from Tom22 for their further transfer to the Tim23 channel (4). For full matrix translocation of preproteins, the TIM23 complex cooperates with PAM. The central subunit of PAM is mtHsp70, which undergoes ATP-dependent cycles of preprotein binding and release to promote polypeptide movement toward the matrix. The activity of mtHsp70 in the translocation process is regulated by four membrane-bound cochaperones, Tim44, the J complex Pam18/Pam16 (Tim14/Tim16), and Pam17. Tim44 provides a binding site for preproteins and mtHsp70 close to the Tim23 channel (1, 17, 22, 36). The J protein Pam18 stimulates the ATPase activity of mtHsp70 (10, 44), whereas the J-related protein Pam16 controls the activity of Pam18 (11, 13, 20). Pam17 plays an organizing role in the TIM23-PAM cooperation (33, 45).The following two different views on the organization of the presequence transport machinery are currently discussed. (i) The TIM23 complex and PAM were proposed to exist in different modular states, termed TIM23^SORT and TIM23-PAM. The TIM23^CORE complex, consisting of Tim23, Tim17 and Tim50, associates with either Tim21 or the subunits of PAM (4, 47, 51). The Tim21-containing form is termed TIM23^SORT since this motor-free form was isolated and shown to mediate membrane insertion of sorted preproteins upon reconstitution (46). The TIM23-PAM form (lacking Tim21) is crucial for mtHsp70-driven preprotein translocation into the matrix (4). (ii) On the other hand, it was proposed that presequence translocase and import motor form a single structural and functional entity. Thus, membrane-integrated TIM23 and import motor would always remain in one complex. This model implies that a motor-free form of the TIM23 complex should not exist (27, 33, 42).To decide between the different views, it is necessary to analyze translocase and motor in their active form, i.e., during their engagement with preproteins. Moreover, the model of modular forms of TIM23 and PAM raises the question whether two strictly separate TIM23 pathways for inner membrane sorting and matrix translocation exist or whether an exchange between the different forms of the presequence translocase occurs. To date, the majority of experimental studies have been performed with the translocases in an inactive, i.e., preprotein-free, state. Studies using preproteins in transit provided only limited information so far and thus did not resolve the controversy, as follows. (i) Mokranjac and Neupert (27) questioned if the in vitro preprotein insertion by purified TIM23^SORT in a proteoliposome assay (46) reflected the in organello situation in intact mitochondria. (ii) Popov-Celeketic et al. (33) accumulated a matrix-targeted preprotein in mitochondrial import sites in vivo and performed pulldown experiments. They copurified TIM23, PAM, and Tim21 and thus concluded that the TIM23 and motor subunits formed a single entity. They did not address the possibility that the accumulated preprotein was associated with different pools of translocase complexes. (iii) Wiedemann et al. (51) made use of the observation that TIM23^SORT associates with the respiratory chain (47). They reported a copurification of inner membrane-sorted preproteins and matrix-targeted preproteins with respiratory chain complexes. This observation raised the possibility that the pathways for inner membrane sorting and matrix translocation are connected at least at the level of respiratory chain interaction; however, the composition of the TIM23 complexes was not analyzed.For this study, we used preproteins with variations in the intramitochondrial sorting signal to monitor the active, preprotein-carrying translocases at distinct stages of mitochondrial import. We observed different forms of active translocases on the presequence pathway. The sorting signals of the preproteins are critical for the selection of specific translocase forms. The motor and sorting forms of the TIM23 complex can be isolated as separate entities in support of the modular model. However, the different TIM23 forms are not permanently separated during preprotein import, but a dynamic exchange between the forms takes place for both matrix-targeted preproteins and inner membrane-sorted preproteins.     

Molecular Insights Revealing Interaction of Tim23 and Channel Subunits of Presequence Translocase

Tim23 is an essential channel-forming subunit of the presequence translocase recruiting multiple components for assembly of the core complex, thereby regulating the protein translocation process. However, understanding of the precise interaction of subunits associating with Tim23 remains largely elusive. Our findings highlight that transmembrane helix 1 (TM1) is required for homodimerization of Tim23, while, together with TM2, it is involved in preprotein binding within the channel. Based on our evidence, we predict that the TM1 and TM2 from each dimer are involved in the formation of the central translocation pore, aided by Tim17. Furthermore, TM2 is also involved in the recruitment of Tim21 and the presequence-associated motor (PAM) subcomplex to the Tim23 channel, while the matrix-exposed loop L1 generates specificity in their association with the core complex. Strikingly, our findings indicate that the C-terminal sequence of Tim23 is dispensable for growth and functions as an inhibitor for binding of Tim21. Our model conceptually explains the cooperative function between Tam41 and Pam17 subunits, while the antagonistic activity of Tim21 predominantly determines the bound and free forms of the PAM subcomplex during import.     

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.     

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.     

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.     

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.     

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.