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.     

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.     

Tim50's presequence receptor domain is essential for signal driven transport across the TIM23 complex

N-terminal targeting signals (presequences) direct proteins across the TOM complex in the outer mitochondrial membrane and the TIM23 complex in the inner mitochondrial membrane. Presequences provide directionality to the transport process and regulate the transport machineries during translocation. However, surprisingly little is known about how presequence receptors interact with the signals and what role these interactions play during preprotein transport. Here, we identify signal-binding sites of presequence receptors through photo-affinity labeling. Using engineered presequence probes, photo cross-linking sites on mitochondrial proteins were mapped mass spectrometrically, thereby defining a presequence-binding domain of Tim50, a core subunit of the TIM23 complex that is essential for mitochondrial protein import. Our results establish Tim50 as the primary presequence receptor at the inner membrane and show that targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner.     

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 and Tim44 act sequentially in protein import into the mitochondrial matrix

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

Signal recognition initiates reorganization of the presequence translocase during protein import

The mitochondrial presequence translocase interacts with presequence‐containing precursors at the intermembrane space (IMS) side of the inner membrane to mediate their translocation into the matrix. Little is known as too how these matrix‐targeting signals activate the translocase in order to initiate precursor transport. Therefore, we analysed how signal recognition by the presequence translocase initiates reorganization among Tim‐proteins during import. Our analyses revealed that the presequence receptor Tim50 interacts with Tim21 in a signal‐sensitive manner in a process that involves the IMS‐domain of the Tim23 channel. The signal‐driven release of Tim21 from Tim50 promotes recruitment of Pam17 and thus triggers formation of the motor‐associated form of the TIM23 complex required for matrix transport.     

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

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

The Mitochondrial ADP/ATP Carrier Associates with the Inner Membrane Presequence Translocase in a Stoichiometric Manner

The majority of mitochondrial proteins are synthesized with amino-terminal signal sequences. The presequence translocase of the inner membrane (TIM23 complex) mediates the import of these preproteins. The essential TIM23 core complex closely cooperates with partner protein complexes like the presequence translocase-associated import motor and the respiratory chain. The inner mitochondrial membrane also contains a large number of metabolite carriers, but their association with preprotein translocases has been controversial. We performed a comprehensive analysis of the TIM23 interactome based on stable isotope labeling with amino acids in cell culture. Subsequent biochemical studies on identified partner proteins showed that the mitochondrial ADP/ATP carrier associates with the membrane-embedded core of the TIM23 complex in a stoichiometric manner, revealing an unexpected connection of mitochondrial protein biogenesis to metabolite transport. Our data indicate that direct TIM23-AAC coupling may support preprotein import into mitochondria when respiratory activity is low.     

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.     

Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria

The TIM23 (translocase of the mitochondrial inner membrane) complex mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane. How the translocase mediates sorting of preproteins into the two different subcompartments is poorly understood. In particular, it is not clear whether association of two operationally defined parts of the translocase, the membrane-integrated part and the import motor, depends on the activity state of the translocase. We established conditions to in vivo trap the TIM23 complex in different translocation modes. Membrane-integrated part of the complex and import motor were always found in one complex irrespective of whether an arrested preprotein was present or not. Instead, we detected different conformations of the complex in response to the presence and, importantly, the type of preprotein being translocated. Two non-essential subunits of the complex, Tim21 and Pam17, modulate its activity in an antagonistic manner. Our data demonstrate that the TIM23 complex acts as a single structural and functional entity that is actively remodelled to sort preproteins into different mitochondrial subcompartments.     

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.     

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.     

Tim23 links the inner and outer mitochondrial membranes

Tim23, a key component of the mitochondrial preprotein translocase, is anchored in the inner membrane by its C-terminal domain and exposes an intermediate domain in the intermembrane space that functions as a presequence receptor. We show that the N-terminal domain of Tim23 is exposed on the surface of the outer membrane. The two-membrane-spanning topology of Tim23 is a novel characteristic in membrane biology. By the simultaneous integration into two membranes, Tim23 forms contacts between the outer and inner mitochondrial membranes. Tethering the inner membrane translocase to the outer membrane facilitates the transfer of precursor proteins from the TOM complex to the TIM23 complex and increases the efficiency of protein import.     

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

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

Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins.

The essential gene TIM44 encodes a subunit of the inner mitochondrial membrane preprotein translocase that forms a complex with the matrix heat-shock protein Hsp70. The specific role of Tim44 in protein import has not yet been defined because of the lack of means to block its function. Here we report on a Saccharomyces cerevisiae mutant allele of TIM44 that allows selective and efficient inactivation of Tim44 in organello. Surprisingly, the mutant mitochondria are still able to import preproteins. The import rate is only reduced by approximately 30% compared with wild-type as long as the preproteins do not carry stably folded domains. Moreover, the number of import sites is not reduced. However, the mutant mitochondria are strongly impaired in pulling folded domains of preproteins close to the outer membrane and in promoting their unfolding. Our results demonstrate that Tim44 is not an essential structural component of the import channel, but is crucial for import of folded domains. We suggest that the concerted action of Tim44 and mtHsp70 drives unfolding of preproteins and accelerates translocation of loosely folded preproteins. While mtHsp70 is essential for import of both tightly and loosly folded preproteins, Tim44 plays a more specialized role in translocation of tightly folded domains.     

Mgr2 promotes coupling of the mitochondrial presequence translocase to partner complexes

Many mitochondrial proteins are synthesized with N-terminal presequences in the cytosol. The presequence translocase of the inner mitochondrial membrane (TIM23) translocates preproteins into and across the membrane and associates with the matrix-localized import motor. The TIM23 complex consists of three core components and Tim21, which interacts with the translocase of the outer membrane (TOM) and the respiratory chain. We have identified a new subunit of the TIM23 complex, the inner membrane protein Mgr2. Mitochondria lacking Mgr2 were deficient in the Tim21-containing sorting form of the TIM23 complex. Mgr2 was required for binding of Tim21 to TIM23(CORE), revealing a binding chain of TIM23(CORE)-Mgr2/Tim21-respiratory chain. Mgr2-deficient yeast cells were defective in growth at elevated temperature, and the mitochondria were impaired in TOM-TIM23 coupling and the import of presequence-carrying preproteins. We conclude that Mgr2 is a coupling factor of the presequence translocase crucial for cell growth at elevated temperature and for efficient protein import.     

Crystal structure of yeast mitochondrial peripheral membrane protein Tim44p C-terminal domain

The protein transports from the cell cytosol to the mitochondria matrix are carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of TIM23 translocon. Tim44p can tightly associate with the inner mitochondrial membrane. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, we have determined the crystal structure of the yeast Tim44p C-terminal domain to 3.2A resolution using the MAD method. The Tim44p C-terminal domain forms a monomer in the crystal structure and contains six alpha-helices and four antiparallel beta-strands. A large hydrophobic pocket was identified on the Tim44p structure surface. The N-terminal helix A1 is positively charged and the helix A1 protrudes out from the Tim44p main body.     

Structural basis for the function of Tim50 in the mitochondrial presequence translocase

Many mitochondrial proteins are synthesized as preproteins carrying amino-terminal presequences in the cytosol. The preproteins are imported by the translocase of the outer mitochondrial membrane and the presequence translocase of the inner membrane. Tim50 and Tim23 transfer preproteins through the intermembrane space to the inner membrane. We report the crystal structure of the intermembrane space domain of yeast Tim50 to 1.83 Å resolution. A protruding β-hairpin of Tim50 is crucial for interaction with Tim23, providing a molecular basis for the cooperation of Tim50 and Tim23 in preprotein translocation to the protein-conducting channel of the mitochondrial inner membrane.     

The TIM17.23 preprotein translocase of mitochondria: composition and function in protein transport into the matrix.

We have analysed the structural organization of the TIM17.23 complex, the preprotein translocase of the mitochondrial inner membrane specific for protein targeting to the matrix. The components Tim17, Tim23 and Tim44 are present in this complex in equimolar amounts. A sub-complex containing Tim23 and Tim44 but no Tim17, or a sub-complex containing Tim23 and Tim17 but no Tim44 was not detected. Tim44 is peripherally associated at the matrix side. Tim44 forms dimers which recruit two molecules of mt-Hsp70 to the sites of protein import. A sequential, hand-over-hand mode of interaction of these two mt-Hsp70.Tim44 complexes with a translocating polypeptide chain is proposed.