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.     

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.     

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.     

Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) from bovine heart is a complicated multisubunit, membrane-bound assembly. Seven subunits are encoded by mitochondrial DNA, and the sequences of 36 nuclear encoded subunits have been described. The subunits of complex I and two subcomplexes (Ialpha and Ibeta) were resolved on one- and two-dimensional gels and by reverse-phase high performance liquid chromatography. Mass spectrometric analysis revealed two previously unknown subunits in complex I, named B14.7 and ESSS, one in each subcomplex. Coding sequences for each protein were identified in data bases and were confirmed by cDNA cloning and sequencing. Subunit B14.7 has an acetylated N terminus, no presequence, and contains four potential transmembrane helices. It is homologous to subunit 21.3b from complex I in Neurospora crassa and is related to Tim17, Tim22, and Tim23, which are involved in protein translocation across the inner membrane. Subunit ESSS has a cleaved mitochondrial import sequence and one potential transmembrane helix. A total of 45 different subunits of bovine complex I have now been characterized.     

The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44.

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70-Tim44 driving system. By blue native electrophoresis, we identify an approximately 90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence-containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.     

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.     

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.     

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.     

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.     

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.     

Conserved N-terminal negative charges in the Tim17 subunit of the TIM23 translocase play a critical role in the import of preproteins into mitochondria

The TIM23 complex of the mitochondrial inner membrane mediates the import of preproteins that contain positively charged targeting signals. This translocase consists of the two phylogenetically related membrane-embedded subunits Tim17 and Tim23 to which four largely hydrophilic subunits, Tim50, Tim44, Tim16, and Tim14, are attached. Whereas in vitro reconstitution experiments have suggested a pore-forming capacity of recombinant Tim23, virtually nothing is known about the properties and function of Tim17. We employed a combined genetic and biochemical approach to address the function of Tim17 in preprotein translocation. Tim17 exposes an N-terminal hydrophilic stretch into the intermembrane space. Truncation of the first 11 amino acid residues of this stretch did not affect the stability or integrity of TIM23 subunits but strongly impaired the import of preproteins. Moreover, expression of the truncated Tim17 variant led to a dominant negative effect on the mitochondrial membrane potential. By an alanine-scanning approach we identified two conserved negative charges in the N terminus of Tim17 as critical for Tim17 function. The replacement of these positions by positively charged residues results in a strong growth defect, which can be cured by reverting two conserved positive charges into aspartate residues between transmembrane domains two and three of Tim17. On the basis of these observations we propose that charged residues in Tim17 are critical for the preprotein-induced gating of the TIM23 translocase.     

A fluorescence assay for peptide translocation into mitochondria

Translocation of the presequence is an early event in import of preproteins across the mitochondrial inner membrane by the TIM23 complex. Import of signal peptides, whose sequences mimic mitochondrial import presequences, was measured using a novel, qualitative, fluorescence assay in about 1h. This peptide assay was used in conjunction with classical protein import analyses and electrophysiological approaches to examine the mechanisms underlying the functional effects of depleting two TIM23 complex components. Tim23p forms, at least in part, the pore of this complex while Tim44p forms part of the translocation motor. Depletion of Tim23p eliminates TIM23 channel activity, which interferes with both peptide and preprotein translocation. In contrast, depletion of Tim44p disrupts preprotein but not peptide translocation, which has no effect on TIM23 channel activity. Two conclusions were made. First, this fluorescence peptide assay was validated as two different mutants were accurately identified. Hence, this assay could provide a rapid means of screening mutants to identify those that fail an initial step in import, i.e., translocation of the presequence. Second, translocation of signal peptides required normal channel activity and disruption of the presequence translocase-associated motor complex did not modify TIM23 channel activity nor prevent presequence translocation.     

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.     

Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM-TIM-preprotein supercomplex

Preproteins with N-terminal presequences are imported into mitochondria at translocation contact sites that include the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Little is known about the functional cooperation of these translocases. We have characterized translocation contact sites by a productive TOM-TIM-preprotein supercomplex to address the role of three translocase subunits that expose domains to the intermembrane space (IMS). The IMS domain of the receptor Tom22 is required for stabilization of the translocation contact site supercomplex. Surprisingly, the N-terminal segment of the channel Tim23, which tethers the TIM23 complex to the outer membrane, is dispensable for both protein import and generation of the TOM-TIM supercomplex. Tim50, with its large IMS domain, is crucial for generation but not for stabilization of the supercomplex. Thus, Tim50 functions as a dynamic factor and the IMS domain of Tom22 represents a stabilizing element in formation of a productive translocation contact site supercomplex.     

The mitochondrial import machinery: preprotein-conducting channels with binding sites for presequences

Mitochondrial preproteins with amino-terminal presequences must cross two membranes to reach the matrix of the organelle. Both outer and inner membranes contain hydrophilic high-conductance channels that are responsible for selective translocation of preproteins. The channels are embedded in dynamic protein complexes, the TOM complex of the outer membrane and the TIM23 complex of the inner membrane. Both channel-forming proteins, Tom40 and Tim23, carry specific binding sites for presequences, but differ in their pore size and response to a membrane potential. Studies with the TOM machinery show that other subunits of the translocase complex also provide specific binding sites for preproteins, modulate the channel activity and are critical for assembly of the channel.     

Membrane binding mechanism of yeast mitochondrial peripheral membrane protein TIM44

The protein translocations across mitochondrial membranes are carried out by specialized complexes, the Translocase of Outer Membrane (TOM) and Translocase of Inner Membrane (TIM). TIM23 translocon is responsible for translocating the mitochondrial matrix proteins across the mitochondrial inner membrane. Tim44 is an essential, peripheral membrane protein in TIM23 complex. Tim44 is tightly associated with the inner mitochondrial membrane on the matrix side. The Tim44 C-Terminal Domain (CTD) functions as an Inner Mitochondrial Membrane (IMM) anchor that recruits the Presequence protein Associated Motor (PAM) to the TIM23 channel. Using X-ray crystallographic and biochemical data, we show that the N-terminal helices A1 and A2 of Tim44 - CTD are crucial for its membrane tethering function. Based on our data, we propose a model showing how the N-terminal A1 and A2 amphipathic helices can either expose their hydrophobic face during membrane binding or conceal it in the soluble form. Therefore, the A1 and A2 helices of Tim44 may function as a membrane sensor.     

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.     

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.     

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

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

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).