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

2008; Springer Nature; Linguagem: Inglês

10.1038/emboj.2008.79

1460-2075

Dusbrevean Popov-Čeleketić, Koyeli Mapa, Walter Neupert, Dejana Mokranjac,

Mitochondrial Function and Pathology

Article17 April 2008free access Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria Dušan Popov-Čeleketić Dušan Popov-Čeleketić Munich Center for Integrated Protein Science, Institute for Physiological Chemistry, University of Munich, Munich, Germany Search for more papers by this author Koyeli Mapa Koyeli Mapa Munich Center for Integrated Protein Science, Institute for Physiological Chemistry, University of Munich, Munich, Germany Search for more papers by this author Walter Neupert Corresponding Author Walter Neupert Munich Center for Integrated Protein Science, Institute for Physiological Chemistry, University of Munich, Munich, Germany Search for more papers by this author Dejana Mokranjac Dejana Mokranjac Munich Center for Integrated Protein Science, Institute for Physiological Chemistry, University of Munich, Munich, Germany Search for more papers by this author Dušan Popov-Čeleketić Dušan Popov-Čeleketić Munich Center for Integrated Protein Science, Institute for Physiological Chemistry, University of Munich, Munich, Germany Search for more papers by this author Koyeli Mapa Koyeli Mapa Munich Center for Integrated Protein Science, Institute for Physiological Chemistry, University of Munich, Munich, Germany Search for more papers by this author Walter Neupert Corresponding Author Walter Neupert Munich Center for Integrated Protein Science, Institute for Physiological Chemistry, University of Munich, Munich, Germany Search for more papers by this author Dejana Mokranjac Dejana Mokranjac Munich Center for Integrated Protein Science, Institute for Physiological Chemistry, University of Munich, Munich, Germany Search for more papers by this author Author Information Dušan Popov-Čeleketić1,‡, Koyeli Mapa1,‡, Walter Neupert 1 and Dejana Mokranjac1 1Munich Center for Integrated Protein Science, Institute for Physiological Chemistry, University of Munich, Munich, Germany ‡These authors contributed equally to this work *Corresponding author. Adolf-Butenandt-Institut, Lehrstuhl für Physiologische Chemie, Ludwig-Maximilians-Universität, Butenandtstr. 5, Munchen 81377, Germany. Tel.: +49 89 2180 77095; Fax: +49 89 2180 77093; E-mail: [email protected] The EMBO Journal (2008)27:1469-1480https://doi.org/10.1038/emboj.2008.79 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Almost all eukaryotic proteins are synthesized on cytoplasmic ribosomes. To reach the sites of their function, roughly half of them are synthesized as preproteins with targeting signals, which lead to their subsequent transport into or across organellar membranes in the cell (Schnell and Hebert, 2003; Wickner and Schekman, 2005). Similarly, in bacterial cells all proteins are synthesized in the cytosol irrespective of where their site of function is. Protein translocases present in the membranes of cell organelles and bacteria are responsible for recognition and translocation of preproteins. Whereas some translocases appear to have specialized for either translocation of proteins across or their insertion into a membrane, some of them can perform both functions (Schnell and Hebert, 2003; Alder and Johnson, 2004). However, very little is known about the mechanisms involved in switching between translocation and insertion modes. The TIM23 (translocase of the mitochondrial inner membrane) complex is one of the translocases in the cell that can operate both in translocation and in insertion modes. It collects preproteins carrying N-terminal, positively charged presequences as soon as they emerge at the outlet of the TOM (translocase of the mitochondrial outer membrane) complex. Using energy of the membrane potential across the inner mitochondrial membrane and ATP in the mitochondrial matrix, it mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane (Endo et al, 2003; Koehler, 2004; Rehling et al, 2004; Neupert and Herrmann, 2007). Tim50 is the receptor subunit of the complex that takes over preproteins from the TOM complex and directs them to the translocation channel in the inner membrane formed by Tim23 and Tim17 (Geissler et al, 2002; Yamamoto et al, 2002; Mokranjac et al, 2003a). Tim23 also has a domain located in the intermembrane space that serves as an additional preprotein receptor of the complex (Bauer et al, 1996). At the matrix face of the channel, preproteins are recognized by Tim44 and mtHsp70 (Neupert and Brunner, 2002). mtHsp70 belongs to the Hsp70 family of chaperones and is the ATP-consuming subunit of the complex. It cycles between ATP- and ADP-bound states, which correspond to low- and high-affinity states for incoming preproteins. mtHsp70 hydrolysis of ATP to ADP and thereby the tight binding of the chaperone to the preprotein is regulated by the J and J-like proteins, Tim14 (Pam18) and Tim16 (Pam16) (D'Silva et al, 2003; Mokranjac et al, 2003b, 2006; Truscott et al, 2003; Frazier et al, 2004; Kozany et al, 2004). The nucleotide exchange factor, Mge1 facilitates the release of ADP from mtHsp70 and thereby starts a new cycle of the chaperone binding. In this way, the energy of ATP hydrolysis is converted into unidirectional transport of preproteins into mitochondria. Operationally, subunits of the TIM23 complex are divided into those which form the membrane-integrated part of the complex (Tim17, Tim23 and Tim50) and those which form the import motor/PAM (presequence translocase-associated motor) (Tim14, Tim16, Tim44, mtHsp70 and Mge1). Recently, two further subunits of the TIM23 complex were discovered. In contrast to all other subunits described above, these are not essential for viability of yeast cells. Tim21 was described as a subunit of the membrane-integrated part of the translocase involved in the cooperation of TOM and TIM23 complexes (Chacinska et al, 2005; Mokranjac et al, 2005). Pam17 was proposed to be part of the import motor (van der Laan et al, 2005), but its function has remained largely unclear. Another protein, Tam41/Mmp37 was recently demonstrated to affect preprotein translocation through the TIM23 complex; however, no direct interaction with the complex has been observed (Gallas et al, 2006; Tamura et al, 2006). Nearly all known substrates of the TIM23 complex have an N-terminal presequence that is necessary and sufficient to target a passenger protein into the mitochondrial matrix (Hurt et al, 1984; Ostermann et al, 1989). Such preproteins contain no other targeting/sorting signals indicating that transport into the matrix is the default mode of the translocase. A group of TIM23 substrates have, in addition to the presequence, a stop-transfer signal which, when recognized by the TIM23 complex, halts the transport into the matrix. The complex then laterally opens to insert the hydrophobic segment into the inner membrane (Glick et al, 1992). Recently, a model was proposed as to how the TIM23 complex manages this differential sorting of preproteins (Chacinska et al, 2005). According to this model, translocation into the matrix requires reversible assembly of the TIM23 complex with the import motor, whereas insertion into the inner membrane is mediated by a motor-free complex. Tim21 was identified as a component specifically present in the TIM23 complex that promotes insertion into the inner membrane. Preprotein translocation into the matrix was suggested to require a switch to a Tim21-free but the motor-bound form of the TIM23 complex. On the other hand, another group showed that Tim21 is present in the TIM23 complex that also contains the components of the import motor (Tamura et al, 2006). Evidently, how the TIM23 complex manages to sort preproteins into two different compartments remains unclear. Here, we describe experiments in which we analysed the TIM23 complex trapped in vivo in different modes of translocation. We found no evidence for the existence of a motor-free form of the translocase. In contrast, our results show that the TIM23 complex undergoes a series of conformational changes in response to the presence and the type of the translocating preprotein. Furthermore, we found that both non-essential components of the TIM23 complex, Tim21 and Pam17, bind to the Tim17–Tim23 core of the translocase. Unexpectedly, we obtained evidence that Tim21 and Pam17 are functionally connected and have antagonistic roles in the TIM23 complex. Our data show that the TIM23 complex functions as a single structural and functional entity that is actively remodelled to sort different types of preproteins into the matrix or the inner membrane. Results Composition of the TIM23 complex during protein translocation To address the question as to how the TIM23 complex sorts preproteins into different mitochondrial subcompartments, we have set out to analyse its composition and conformation in different states of activity. To this end, we developed a method to in vivo trap the TIM23 complex in different translocation states. First, we generated the empty state of the translocase by treating yeast cells with puromycin to terminate protein synthesis and allow the truncated polypeptide chains to be completely imported (+PUR) (Figure 1A). Mitochondria were isolated also from cells grown under standard conditions, that is, without any further treatment. This served as a control for the state of the TIM23 complex prevailing under the usual conditions of analysis of preprotein import (STD). To investigate the effects of translocating preproteins on the TIM23 complex, we trapped in the complex different hybrid preproteins whose import pathways were described previously (Geissler et al, 2000 and references therein). The first one, abbreviated as b2, consists of the N-terminal 167 amino-acid residues of yeast cytochrome b2 fused to full-length dihydrofolate reductase (DHFR) from mouse. This preprotein has a matrix-targeting signal at its N terminus followed by a hydrophobic stop-transfer signal, which directs it into the inner membrane. The second preprotein, indicated as b2Δ, lacks the hydrophobic-sorting signal and the preprotein is completely translocated into the matrix. When expression of these proteins is induced in the presence of the folate analogue, aminopterine, the DHFR moieties fold stably in the cytosol (Wienhues et al, 1991). This prevents complete import into mitochondria leading to accumulation of both types of preproteins as intermediates that span and connect both TOM and TIM23 complexes (Figure 1A). In case of b2, the TIM23 complex is locked in the state of lateral sorting and in the case of b2Δ in the state of translocation into the matrix. Both preproteins were expressed though the expression levels were somewhat higher for b2Δ (Supplementary Figure S1A). The import of radioactive preproteins by the TIM23 complex was strongly inhibited, demonstrating that both preproteins were efficiently arrested and that the majority of complexes contained trapped preproteins (Supplementary Figure S1B). Interestingly, import of both laterally sorted and matrix-targeted preproteins was inhibited to a similar extent, indicating that the same pool of the TIM23 complexes mediates both functions. In contrast, import of TIM23-independent substrates, such as ADP–ATP carrier, was affected only mildly, likely due to the partial occupancy of the TOM complexes. Figure 1.Composition of the TIM23 complex during translocation of preproteins. (A) Schematic representation of the different states of the TIM23 complex analysed. See text for details. OM, outer mitochondrial membrane; IMS, intermembrane space; IM, inner mitochondrial membrane. (B) Mitochondria were solubilized with digitonin and immunoprecipitated with affinity-purified antibodies to Tim16 or Tim17 or preimmune (PI) serum as a control. Samples were analysed by SDS–PAGE and immunodecoration. Here, 20% of the material used for immunoprecipitations was loaded as total. The amounts of Tim23 and Tim14 precipitated with antibodies to Tim16 and Tim17, respectively, under these conditions were quantified from three independent experiments (lower panels). Precipitation in STD, 100%. (C) Mitochondria were solubilized with digitonin and analysed by BN-PAGE and immunodecoration with antibodies to Tim17. Download figure Download PowerPoint To analyse possible changes in the composition of the TIM23 complex in response to protein translocation, we performed immunoprecipitation experiments. Mitochondria were solubilized with digitonin and incubated with affinity-purified antibodies to Tim17 and Tim16 or preimmune immunoglobulins bound to ProteinA-Sepharose. We have chosen antibodies to Tim17 and Tim16, as we have previously shown that they are able to immunodeplete their respective antigens and also to precipitate all other known components of the TIM23 complex with, however, different efficiencies due to the reported instability of the complex upon solubilization (Kozany et al, 2004). Under these conditions, the precipitation patterns of the essential components of the TIM23 complex, Tim50, Tim23, Tim17, Tim44, Tim14 and Tim16 were essentially identical in all four types of mitochondria analysed (Figure 1B). In the empty state of the translocase, the import motor and the membrane-integrated part were clearly present in one complex. Apparently, the translocating chain is not required for association of the two parts of the complex. Furthermore, translocating preproteins did neither stabilize nor destabilize the association of various TIM23 components. Translocating preproteins were, however, required to connect TIM23 and TOM complexes. In the presence of b2Δ, TOM and TIM23 complexes were associated stably enough to be detectable as a supercomplex both by coimmunoprecipitation (Figure 1B) and by Blue Native electrophoresis (BN-PAGE) (Figure 1C). Interestingly, the interaction of b2 with the TIM23 complex is not strong enough to allow its coisolation with the complex upon solubilization of mitochondria. This was not unexpected in view of the partitioning of such substrates between the TIM23 channel and the lipid phase of the membrane. This behaviour did not depend on the length of the laterally sorted preprotein in front of the DHFR passenger; none of the tested b2 constructs ranging from 87 to 220 residues was coisolated with the TIM23 complex. However, laterally sorted preproteins were found stably associated with the TOM complex (see below) and they were apparently present in close vicinity to the TIM23 complex as they could be crosslinked to it (data not shown). These results strongly argue against any model according to which the membrane part and the motor associate only in the presence of a preprotein in transit into the matrix. Rather, they support a mechanistic model of the TIM23 complex in which all essential components form a non-transient assembly with the various components undergoing dynamic rearrangements depending on the state of activity. Tim23 inserts into the outer membrane in response to preprotein translocation Can one use mitochondria containing the TIM23 complex trapped in various states of its function to show that these states are indeed different and if so, to obtain insights into how they look like? We have previously shown that the N-terminal segment of Tim23 is exposed on the surface of mitochondria and have suggested that it contributes to the coordinated action of TOM and TIM23 complexes during translocation of preproteins into mitochondria (Donzeau et al, 2000). However, this view has been challenged (Chacinska et al, 2005). We have therefore analysed whether the association of Tim23 with the outer membrane is influenced by the translocation activity of the TIM23 complex. After 10-min incubation with proteinase K, ca. 5% of Tim23 was accessible to protease added to mitochondria isolated from puromycin-treated cells and ca. 10% in control mitochondria (Figure 2A). In contrast, in mitochondria with arrested b2 roughly 35% of Tim23 was clipped by externally added protease and ca. 45% in mitochondria with arrested b2Δ. The intactness of mitochondria was not compromised under these conditions as the accessibilities of marker proteins of the outer membrane (Tom70), the intermembrane space (Tim50) and the matrix (Hep1) were not changed. To confirm that the accessibility of Tim23 correlates with an increased translocation load, we incubated mitochondria isolated from puromycin-treated cells with recombinant preprotein b2Δ. Indeed, addition of increasing amounts of purified b2Δ to mitochondria led to increased insertion of Tim23 into the outer membrane as documented by the strongly increased efficiency of clipping of Tim23 (Figure 2B). Upon removal of the translocating chain from the TIM23 complex, clipping of Tim23 returned to the level observed with mitochondria containing empty TIM23 complex (Figure 2C). Taken together, these data clearly show that the N-terminal segment of Tim23 actively responds to translocation of preproteins through the TIM23 complex (Figure 2D). Figure 2.Exposure of Tim23 on the mitochondrial surface during translocation of preproteins. (A) Mitochondria were treated with proteinase K (PK) and analysed by SDS–PAGE and immunodecoration. The percentage of Tim23 clipped under these conditions was quantified from three independent experiments (right panel). (B) Mitochondria isolated from puromycin-treated cells were incubated with increasing amounts of recombinant preprotein b2Δ (0–100 μg), treated with PK and analysed as described in (A). (C) Mitochondria isolated from puromycin-treated cells were incubated with or without recombinant preprotein b2Δ in the presence of dihydrofolate and NADPH. Dihydrofolate and NADPH were subsequently removed from one portion of the sample (chase) and further incubated to clear the TIM23 complex from preproteins. Samples were then treated with PK and analysed as described in (A). (D) Schematic presentation showing the dynamic interaction of the N-terminal segment of Tim23 with the outer membrane during translocation of preproteins. Download figure Download PowerPoint Conformational changes of the TIM23 complex during translocation of preproteins The different exposure of Tim23 on the mitochondrial surface in the empty and occupied states of the TIM23 complex demonstrates that the translocase reacts to the presence of the translocating preprotein. However, does the response differ if the translocating preprotein is destined to the matrix or to the inner membrane? We used protein crosslinking in intact mitochondria to probe the molecular environment of TIM23 components in the empty and occupied states of the translocase. In mitochondria under standard condition of analysis, Tim23 gives one major crosslinking adduct to a protein of ca. 17 kDa (Figure 3A; Bauer et al, 1996). Using mitochondria containing His-tagged Pam17, we identified this major crosslinking product as an adduct of Tim23 to Pam17 (Supplementary Figure S2), an unexpected result in view of the recently reported role of Pam17 as a component of the import motor (van der Laan et al, 2005). Does this crosslinking pattern change in different functional states of the TIM23 complex? Crosslinking of Tim23 in mitochondria from control cells and cells treated with puromycin gave essentially the same result. Together with the above-described accessibility of Tim23 to the protease this shows that the TIM23 complex is largely empty in mitochondria isolated under standard conditions (Figure 2B). In contrast, clear differences were visible in mitochondria containing arrested preproteins. Arrest of laterally sorted b2 led to a pronounced increase in the visibility of previously observed Tim23 dimers (Bauer et al, 1996). On the other hand, Tim23 was not crosslinked to any protein upon arrest of matrix-targeted b2Δ. The TIM23 complex thus clearly undergoes conformational changes in response to the translocating chain. Furthermore, these changes seem to depend on the type of the translocating preprotein. Figure 3.Conformation of the TIM23 complex during translocation of preproteins. (A–D) Mitochondria containing TIM23 complex in different translocation states were crosslinked with disuccinimidyl glutarate (DSG) (A, B, D) or disuccinimidyl suberate (DSS) (C). Samples were analysed by SDS–PAGE and immunodecoration with affinity-purified antibodies to Tim23 (A, B), Tim44 (C) or Tim16 (D). Mitochondria containing accumulated b2 and b2Δ (A, C, D) or Cox5a and Cox5aΔTM (B) were used. Download figure Download PowerPoint To confirm that these various crosslinking patterns of Tim23 indeed represent different conformations of the complex in different modes of translocation and are not just a reflection of specific interactions of a certain preprotein with the complex, we analysed another couple of differentially sorted preproteins. The hybrid protein Cox5aDHFR, abbreviated Cox5a, is laterally sorted into the inner membrane by the TIM23 complex, whereas Cox5a(ΔTM)DHFR, abbreviated Cox5aΔTM, lacks the transmembrane domain and is targeted to the matrix (Gärtner et al, 1995). Arrest of these two proteins induced essentially the same changes in the crosslinking patterns of Tim23 as the arrest of b2 and b2Δ (Figure 3B). This clearly demonstrates that the observed changes represent genuine differences in the conformation of the complex due to the different translocation modes. Are other components of the TIM23 complex rearranging during translocation of preproteins as well? We analysed the molecular environments of Tim44 and Tim16, two proteins whose crosslinking patterns we have previously characterized. Analysis of the environment of Tim44 revealed several changes in the active translocase as compared with its empty state (Figure 3C). In mitochondria containing arrested b2 or b2Δ, crosslinks between two Tim44, crosslinks between Tim44 and Tim14 as well as Tim44 and Tim16 were strongly decreased. The prominent crosslinking product of ca. 80 kDa in mitochondria containing arrested b2Δ represented an adduct of Tim44 to the preprotein. This was demonstrated by arresting His-tagged b2Δ in vitro followed by crosslinking and NiNTA-Agarose pull down (Supplementary Figure S3). In case of Tim16, the most prominent difference between control mitochondria and mitochondria saturated with preproteins was the reduced crosslinking to Tim14, in particular in mitochondria containing arrested b2Δ (Figure 3D). Taken together, the crosslinking experiments demonstrate that the TIM23 complex actively responds to the translocating preproteins and that the nature of this response depends on the type of preprotein being translocated. Behaviour of Tim21 and Pam17 during translocation of preproteins through the TIM23 complex So far we have analysed only the components of the TIM23 complex that are essential for the function of the translocase and therefore are all also essential for the viability of yeast cells. What is happening with the two non-essential components, Tim21 and Pam17? Conflicting results were obtained concerning the presence of Tim21 in the TIM23 complex actively involved in translocation of preproteins into the matrix. In one study, Tim21 was absent from such a complex (Chacinska et al, 2005), whereas in the other it was present (Mokranjac et al, 2005). As in both cases tagged forms of Tim21 were analysed, we have reassessed the issue. We used immunoprecipitation experiments to analyse the association of Tim21 and Pam17 with the TIM23 complex in various states of its function. We found Tim21 associated with the complex in all states of activity tested (Figure 4A). In contrast, Pam17 was present in the empty complex and in the complex saturated with b2, but was absent in the complex saturated with b2Δ. This is in agreement with the observed crosslinking pattern of Tim23 to Pam17 under the same conditions (Figure 3A). Figure 4.Association of Tim21 and Pam17 with the TIM23 complex in different states of translocation. (A) Mitochondria containing the TIM23 complex in various states of activity were solubilized with digitonin and subjected to immunoprecipitation with affinity-purified antibodies to Tim16, Tim17 or preimmune (PI) serum as a control. Total (20%) and immunoprecipitated material were analysed by SDS–PAGE and immunodecoration with antibodies to Tim21 and Pam17. Right panel: amounts of Tim21 and Pam17 precipitated under these conditions were quantified from three independent experiments. Precipitation in STD, 100%. (B) Mitochondria containing in vivo-arrested His-tagged versions of b2 or b2Δ were solubilized with digitonin and incubated with NiNTA-Agarose beads. T, total; S, supernatant and B, bound fractions were analysed by SDS–PAGE and immunodecoration. Total and supernatant, 5%. Download figure Download PowerPoint To further study the behaviour of Tim21 and Pam17 in response to the presence of a translocating preprotein, we expressed and arrested His-tagged versions of b2 and b2Δ in intact cells, isolated the mitochondria, lysed them with detergent and analysed for interacting components by binding to NiNTA-Agarose. Indeed, Tim21 was specifically retained on the beads together with the components of the TOM–TIM23 supercomplex linked by a translocating matrix-destined preprotein. Pam17 was absent from the TIM23 complex actively translocating preproteins into the matrix also when analysed by this procedure (Figure 4B). On the other hand, the TOM–TIM23 supercomplex generated in the presence of laterally sorted preprotein b2 is apparently less stable as compared with the one generated in the presence of b2Δ, in agreement with above-described results obtained by coimmunoprecipitation and BN-PAGE. In this case, the TIM23 components were recovered on the beads only slightly above background. Interestingly, the TOM complex components, exemplified here by TOM40, were equally efficiently associated with both types of preproteins, demonstrating that it is the association of the laterally sorted preprotein with the TIM23 complex that is labile. This further supports the notion of different conformations of the TIM23 complex involved in lateral insertion and matrix translocation. Both Tim21 and Pam17 interact with the Tim17–Tim23 core of the TIM23 complex In view of a rather surprising absence of Pam17 from TIM23 complex engaged in translocation into the matrix, a process which requires the activity of the import motor, and of the observed crosslink of Pam17 to Tim23, we decided to analyse the association Pam17 and Tim21 with the TIM23 complex in more detail. Wild-type mitochondria were lysed with digitonin and subjected to immunoprecipitation with antibodies to Tim16, Tim17 and Tim23 (Figure 5A). The majority of Tim21 in the mitochondrial extract was co-precipitated with antibodies to Tim17 and Tim23. Small but detectable amounts could also be precipitated with antibodies to Tim16. In contrast, the majority of Pam17 remained in the supernatant, irrespective of which antibody was used for precipitation, and only small amounts were detected in the pellets after precipitation with antibodies to Tim17 and Tim23, but not with antibodies to the motor component Tim16. Thus, it appears that the majority of Tim21 but only minor amounts of Pam17 are associated with the TIM23 complex or that the association of the latter one is very weak. Figure 5.Tim21 and Pam17 interact with the Tim17–Tim23 core of the TIM23 complex. (A) Wild-type mitochondria were solubilized with digitonin and immunoprecipitated with the affinity-purified antibodies to Tim16, Tim17 or antibodies from preimmune (PI) serum as a control. Samples were analysed by SDS–PAGE and immunodecoration. For simplicity reasons, only decorations with the antibodies to Tim21 and Pam17 are shown. Total and supernatant fractions represent 20% of the material used for immunoprecipitations. (B–G) Mitochondria were isolated from cells depleted of the indicated TIM23 components and analysed by immunoprecipitation as described under (A). Only total and immunoprecipitated materials are shown. (H) Mitochondria, isolated from wild-type cells and cells expressing Tim21 with C-terminal His or ProteinA tag, were solubilized with digitonin and incubated with NiNTA-Agarose or IgG-Sepharose beads. Samples were analysed by SDS–PAGE and immunodecoration. T, total and S, supernatant fractions contain 5% of the material present in B, bound fraction. (I) Mitochondria isolated from wild type and ce