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. 2008 Jun;19(6):2642–2649. doi: 10.1091/mbc.E07-12-1226

Mitochondrial Protein Import Motor: Differential Role of Tim44 in the Recruitment of Pam17 and J-Complex to the Presequence Translocase

Dana P Hutu *,†,‡, Bernard Guiard §, Agnieszka Chacinska *, Dorothea Becker *,†, Nikolaus Pfanner *,✉, Peter Rehling *,‡,✉, Martin van der Laan *
Editor: Benjamin Glick
PMCID: PMC2397324  PMID: 18400944

Abstract

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.

INTRODUCTION

The vast majority of mitochondrial proteins are synthesized on cytosolic ribosomes and subsequently imported into the organelle. Virtually all precursor proteins initially enter the mitochondria via the general translocase of the outer membrane, the TOM complex. At the intermembrane space side of the TOM complex several import pathways diverge. The presequence translocase of the inner membrane (TIM23 complex) is dedicated to the import of preproteins with amino-terminal presequences (Jensen and Johnson, 2001; Koehler, 2004; Dolezal et al., 2006; Bohnert et al., 2007; Neupert and Herrmann, 2007). The channel-forming Tim23 protein and its partner protein Tim17 constitute the membrane-embedded core of the TIM23 complex (Dekker et al., 1997; Truscott et al., 2001). Tim21 and Tim50 expose domains to the intermembrane space and are involved in the transfer of preproteins from the TOM complex to the TIM23 complex (Geissler et al., 2002; Yamamoto et al., 2002; Mokranjac et al., 2003a; Chacinska et al., 2003, 2005; Oka and Mihara, 2005; Perry and Lithgow, 2005; Albrecht et al., 2006). In the absence of a preprotein substrate, Tim50 maintains the Tim23 channel in a closed state (Meinecke et al., 2006).

Translocation of the presequences across the inner membrane depends on the electrochemical gradient (Δp) that activates the Tim23 channel and exerts an electrophoretic effect on the positively charged presequences (Geissler et al., 2000; Truscott et al., 2001; Huang et al., 2002). The ATP-driven presequence translocase-associated motor (PAM) is essential for full translocation of preproteins into the matrix (Jensen and Johnson, 2001; Endo et al., 2003; Koehler, 2004; Dolezal et al., 2006; Bohnert et al., 2007; Dudek et al., 2007; Neupert and Herrmann, 2007). Recent studies have demonstrated that the TIM23 complex exists in two different forms. 1) For preproteins, which carry an inner membrane-sorting signal and are laterally released from the TIM23 complex into the lipid phase, Δp can be used as the only external energy source (van der Laan et al., 2007). This form of the presequence translocase consists of Tim23/Tim17, Tim50, and Tim21 and is termed TIM23SORT (Chacinska et al., 2005; Oka and Mihara, 2005; Perry and Lithgow, 2005; van der Laan et al., 2006, 2007). Tim21 binds to the proton-pumping respiratory chain complexes III and IV to stimulate the Δp-driven preprotein insertion into the inner membrane (van der Laan et al., 2006; Wiedemann et al., 2007). 2) The majority of presequence-carrying preproteins, however, are fully translocated into the matrix. For these preproteins, the motor PAM associates with the TIM23 complex, whereas Tim21 is released, and ATP is used as external energy source in addition to Δp (Chauwin et al., 1998; Chacinska et al., 2005; Oka and Mihara, 2005; Perry and Lithgow, 2005; Bohnert et al., 2007; Neupert and Herrmann, 2007).

The central component of PAM is the mitochondrial heat shock protein 70 (mtHsp70), which generates an inward-directed import driving activity at the expense of ATP. MtHsp70 binds to the TIM23 complex via the adaptor protein Tim44 (Kronidou et al., 1994; Rassow et al., 1994; Schneider et al., 1994). Pam18 belongs to the J-protein family, a family of cochaperones that stimulate the ATPase activity of Hsp70 proteins (Walsh et al., 2004; Young et al., 2004; Bukau et al., 2006). Pam18 is an integral membrane protein, which exposes its J-domain to the mitochondrial matrix to stimulate the activity of mtHsp70 at protein import sites (D'Silva et al., 2003; Mokranjac et al., 2003b; Truscott et al., 2003; Li et al., 2004). Pam18 is associated with the partner protein Pam16 (Frazier et al., 2004; Kozany et al., 2004), which has been classified as J-like protein, as it shows significant homology to members of the J-protein family, but lacks the conserved signature sequence HPD (Walsh et al., 2004). Pam18 and Pam16 form a heterodimeric complex, which is defined here as J-complex (Frazier et al., 2004; Kozany et al., 2004; Li et al., 2004; D'Silva et al., 2005; Mokranjac et al., 2006). Another regulatory subunit of the PAM complex, Pam17, is required for efficient protein import and influences the association of Pam18 and Pam16 in the J-complex (van der Laan et al., 2005).

Different views have been reported on how the motor PAM is recruited to the TIM23 complex. Only one direct interaction site between PAM and the TIM23 complex has been reported so far, the binding of the intermembrane space domain of Pam18 to Tim17 (Chacinska et al., 2005). The significance of this interaction has been questioned by Mokranjac et al. (2006, 2007). A recent study, however, demonstrated that Pam18 binds to Tim17 and additionally interacts with the presequence translocase via a second site that involves Pam16 and likely Tim44 (D'Silva et al., 2008). Tim44 is thought to function as a scaffold, which does not only bind mtHsp70 but also the further PAM subunits, in particular the J-complex (Kozany et al., 2004; Mokranjac et al., 2007; D'Silva et al., 2008). Direct experimental evidence that the activity of Tim44 is needed for the recruitment of Pam proteins has not been obtained so far. Moreover, it is unknown how Pam17 interacts with the presequence translocase.

For this study, we have screened for conditional mutants of TIM44 in order to assess the function of Tim44 for the organization of the PAM complex. We describe a conditional allele, termed tim44-804, which displays a selective import defect for precursors destined for the mitochondrial matrix. The inactivation of Tim44 leads to a reorganization within the TIM23-PAM machinery. The J-complex dissociates from the translocase, while Pam17 binding to Tim23 is strongly enhanced. We propose that Tim44 fulfills a critical function in the dynamics of the mitochondrial import motor, while Tim23 not only functions as protein import channel but also as binding site for the regulatory subunit Pam17.

MATERIALS AND METHODS

Yeast Strains and Growth Conditions

Saccharomyces cerevisiae strain YPH499 (Sikorski and Hieter, 1989) was used as wild-type strain throughout this study. For generation of the mutant strain tim44-804 (YPH-BG-0804) (MATa, ade2-101_ochre, his3-Δ200, leu2-Δ1, ura3-52, trp1-Δ63, lys2-801_amber, tim44::ADE2, [pBG-TIM44-0804]) we constructed a tim44Δ strain that was complemented by a plasmid-borne copy of TIM44 under control of the MET25 promotor and followed by the CYC1 terminator. PCR mutagenesis was used to introduce random nucleotide sequence alterations into the TIM44 gene (Leung et al., 1989). The mutated PCR fragment was transformed together with a gapped plasmid into the complemented tim44Δ strain. Transformants were selected and subsequently cured from plasmids carrying the wild-type TIM44 by selection on plates containing 5-fluoroorotic acid (5-FOA; Sikorski and Boeke, 1991). Plasmids that led to a temperature-sensitive growth phenotype were isolated from the transformants and reintroduced into the tim44Δ strain to confirm the authenticity of the phenotype. Selected mutants were subjected to a biochemical screening protocol. Mitochondria were isolated from cells that were grown under permissive conditions. Isolated mitochondria were analyzed for steady-state protein composition, wild-type-like membrane potential, import of mitochondrial precursor proteins into various subcompartments, stability of mitochondrial protein complexes, and intactness of mitochondria by protease treatment. Sequence analysis of the tim44-804 allele revealed the following amino acid alterations compared with the wild-type Tim44 protein: K59E, Q66R, N240I, P249S, F276S, and V397A.

pam16-1, tom22-2, pam17Δ, and tim21Δ yeast mutant strains have been described (Frazier et al., 2003, 2004; Chacinska et al., 2005; van der Laan et al., 2005). Mutant and corresponding wild-type cells were grown at 24°C (tim44-804, pam16-1, pam17Δ) or 30°C (tom22-2, tim21Δ) in YPG medium (1% [wt/vol] yeast extract, 2% [wt/vol] Bacto Peptone, and 3% [vol/vol] glycerol).

Isolation of Mitochondria and In Vitro Import of 35S-labeled Preproteins

Mitochondria were isolated by differential centrifugation (Meisinger et al., 2006) and resuspended in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS-KOH, pH 7.2) at a protein concentration of 10 mg/ml. For preincubation at high temperature mitochondria were incubated for 15 min at 37°C. 35S-labeled preproteins were synthesized by in vitro transcription and translation using the TNT SP6 Quick Coupled kit (Promega, Madison, WI) in the presence of [35S]methionine (GE Healthcare, Waukesha, WI) and imported into isolated mitochondria in BSA import buffer (250 mM sucrose, 80 mM KCl, 3% [wt/vol] bovine serum albumin, 5 mM MgCl2, 2 mM KH2PO4, 5 mM methionine, 10 mM MOPS-KOH, pH 7.2) supplemented with 2 mM ATP and 2 mM NADH. Where indicated the proton-motive force (Δp) was dissipated by addition of 1 μM valinomycin, 8 μM antimycin, and 20 μM oligomycin. For protease treatment mitochondria were incubated with 50 μg/ml proteinase K for 15 min on ice. The protease was subsequently inhibited by the addition of 2 mM phenylmethylsulfonyl fluoride.

Coimmunoprecipitation

Polyclonal antibodies raised against Tim23 were covalently coupled to protein A-Sepharose beads using dimethyl pimelimidate. For coimmunoprecipitations, mitochondria were resuspended in lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM EDTA, and 1% digitonin) and gently shaken for 20 min at 4°C. After a clarifying spin, supernatants were incubated with antibody-coated beads for 1 h at 4°C. Bound proteins were eluted with 100 mM glycine, pH 2.5, precipitated with 10% trichloroacetic acid (TCA), and analyzed by SDS-PAGE and Western blotting.

Chemical Cross-Linking

The homobifunctional, amine-reactive agents disuccinimidyl glutarate (DSG, 0.5 mM final concentration) and ethylene glycol bis-succinimidyl succinate (EGS, 0.25 mM final concentration) were used for chemical cross-linking experiments. Mitochondria were resuspended in import buffer without BSA for DSG cross-linking or KPS buffer (250 mM sucrose, 50 mM potassium phosphate, pH 8.5) for EGS treatment. Cross-linking reagents were added from 100-fold stock solutions in dimethylsulfoxide (DMSO). Control reactions without cross-linker received the identical amount of solvent. Samples were incubated for 30 min at 4°C, and reactions were stopped by addition of glycine, pH 8.0 (0.1 M final concentration).

Miscellaneous

Mitochondrial protein complexes were analyzed by blue native electrophoresis essentially as described (Dekker et al., 1997). After protein separation on SDS polyacrylamide gels, proteins were transferred to PVDF membranes. Standard techniques were applied for Western blotting and protein–antibody complexes were detected by enhanced chemiluminescence (GE Healthcare).

RESULTS

A Conditional Mutant of TIM44 with a Selective Defect in Preprotein Import into the Matrix

Cross-linking experiments showed that Pam18/Pam16 are in close proximity to Tim44 (Kozany et al., 2004; Mokranjac et al., 2007), yet the function of Tim44 in recruiting Pam proteins has so far only been studied by the use of Tim44-depletion mutants (Mokranjac et al., 2003b; Kozany et al., 2004) and suppressor mutants of TIM44 in a pam16 mutant background (D'Silva et al., 2008). Because in the depletion experiments the cells have to grow for many hours to gradually decrease the amounts of Tim44, indirect effects of the loss of Tim44 cannot be excluded. We thus screened for temperature-conditional mutants of the essential gene TIM44. The mutants should allow growth of the cells at permissive (low) temperature to minimize indirect effects on mitochondrial composition and function. The mutant phenotype would then be induced by a short temperature shift of the isolated mitochondria.

We generated mutants of TIM44 in the yeast S. cerevisiae by error-prone PCR and plasmid shuffling (Leung et al., 1989; Sikorski and Boeke, 1991). Mutants were tested for their growth behavior at 24 versus 37°C by replica plating. Mitochondria were isolated from different mutant strains and analyzed for steady-state levels of proteins and protein complexes, for integrity of the membrane potential across the inner mitochondrial membrane, and for preprotein import activity (data not shown). We selected the tim44-804 allele, which conferred a temperature-sensitive growth phenotype. tim44-804 cells grew similar to wild-type cells at 24°C, but were strongly impaired in growth at 37°C (Figure 1A). When the mutant cells were grown at permissive temperature, isolated mitochondria contained a steady-state protein composition that was similar to that of wild-type mitochondria, including the subunits of PAM, TIM23 complex, and the control proteins Tom40 of the outer membrane and ADP/ATP carrier of the inner membrane (Figure 1B). Because the mutant Tim44 protein, as well as the other subunits of PAM and TIM23, remained stable under these growth conditions, we could use the mutant mitochondria to analyze the function of Tim44.

Figure 1.

Figure 1.

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A temperature-conditional yeast mutant of TIM44. (A) Ten-fold serial dilutions of wild-type (WT) and tim44-804 cells were spotted on YPD plates and incubated at the indicated temperatures. (B) Steady-state protein levels. Mitochondria (10 and 20 μg of protein) isolated from WT and tim44-804 cells were analyzed by SDS-PAGE and Western blotting. AAC, ADP/ATP carrier.

To examine the activity of the PAM machinery in tim44-804 mitochondria, we analyzed the import of the matrix-targeted model preprotein b2(167)Δ-DHFR that consists of the N-terminal portion of cytochrome b2, in which the inner membrane sorting signal is inactivated by deletion of 19-amino acid residues, and mouse dihydrofolate reductase (Koll et al., 1992; Voos et al., 1993). The Δp-dependent import of b2(167)Δ-DHFR, determined by processing of the N-terminal presequence and transport to a protease-protected location, was strongly inhibited in tim44-804 mitochondria compared with wild-type mitochondria (Figure 2A). A similar defect was observed, when the matrix-targeted preprotein of the β-subunit of the F1Fo-ATPase was imported (Figure 2B).

Figure 2.

Figure 2.

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tim44-804 mutant mitochondria are defective in preprotein import into the matrix. 35S-labeled precursors of b2(167)Δ-DHFR (A) and the β-subunit of F1Fo ATPase (F1β; B) were imported into wild-type (WT) and tim44-804 mutant mitochondria in the presence or absence of a proton-motive force (Δp). Where indicated, mitochondria were subsequently treated with proteinase K (Prot. K). Samples were analyzed by SDS-PAGE and digital autoradiography. p, precursor; i, intermediate; m, mature.

To determine if tim44-804 mutant mitochondria displayed a general import defect, we studied the transport of the inner membrane-sorted preproteins b2(167)-DHFR and cytochrome c1, which are transported by the TIM23SORT complex in a PAM-independent manner (Glick et al., 1992; Voos et al., 1993; Chacinska et al., 2005; van der Laan et al., 2007). In contrast to the matrix-destined precursor proteins, Δp-dependent import of b2(167)-DHFR and cytochrome c1 was similar in wild-type and tim44-804 mutant mitochondria (Figure 3, A and B). We conclude that the tim44-804 mutant mitochondria are selectively impaired in the import of matrix-targeted precursor proteins.

Figure 3.

Figure 3.

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Sorting of preproteins into the inner membrane is not affected in tim44-804 mitochondria. 35S-labeled precursors of b2(167)-DHFR and cytochrome c1 (Cyt. c1) were incubated with wild-type (WT) and tim44-804 mitochondria in the presence or absence of a proton-motive force (Δp). After the import reactions mitochondria were treated with proteinase K (Prot. K) as indicated. Samples were analyzed by SDS-PAGE and digital autoradiography. p, precursor; i, intermediate; m, mature.

The J-Complex Is Stable in tim44 Mutant Mitochondria But Impaired in Association with the TIM23 Complex

To analyze the organization of the TIM23 complex in tim44-804 mutant mitochondria, we used blue native electrophoresis. In wild-type mitochondria, lysed with the nonionic detergent digitonin, the Tim21-containing but motor-free TIM23SORT form migrates in two high-molecular-weight species (Figure 4A, lanes 1 and 3; Chacinska et al., 2005; van der Laan et al., 2007). The motor PAM is released from the TIM23 complex during blue native electrophoresis and thus the Tim21-free core of the TIM23 complex migrates as a 90-kDa subcomplex (TIM23CORE; Figure 4A, lane 1; Dekker et al., 1997; Chacinska et al., 2005; van der Laan et al., 2007). In tim44-804 mutant mitochondria, both TIM23CORE and TIM23SORT complexes were present (Figure 4A, lanes 2 and 4), indicating that the general architecture of the presequence translocase was not disrupted by the mutation of TIM44. tim44-804 mitochondria contained an additional species migrating at ∼130 kDa, which was detected with antibodies against Tim23 but not with antibodies against Tim21 (Figure 4A, lanes 2 and 4). The Pam18/Pam16 J-complex forms a separate complex on blue native electrophoresis (Figure 4A, lane 5; Frazier et al., 2004; van der Laan et al., 2005). The J-complex was indistinguishable between wild-type and tim44-804 mitochondria (Figure 4A, lanes 5 and 6). Thus, the additional 130-kDa complex detected with Tim23 antibodies did not include the J-complex.

Figure 4.

Figure 4.

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Interaction between Tim44 and Pam18/Pam16 is inhibited in tim44-804 mitochondria. (A) Wild-type (WT) and tim44-804 mitochondria were solubilized in buffer containing 1% digitonin and subjected to blue native electrophoresis and Western blotting. TIM23′, additional form of the TIM23 complex in tim44-804 mitochondria. (B) Mitochondria from WT and tim44-804 mitochondria were incubated in the presence or absence of 0.5 mM DSG and subsequently analyzed by SDS-PAGE and Western blotting. Asterisks, cross-linking products.

We used two approaches to study the association of the J-complex with the TIM23 complex, chemical cross-linking and coimmunoprecipitation. 1) In wild-type mitochondria, Pam18 and Pam16 can be cross-linked to Tim44 (Kozany et al., 2004; Mokranjac et al., 2007), as shown here with the cross-linking reagent DSG and immunodecoration with antibodies against Pam16, Pam18, and Tim44 (Figure 4B, lanes 1, 3, and 5). In tim44-804 mitochondria, cross-linking of Tim44 to Pam16 and Pam18 was almost completely abolished (Figure 4B, lanes 8 and 10). 2) We lysed the mitochondria with digitonin and performed coimmunoprecipitation under mild conditions with antibodies directed against Tim23. Tim17 was efficiently coprecipitated with Tim23 in both wild-type and tim44-804 mitochondria (Figure 5, lanes 3 and 4). The amount of Tim44 coprecipitated with Tim23 was only moderately reduced in tim44-804 mitochondria, whereas the coprecipitation of Pam18 was strongly diminished compared with wild-type mitochondria (Figure 5, lanes 3 and 4).

Figure 5.

Figure 5.

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Tim44 differentially affects the association of the J-complex and Pam17 with the TIM23 complex. Mitochondria from wild-type (WT) and tim44-804 mitochondria were solubilized in digitonin buffer and subjected to immunoprecipitation with antibodies against Tim23. Samples were analyzed by SDS-PAGE and immunodecoration with the indicated antibodies. Mito, 5% total mitochondrial extract; Tim23-precipitate, 100%.

Taken together, the results described in Figures 4 and 5 indicate that the interaction of Pam18 and Pam16 in the J-complex is stable in tim44-804 mitochondria, but that the association of the J-complex with the TIM23 complex is strongly impaired.

Pam17 Accumulates at the TIM23 Complex in tim44 Mutant Mitochondria

The coprecipitation with antibodies against Tim23 revealed an unexpected and massive effect on Pam17. The coprecipitate from tim44-804 mitochondria contained considerably larger amounts of Pam17 than that from wild-type mitochondria (Figure 5, lanes 3 and 4), although the total amount of Pam17 was similar in wild-type and mutant mitochondria (Figures 1B and 5, lanes 1 and 2).

Thus, the alteration of Tim44 led to opposite effects on the association of the J-complex and Pam17 with the TIM23 complex. We asked if a reduced binding of the J-complex to TIM23 induced an increase in the binding of Pam17. We utilized pam16 mutant mitochondria that are characterized by loss of the J-complex from the TIM23 complex (Figure 6, lane 4; Frazier et al., 2004). However, Tim44 and Pam17 were recovered in the immunoprecipitates from pam16-1 mitochondria in similar amounts as from wild-type mitochondria, i.e., the yield of copurification of Pam17 was not increased (Figure 6). We conclude that the loss of the J-complex per se does not affect the association of Pam17 with the TIM23 complex, whereas the inactivation of Tim44 causes a dual effect, diminished J-complex association but strongly increased association of Pam17 with the TIM23 complex.

Figure 6.

Figure 6.

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Inactivation of the J-complex does not affect Pam17 binding to the TIM23 complex. Wild-type (WT) and pam16-1 mutant mitochondria were solubilized in digitonin-containing buffer and TIM23 complexes were immunoprecipitated with antibodies against Tim23. Samples were analyzed by SDS-PAGE and Western blotting. Mito, 5% total mitochondrial extract; Tim23-precipitate, 100%.

Pam17 Is in Proximity to Tim23

Because the association of Pam17 with the TIM23 complex was increased in tim44-804 mitochondria in which the function of Tim44 was compromised and Pam18/Pam16 were released from the translocase, we wondered which binding partner mediated the interaction of Pam17 with the translocase. We performed chemical cross-linking in intact mitochondria and searched for cross-linking products of the TIM23 constituents with a protein of ∼15–20 kDa. Using the cross-linking reagent EGS, we identified a product of Tim23 that migrated at 40 kDa (Figure 7A, lane 1). Tom22 and Tim21 were potential candidates for the cross-linking partners of Tim23. We thus used tom22-2 mitochondria, which contain a truncated form of Tom22 (Frazier et al., 2003), as well as tim21Δ mitochondria (Chacinska et al., 2005). In both mutants, the 40-kDa cross-linking product was present and not altered in its size (Figure 7A, lanes 2 and 4). However, when cross-linking was performed in pam17Δ mitochondria (van der Laan et al., 2005), the cross-linking product was lost (Figure 7B, lane 6 vs. lane 5), suggesting that Tim23 was cross-linked to Pam17. To verify that Pam17 was a component of the cross-linking product and did not indirectly affect cross-linking of Tim23 to another protein, immunodecoration with anti-Pam17 antibodies was carried out. Indeed, antibodies against Pam17 specifically decorated the 40-kDa cross-linking product in wild-type mitochondria but not in pam17Δ mitochondria (Figure 7B, lanes 3 and 4). We conclude that Pam17 is in close proximity to Tim23 in intact mitochondria.

Figure 7.

Figure 7.

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Cross-linking of Pam17 to Tim23. (A) Wild-type (WT), tom22-2 and tim21Δ mitochondria were incubated in the presence or absence of 0.25 mM EGS. Samples were analyzed by SDS-PAGE and immunodecoration with antibodies against Tim23. (B) Mitochondria from WT and pam17Δ cells were treated with EGS and analyzed as described for A with the indicated antibodies. (C) WT and tim44-804 mitochondria were subjected to EGS treatment and analyzed as described in A. Asterisks, cross-linking products.

The Pam17-Tim23 cross-linking product provided a direct assay to determine if the coimmunoprecipitation data from detergent-lysed mitochondria, where the yield of association of Pam17 with the TIM23 complex was strongly increased (Figure 5), reflected the in organello situation. We thus performed cross-linking in tim44-804 mutant mitochondria and wild-type mitochondria in parallel. Indeed, the formation of the Tim23-Pam17 cross-linking product was significantly enhanced in the mutant mitochondria (Figure 7C, lane 2 vs. lane 1). We conclude that inactivation of Tim44 enhances the association of Pam17 with the TIM23 complex. Pam17 binds to Tim23 or in close proximity of this channel-forming subunit of the TIM23 complex.

DISCUSSION

Our study extends the view of the function of Tim44 in the mitochondrial protein import machinery. Although it has been assumed that Tim44 simply functions as adaptor/scaffold for the association of mtHsp70 and further PAM subunits with the presequence translocase (Kozany et al., 2004; Dudek et al., 2007; Neupert and Herrmann, 2007; Mokranjac et al., 2007; D'Silva et al., 2008), we report that Tim44 plays a differential role in the recruitment of distinct PAM modules to the TIM23 complex.

We generated a conditional yeast mutant of TIM44 that allowed the analysis of Tim44 function with isolated mitochondria, whereas the mutant cells were grown at permissive conditions and thus indirect effects of the tim44 mutation on mitochondrial composition and function were minimized. The tim44 mutant mitochondria were impaired in the association of the Pam18/Pam16 J-complex with the TIM23 complex, providing direct evidence for the view that Tim44 is important for recruiting the J-complex to the translocation channel (Kozany et al., 2004; Mokranjac et al., 2007; D'Silva et al., 2008). Together with the work by D'Silva et al. (2008), our study thus sheds light on the controversially discussed issue of how Pam18 associates with the TIM23 complex. We conclude that three distinct interactions contribute to this association: 1) the amino-terminal intermembrane space domain of Pam18 interacts with Tim17 (Chacinska et al., 2005; D'Silva et al., 2008); 2) the transmembrane domain of Pam18 stabilizes the interaction with the presequence translocase (Mokranjac et al., 2007); and 3) the J-complex interacts with Tim44 most likely via Pam16 (this study; D'Silva et al., 2008). If only one of these interactions is disturbed, reduced binding of Pam18 is observed, whereas interfering with two interaction sites appears to be deleterious (Mokranjac et al., 2007; D'Silva et al., 2008).

Surprisingly, a further regulatory factor of the motor, Pam17, was strongly enhanced in binding to the TIM23 complex in tim44 mutant mitochondria. Because Pam17 has been shown to be involved in the association of the J-complex with the TIM23 complex (van der Laan et al., 2005), we wondered if the increased binding of Pam17 to the TIM23 complex was not directly caused by the inactivation of Tim44 but an indirect consequence of the impaired interaction of the J-complex with the TIM23 complex, i.e., the increased binding of Pam17 would represent a futile attempt to rescue the disturbed interaction of the J-complex with the TIM23 complex. We thus made use of pam16 mutant mitochondria, where the J-complex was blocked in its association with the TIM23 complex, whereas the binding of Tim44 to the translocase was not affected (Frazier et al., 2004). Importantly, in pam16 mutant mitochondria, the binding of Pam17 to the TIM23 complex was not affected, i.e., was not enhanced in contrast to the situation in tim44 mutant mitochondria. We conclude that the increased binding of Pam17 to the TIM23 complex in tim44 mutant mitochondria was not indirectly caused by the release of the J-complex. Thus, Tim44 is not simply a platform for binding of other motor subunits but shows both stimulatory and inhibitory influence on the association of PAM modules with the TIM23 complex. Tim44 binds mtHsp70 and the J-complex and thus promotes the interaction of these motor modules with the TIM23 complex. In contrast, Tim44 keeps the level of TIM23-bound Pam17 low, whereas inactivation of Tim44 stimulates binding of Pam17 to the translocase.

Our findings shed new light on a puzzling observation regarding the relation of Pam17 and the J-complex. Wiedemann et al. (2007) showed that a fraction of the J-complexes but not Pam17 were recruited to the respiratory chain in early steps of preprotein translocation. This observation implied that the localization of Pam17 and J-complex were differently regulated; however, it was unclear whether this just represented a special situation concerning the recruitment to the respiratory chain or whether a separate localization of Pam17 and J-complex was a general principle of the motor function. We report that Pam17 binds to a new interaction site at the presequence translocase, at or in close proximity to the channel-forming protein Tim23. Thus, Pam17 and J-complex bind to the TIM23 complex via different sites and show an opposite dependence on the functionality of Tim44.

We propose that the assembly of the mitochondrial protein import motor involves a regulated interplay of several membrane-bound cochaperones: Tim44, Pam18/Pam16, and Pam17. The cochaperones do not form one stable complex but differently interact with the presequence translocase. Interestingly, the cochaperones not only possess stimulatory activities but also in part inhibitory characteristics. Pam16 was shown to promote association of Pam18 with the presequence translocase (Frazier et al., 2004; Kozany et al., 2004; D'Silva et al., 2005, 2008; Mokranjac et al., 2007) but also to reduce the stimulatory function of Pam18 on the ATPase activity of mtHsp70 (Li et al., 2004; D'Silva et al., 2005). We found that Tim44 promotes the binding of the J-complex to the presequence translocase but impairs the binding of Pam17. The multistep regulation of mtHsp70 function at the protein translocation channel by four membrane-bound cochaperones identifies the mitochondrial protein import motor as one of the most complicated chaperone systems known.

ACKNOWLEDGMENTS

We are grateful to Drs. N. Wiedemann, W. Voos, J. Dudek, and C. Meisinger for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich 746, Gottfried Wilhelm Leibniz Program, Max Planck Research Award, and the Fonds der Chemischen Industrie.

Abbreviations used:

Δp

proton-motive force

J-complex

Pam18-Pam16 complex of mitochondria

mtHsp70

mitochondrial heat shock protein 70

PAM

presequence translocase-associated motor

TIM

presequence translocase of inner mitochondrial membrane

Footnotes

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-12-1226) on April 9, 2008.

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