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Originally published In Press as doi:10.1074/jbc.M313037200 on January 13, 2004

J. Biol. Chem., Vol. 279, Issue 13, 12396-12405, March 26, 2004
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The Tim8-Tim13 Complex of Neurospora crassa Functions in the Assembly of Proteins into Both Mitochondrial Membranes*

Suzanne C. Hoppins and Frank E. Nargang{ddagger}

From the Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada

Received for publication, December 1, 2003 , and in revised form, January 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Tim8 and Tim13 proteins in yeast are known to exist in the mitochondrial intermembrane space and to form a hetero-oligomeric complex involved in the import of the mitochondrial inner membrane protein Tim23, the central component of the TIM23 translocase. Here, we have isolated tim8 and tim13 mutants in Neurospora crassa and have shown that mitochondria lacking the Tim8-Tim13 complex were deficient in the import of the outer membrane {beta}-barrel proteins Tom40 and porin. Cross-linking studies showed that the Tom40 precursor contacts the Tim8-Tim13 complex. The complex is involved at an early point in the Tom40 assembly pathway because cross-links can only be detected during the initial stages of Tom40 import. In mitochondria lacking the Tim8-Tim13 complex, the Tom40 precursor appears in a previously characterized early intermediate of Tom40 assembly more slowly than in wild type mitochondria. Thus, our data suggest a model in which one of the first steps in Tom40 assembly may be interaction with the Tim8-Tim13 complex. As in yeast, the N. crassa Tim23 precursor was imported inefficiently into mitochondria lacking the Tim8-Tim13 complex when the membrane potential was reduced. Tim23 import intermediates could also be cross-linked to the complex, suggesting a dual role for the Tim8-Tim13 intermembrane space complex in the import of proteins found in both the outer and inner mitochondrial membranes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Most mitochondrial proteins are encoded in the nucleus and translated on cytosolic ribosomes as precursor proteins. Targeting and sorting signals within these precursors are decoded by multisubunit translocases located in the mitochondrial outer and inner membranes so that the proteins are recognized and imported to the proper compartment within the organelle (1–5). The translocase of the outer membrane (TOM)1 complex contains receptors for all proteins targeted to mitochondria and forms the pore through which they cross the outer membrane. Proteins destined for either the inner membrane or the matrix require the action of one of two translocases of the inner membrane (TIM) complexes. The TIM23 complex recognizes precursors containing cleavable N-terminal targeting signals and forms a pore through which they are translocated across the inner membrane into the matrix. A number of precursor proteins do not contain a cleavable presequence but carry their targeting information within the mature protein sequence. A subset of such precursors, with multiple membrane spanning domains, is inserted into the inner membrane by the TIM22 complex.

Precursors inserted by the TIM22 complex include the mitochondrial carrier proteins and certain components of the TIM complexes. As these precursors emerge from the TOM complex, their subsequent interaction with the TIM22 complex is mediated by a group of small structurally related proteins including Tim8, Tim9, Tim10, Tim12, and Tim13, which exist in the intermembrane space (6, 7). The Tim9 and Tim10 proteins are essential gene products in Saccharomyces cerevisiae which form a soluble 70-kDa complex containing three subunits of each protein (8–11). The Tim9-Tim10 complex interacts with the membrane spanning domains of carrier protein precursors as they emerge from the TOM complex and delivers the precursors to the TIM22 translocase for insertion into the membrane (8, 9, 12–16). The TIM22 complex contains the integral membrane proteins Tim22, Tim54, and Tim18 (17–20). These proteins can be isolated in a 300-kDa complex that also contains the peripheral membrane protein Tim12 and small amounts of Tim9 and Tim10 (8, 9, 12, 13). Like Tim9 and Tim10, Tim12 is essential for the viability of yeast cells. The Tim9-Tim10 complex probably delivers its cargo precursor molecule to the TIM22 translocase via interaction with Tim12 (8, 9).

The Tim8 and Tim13 proteins also form a soluble, 70-kDa hetero-oligomeric complex containing three subunits of each protein (21, 22). In contrast to the other small Tim proteins, loss of Tim8 and/or Tim13 does not have severe effects on the growth of yeast cells. A deletion of Tim8 was found to be synthetically lethal with a temperature-sensitive allele of Tim10 (21), and a strain lacking both Tim8 and Tim13 did not grow at low temperatures on glucose (23). Several groups have shown an effect on Tim23 import in mitochondria lacking the Tim8-Tim13 complex in yeast, but there are minor differences in the nature of the defect reported. Certain studies have shown that the precursor of yeast Tim23 was imported efficiently in the absence of Tim8-Tim13 under conditions where the potential across the inner membrane was high (23–25), although the complex was required for efficient import of the precursor when the membrane potential was low (23, 25). Others have reported that the import of yeast Tim23 into mitochondria lacking the Tim8-Tim13 complex is reduced even under normal, high membrane potential conditions (26, 27). Abundant cross-linking between the Tim8-Tim13 complex and the hydrophilic N-terminal region of Tim23 was observed when the translocation of the protein across the outer membrane was arrested (23, 24). However, peptide scanning showed that Tim8–13 also binds to hydrophobic transmembrane domains in the C-terminal region of Tim23 (22) where import signals necessary for its membrane potential-dependent insertion into the inner membrane have been identified (28, 29). Furthermore, significant cross–linking was also seen between the C-terminal region of Tim23 and the Tim9-Tim10 complex (24), although peptide scans revealed no affinity of Tim9-Tim10 for Tim23 (16). Thus, it is unclear whether the Tim9-Tim10 complex plays a role in the import of yeast Tim23 and whether the Tim8-Tim13 complex is required to facilitate the import of the protein under all conditions or only at times when the membrane potential is reduced.

The human homolog of Tim8 is the DDP1 protein (deafness-dystonia peptide 1) encoded by the Tim8a gene (21). Mutations in the gene result in a neurodegenerative disorder called Mohr-Tranebjaerg syndrome (30, 31). The mammalian homologs of Tim8 and Tim13 also form a 70-kDa complex in the intermembrane space that interacts with Tim23 precursor in mammalian mitochondria. The normal human proteins can rescue the defects seen in yeast cells lacking both proteins (25, 27), but a DDP1 mutant protein containing an amino acid substitution found in a full blown disease case could not substitute for yeast Tim8 (27, 32). Efficient import of human Tim23 into isolated yeast mitochondria has a dependence on the Tim8-Tim13 complex even in the presence of a high membrane potential, suggesting that human Tim23 may have a weaker C-terminal import signal than the one identified in the yeast protein (23, 25). Thus, the present model for the phenotype of Mohr-Tranebjaerg syndrome suggests that reduced assembly of the Tim23 precursor protein into the TIM23 complex results from the deficiency of Tim8. This, in turn, leads to reduced import of presequence containing precursors through the TIM23 complex and results in mitochondrial defects (23, 25, 27, 33). Despite these findings, no defects in mitochondrial function have been found in cultured cells or tissues of deafness-dystonia patients (27, 34).

To elucidate further the function of Tim8-Tim13, we have created mutants in Neurospora crassa which lack the proteins. Our findings define a new role for the Tim8-Tim13 complex in the biogenesis of mitochondrial membrane proteins and suggest that the complex functions during the import of different classes of proteins entering either the inner or outer mitochondrial membrane.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Growth of N. crassa and Strains Used—Growth and handling of N. crassa strains was carried out as described previously (35). Strains used in this study are listed in Table I.


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  		TABLE I
Strains used in this study

 
Cosmids and Plasmids—An expressed sequence tag for N. crassa tim8 was identified in a BLAST search of the University of Oklahoma expressed sequence tag data base using the sequence of the S. cerevisiae Tim8 protein as the query. Primers designed from the expressed sequence tag were used to generate PCR products to probe a N. crassa cosmid library. Several positive clones were identified. The upstream and downstream sequences were determined from a single cosmid. Plasmid pT8HL was constructed by cloning a 2-kb PCR fragment containing the tim8 gene plus flanking sequences into the NotI site of the hygromycin-resistant plasmid pCSN44 (36).

The N. crassa tim13 gene was identified by a BLAST search of the Neurospora genome (WICGR; www-genome.wi.mit.edu/) using the S. cerevisiae Tim13 protein sequence as the query. From this sequence, tim13-specific primers were designed to amplify the gene for cloning. Plasmid p44T13 was constructed by cloning a 2.1-kb PCR-derived fragment containing the tim13 gene plus flanking DNA into the NotI sites of the hygromycin-resistant plasmid pCSN44. The complete DNA sequences of the tim8 and tim13 genes are available at the Neurospora genome website (www-genome.wi.mit.edu/annotation/fungi/neurospora/).

Creation of Repeat-induced Point (RIP) Mutants—The tim8 and tim13 genes were inactivated by the process of RIP mutation. RIP inactivates duplications in the genome by creating GC to AT transition mutations in both copies of the duplication during a sexual cross (37). Thus, the procedure requires the use of a strain containing a duplication of the gene to be inactivated. For tim8, the plasmid pT8HL was transformed into strain 76-26 to create the tim8 RIP substrate. Transformants were selected on hygromycin-containing medium, taken through one round of purification on hygromycin-containing plates, and examined by Southern analysis for evidence of ectopic integration of a single copy of tim8. The tim8 duplication-containing strain, T8HL-7, was crossed as the male parent to strain NCN251 in the RIP cross. To identify progeny containing only RIP mutated alleles of tim8, mitochondria were isolated from several strains and screened by Western blot analysis for the absence of the protein. Strain T8HL7-7 was chosen for further analysis.

The procedure for isolation of tim13 RIP mutants was as for tim8 except that the plasmid p44T13 was transformed into strain 76-26 to generate the RIP substrate. The tim13 duplication-containing strain T13-16 was chosen to act as the male in the RIP cross with NCN251. Strain T13R16–45 was chosen for further analysis based on absence of the Tim13 protein.

Antibody Production—Antisera were raised against His6-tagged fusion proteins comprised of full-length mouse dihydrofolate reductase and the C terminus of either Tim8 (residues 35–92) or Tim13 (residues 31–86). Fusion proteins were purified on nickel-nitrilotriacetic acid columns (Qiagen) in 8 M urea according to the manufacturer's instructions except that the proteins were eluted in 0.1% SDS, 10 mM Tris-Cl, pH 7.4.

Import of Radiolabeled Proteins into Isolated Mitochondria and Cross-linking—In vitro import studies were as described previously (38). In some cases membrane potential was reduced by preincubation of the mitochondria with 8 µM antimycin and 20 µM oligomycin at 25 °C for 2 min. For imports analyzed by blue native gel electrophoresis (BNGE), the usual proteinase K treatment after import was omitted, and mitochondria were washed in SEMPK buffer (0.25 M sucrose, 1 mM EDTA, 10 mM MOPS, pH 7.2, 1 mM phenylmethylsulfonyl fluoride, 80 mM KCl) prior to solubilization.

Cross-linking studies were performed following the import of the precursors of Tom40 or Tim23 into wild type mitochondria. For Tom40, import was performed under normal conditions at different temperatures. For Tim23, the precursor was arrested as a translocation intermediate by adding 25 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and 1 µM valinomycin prior to import to dissipate the membrane potential. After import, cross-linking was performed with 300 µM disuccinimidylglutarate (DSG) for 30 min in an ice bath. The reactions were quenched with 100 mM Tris-HCl, pH 7.5. Mitochondria were washed with SEMPK buffer and reisolated. For immunoprecipitation, reisolated mitochondria were lysed in 10 mM Tris-HCl, pH 7.4, 300 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100.

Sephacryl Column Chromatography—Mitochondria (0.5 mg) were solubilized in 500 µl of column buffer (20 mM HEPES, pH 7.2, 50 mM NaCl, 2.5 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride), containing 1% n-dodecyl maltoside, for 30 min at 4 °C. After centrifugation at 180,000 x gmax for 30 min at 4 °C, the cleared lysate was loaded onto a Sephacryl-12 column, and the proteins were separated in column buffer containing 0.1% n-dodecyl maltoside. One-ml fractions were collected, and the proteins were precipitated with trichloroacetic acid at a final concentration of 9%. Proteins were resuspended in cracking buffer (0.06 M Tris-HCl, pH 6.7, 2.5% SDS, 0.01% 2-mercaptoethanol, 5% sucrose) and analyzed by SDS-PAGE and immunoblotting with antibodies against Tim8 or Tim13. For calibration, apoferritin (440 kDa), {beta}-amylase (200 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa) were used as standards.

Mitochondrial Subfractionation—Mitochondria were isolated and resuspended in swelling buffer (5 mM KPO4, pH 7.2, 100 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) and incubated on ice for 1 h. A mitoplast fraction enriched for mitochondrial membrane and matrix contents was collected as a pellet after centrifugation at 12,000 rpm for 20 min at 4 °C in an SS-34 rotor. The supernatant, which was enriched for intermembrane space proteins, was centrifuged again at 28,000 rpm in a TLA-55 rotor for 1 h at 4 °C to remove remaining contaminants. Proteins in the intermembrane space fraction were precipitated with trichloroacetic acid, resuspended in cracking buffer, and the protein concentration was determined. Samples (5 µg) of both the pellet and intermembrane space fraction were analyzed by SDS-PAGE and immunoblotting.

Other Techniques—Standard molecular biology techniques were performed as described (39). BNGE was performed as described (40, 41), except that the Tim8-Tim13 complex was transferred to nitrocellulose membrane rather than polyvinylidene fluoride membrane. Genomic DNA was isolated as described previously (42). DyeNamic sequencing kits (Amersham Biosciences) and a model 373 stretch sequencer separation system (Applied Biosystems, Foster City, CA) were used for DNA sequencing according to the supplier's instructions. Radioactive precursor proteins for import were generated by coupled in vitro transcription and translation with the Promega (Madison, WI) TNT reticulocyte lysate system in the presence of [35S]methionine (ICN, Costa Mesa, CA). PhosphorImaging and quantitation of radiolabeled bands on blots were done using a PhosphorImager 445 SI with Molecular Dynamics ImageQuantTM software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
N. crassa Strains Lacking Tim8 and Tim13—Tim8 and Tim13 were identified in N. crassa data bases using the yeast homologs as queries (Fig. 1). Both proteins contain the conserved twin CX3C motif characteristic of all the small intermembrane space Tim proteins (6, 7). The procedure of RIP was utilized to generate tim8 and tim13 mutants in N. crassa (see "Experimental Procedures"). In the tim8RIP strain the Tim8 protein is not detectable (Fig. 2A). Small amounts of Tim13 were detectable in this strain, which was more evident when the film was overexposed (Fig. 2B). The tim13RIP strain completely lacks the Tim13 protein and is also virtually devoid of Tim8 (Fig. 2, A and B). The observation that lack of one protein results in severe deficiency of the other suggests that the two proteins exist in a heteromeric complex, the formation of which stabilizes both proteins, as found in other organisms (21, 27, 32). The levels of other mitochondrial proteins examined in both mutant strains were similar to controls (Fig. 2A). No growth defect was observed in either mutant strain after incubation at 15, 22, or 37 °C with either sucrose or glycerol as the carbon source (data not shown).



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  		FIG. 1.
Alignment of Tim8 and Tim13 proteins. The Tim8 (A) and Tim 13 (B) proteins of N. crassa (N.c.), S. cerevisiae (S.c.), and H. sapiens (H.s.) are shown. There are two genes known to encode Tim8 in humans, but only the Tim8a gene product, which encodes the DDP1 protein, is shown here. The number of residues in each protein is indicated at the right. Black shading indicates amino acid identity between at least two species. Arrows indicate the location of the introns in the N. crassa genes. Black bars above the sequences show the conserved CX3C motifs.

 



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  		FIG. 2.
Steady-state levels of mitochondrial proteins in the tim8RIP and tim13RIP mutants. A, mitochondria were isolated from a control strain (NCN251), the strains containing the tim8 and tim13 duplications used in the RIP crosses (tim8 dupl and tim13 dupl, respectively), as well as from the tim8RIP and tim13RIP strains. Mitochondrial proteins (20 µg) were separated by SDS-PAGE, blotted to nitrocellulose, and immunodecorated with antisera against the indicated proteins. B, overexposure of the bottom two rows from A.

 
Tim8 and Tim13 Form a Soluble 80-kDa Hetero-oligomeric Complex in the Intermembrane Space of N. crassa Mitochondria—To determine the size of the Tim8-Tim13 complex in N. crassa, mitochondria were lysed in n-dodecyl maltoside, and the solubilized proteins were separated by gel filtration on a Sephacryl S-200 column. The Tim8 and Tim13 proteins coeluted as a complex with an estimated size of 80 kDa (Fig. 3A). Mitochondria solubilized in n-dodecyl maltoside were also analyzed by BNGE. Tim8 and Tim13 comigrated to an identical position on the blue native gel with an apparent molecular mass of 80 kDa (Fig. 3B). These results suggest that Tim8 and Tim13 form a hetero-oligomeric complex. Tim8 was coimmunoprecipitated with Tim13 antiserum, but surprisingly, no Tim13 was coimmunoprecipitated with Tim8 antiserum (Fig. 3C). Because Tim8 may form the core of the Tim8-Tim13 complex (22) it is conceivable that the conditions of immunoprecipitation result in dissociation of Tim13 when the antibodies interact with Tim8. Antiserum to Tim8 or Tim13 did not immunoprecipitate Tim9 or Tim10 (Fig. 3C).



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  		FIG. 3.
Characterization of the N. crassa Tim8-Tim13 complex. A, gel filtration analysis of N. crassa Tim8-Tim13. Wild type mitochondria were solubilized with 1% n-dodecyl maltoside and analyzed on a Sephacryl S-200 column. Fractions were analyzed by SDS-PAGE, blotted to nitrocellulose, and immunodecorated with either Tim8 or Tim13 antiserum (shown in the inset). Tim8 and Tim13 coeluted in fractions corresponding to a molecular mass of 80 kDa. B, BNGE analysis of the Tim8-Tim13 complex. Mitochondria isolated from the same strains used in Fig. 2 were solubilized in 1% n-dodecyl maltoside, loaded in two sets for Tim8 and Tim13 immunodetection, separated by BNGE, and transferred to nitrocellulose. The blot was cut lengthwise, and the halves were immunodecorated with either Tim8 or Tim13 antiserum. The Tim8-Tim13 complex (indicated by an arrow) was detected in an identical position corresponding to an apparent molecular mass of 80 kDa with both antisera. The positions of standards apoferritin (440 kDa), {beta}-amylase (200 kDa), and bovine serum albumin (66 kDa) are indicated on the left. Small amounts of nonspecific bands detected by the sera are visible on the blots. C, coimmunoprecipitation. Mitochondria isolated from wild type strain NCN251 were solubilized with 1% Triton X-100 and incubated with antibodies against Tim8 ({alpha}Tim8), Tim13 ({alpha}Tim13), or preimmune serum (PIS) bound to protein A-Sepharose beads. The beads were collected by centrifugation, washed, and bound proteins were eluted with Laemmli gel cracking buffer. Samples were analyzed by SDS-PAGE, blotted to nitrocellulose, and immunodecorated with the indicated antisera. Total represents 100% of the starting material in the immunoprecipitation reactions. D, alkaline carbonate extraction of N. crassa mitochondria. Wild type (NCN251) mitochondria were treated with 0.1 M sodium carbonate for 30 min on ice. The membrane fraction was pelleted by centrifugation, and soluble proteins in the supernatant were precipitated with trichloroacetic acid. Samples were analyzed by SDS-PAGE and immunodecoration with the indicated antisera. E, submitochondrial localization of Tim8-Tim13. Wild type (NCN251) mitochondria were isolated and suspended in swelling buffer followed by incubation on ice for 1 h. Mitoplasts were pelleted by centrifugation, and the supernatant was collected as a fraction enriched for intermembrane space proteins. Samples (5 µg) were analyzed by SDS-PAGE and immunodecoration with the indicated antisera.

 
After alkaline carbonate extraction of wild type mitochondria both Tim8 and Tim13 were found in the soluble supernatant fraction as were the matrix proteins Hsp60 and Hsp70 (Fig. 3D). Tom40 and porin, known outer membrane proteins, were found in the pelleted membrane fraction (Fig. 3D). Thus, Tim8-Tim13 forms a soluble protein complex. To localize the complex either to the matrix or the intermembrane space, mitochondria were isolated and swollen in hypotonic buffer containing 100 mM NaCl to create mitoplasts and release the contents of the intermembrane space. The mitoplast fraction was pelleted by centrifugation, and the supernatant was taken as a fraction enriched in intermembrane space proteins. The contents of the membrane and supernatant fractions were compared by Western blot analysis. Tim8 and Tim13 were enriched in the intermembrane space fraction as was cytochrome c, a protein known to exist in this compartment (Fig. 3E). The matrix protein Hsp60 was enriched in the mitoplast pellet, and the mitochondrial membrane proteins Tom40 and the ADP/ATP carrier were found exclusively in the pellet. Taken together, these data demonstrate that N. crassa Tim8 and Tim13 form a soluble hetero-oligomeric complex of ~80 kDa which exists in the mitochondrial intermembrane space.

Function of the Tim8-Tim13 Complex in N. crassa—A variety of mitochondrial precursor proteins were imported into mitochondria isolated from the tim8RIP and tim13RIP strains. Fig. 4 shows results obtained using the tim13RIP strain. Virtually identical results were obtained with the tim8RIP strain (data not shown). The matrix-targeted precursors for the {beta}-subunit of mitochondrial ATP synthase (F1{beta}), the intermembrane space protein cytochrome c heme lyase, and ADP/ATP carrier of the inner membrane were all imported at rates indistinguishable from control strains (Fig. 4, A–C). It has been reported that the S. cerevisiae Tim8-Tim13 complex is involved in importing Tim23 into mitochondria, particularly when the membrane potential is low (23, 24, 26). We examined the import of Tim23 into both energized mitochondria and mitochondria that had been treated with antimycin A and oligomycin to reduce the membrane potential. Import of Tim23 into energized mitochondria from the tim13RIP strain did not differ from the control (Fig. 4D) but was decreased relative to wild type when the membrane potential was reduced (Fig. 4E). As a control for the reduction of the membrane potential we analyzed the import of F1{beta}, a matrix-targeted precursor that is strongly dependent on the membrane potential for import. Under conditions of membrane potential depletion, import of the F1{beta} precursor was virtually undetectable (Fig. 4F).



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  		FIG. 4.
Import of precursor proteins into mitochondria lacking Tim8-Tim13. Radiolabeled mitochondrial precursors were incubated with mitochondria isolated from either the T13-16 strain (control) or the tim13RIP strain at 25 °C (for F1{beta}, cytochrome c heme lyase, ADP/ATP carrier, Tom22) or 10 °C (for Tim23, Tom40, porin) for the indicated times. After a postimport proteinase K treatment, mitochondria were reisolated and analyzed by SDS-PAGE. The gels were transferred to nitrocellulose and exposed to x-ray film (insets) and then a PhosphorImager screen for quantitation. One sample from each strain was treated with trypsin prior to import (pre trp) to demonstrate receptor-dependent import. A–C, Tim8-Tim13-deficient mitochondria are not defective in import of the precursors for the matrix-targeted {beta}-subunit of the F1-ATP synthase (A, F1{beta}),the intermembrane space-targeted cytochrome c heme lyase (B, CCHL), or the inner membrane-targeted ADP/ATP carrier (C, AAC). D–F, Tim8-Tim13-deficient mitochondria are defective in import of Tim23 when membrane potential is reduced. Radiolabeled Tim23 precursor was imported in the absence (D) or presence (E)of20 µM antimycin A, 2.5 mM ATP, and 8 µM oligomycin to reduce the membrane potential. As a control for membrane potential reduction, import was performed as in D and E with radiolabeled F1{beta} precursor in the absence (F, top) and the presence (F, bottom) of antimycin A, ATP, and oligomycin. G and H, Tim8-Tim13-deficient mitochondria are defective in import of Tom40 and porin. In vitro import was performed with radiolabeled Tom40 (G) or porin (H) precursor proteins. Treatment of mitochondria with proteinase K after import of Tom40 results in the generation of characteristic fragments of the protein with apparent molecular masses of 26 and 12 kDa from a portion of the Tom40 population that is fully assembled in the membrane (44). The quantitation shown was based on the levels of the 26-kDa fragment. I, Tim8-Tim13-deficient mitochondria are not defective in import of Tom22. In vitro import was performed with radiolabeled Tom22 precursor protein. Post import proteinase K treatment results in the generation of cleavage products because Tom22 is exposed on the cytosolic side of the outer membrane. The highest molecular mass cleavage product was quantified.

 
We also examined the import of the outer membrane proteins Tom40, porin, and Tom22. Surprisingly, the import of the {beta}-barrel proteins Tom40 and porin into mitochondria from the tim13RIP (Fig. 4, G and H) strain and the tim8RIP strain (data not shown) was reduced. Import of Tom22 was found to occur normally in the mutant strain so that the Tim8-Tim13 complex is not necessary for import of all outer membrane proteins (Fig. 4I).

Porin Assembly Defects in Tim8-Tim13 Mutants—Relatively little is known about the biogenesis of the porin complex in the mitochondrial outer membrane. S. cerevisiae porin exists in three distinct complexes with apparent molecular masses of 440, 400, and 200 kDa, although only the 200-kDa species was resistant to alkaline extraction. When radiolabeled porin was imported into isolated mitochondria, it assembled into each of these preexisting complexes and was also found in a 100-kDa form that may represent an assembly intermediate (43). To investigate the role of the Tim8-Tim13 complex in the assembly of porin in N. crassa, the precursor was imported into mitochondria isolated from a control and the tim13RIP strain, at 0 and 25 °C for different times. BNGE revealed the accumulation of the precursor into three distinct complexes with apparent molecular masses of ~270, 115, and 66 kDa (Fig. 5A). The amount of precursor in all three forms was reduced after import into mitochondria isolated from the tim13RIP strain under all import conditions examined, suggesting an important role of the Tim8-Tim13 complex in the assembly of porin.



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  		FIG. 5.
Import and assembly of porin and Tom40 into Tim8-Tim13-deficient mitochondria. Radiolabeled precursors of porin (A) or Tom40 (B) were incubated with mitochondria isolated from a control strain (Ct, strain T13-16) or the tim13RIP strain (RIP) for the indicated times at the indicated temperatures. Mitochondria were reisolated, washed, and lysed in blue gel sample buffer containing 1% digitonin. The samples were subjected to BNGE, blotted to polyvinylidene difluoride membrane, and analyzed by autoradiography. Standards used were apoferritin (440 kDa), {beta}-amylase (200 kDa), and bovine serum albumin (66 kDa) and are shown on the left. The apparent sizes of the porin species detected after import (A) and the known Tom40 assembly intermediates (B) are shown on the right.

 
Tim8-Tim13 Is Involved in Early Steps of Tom40 Assembly— The assembly of Tom40 precursor into the TOM complex is well characterized and involves the formation of assembly intermediates that can be visualized on blue native gels (38, 44–46). After targeting to mitochondria, the Tom40 precursor associates with the sorting and assembly machinery to give a 250-kDa complex. The precursor then progresses to a 100-kDa complex before being integrated into the fully assembled 400-kDa TOM complex. The assembly pathway of Tom40 was examined in mitochondria from the tim13RIP strain and a control strain by BNGE. In both strains the Tom40 precursor was most abundant in the 250-kDa intermediate when import was performed at 25 °C for 1 min or at 0 °C for 15 min, but the level of this intermediate was reduced in the tim13RIP mutant, about 2-fold after 15 min at 0 °C and about 3-fold after 1 min at 25 °C (Fig. 5A). When import was allowed to proceed for 15 min at 25 °C, the Tom40 precursor was found predominantly in the fully assembled 400-kDa complex, and the amount of Tom40 in all forms was about the same in the control and mutant mitochondria (Fig. 5B).

The Tim8-Tim13 Complex Is Close to the Tom40 Precursor during Import—To determine whether the Tim8-Tim13 complex is in close vicinity to Tom40 precursors as they are imported, radiolabeled precursor was imported into wild type mitochondria followed by the addition of cross-linking reagents. Cross-linking of mitochondrial precursor proteins to components of different mitochondrial translocase complexes is typically enhanced by arresting the precursor in the process of translocation by eliminating the membrane potential required for import. However, because the import of outer membrane proteins is not dependent on the potential across the inner membrane, we used relatively short import times or reduced temperature in an attempt to detect Tom40 precursors as translocation intermediates. After import and cross-linking, mitochondria were reisolated, dissolved in buffer containing 1% Triton X-100, and subjected to immunoprecipitation with antiserum either to Tim8 or Tim13. Cross-linked products with Tom40 were detectable with both Tim8 and Tim13 when import was done at 0 °C for 15 min or for 1 min at 25 °C (Fig. 6A). The apparent size of the adducts was ~50 kDa, in good agreement with the expected size of a cross-linked product between Tom 40 (38.1 kDa) and Tim8 (10.5 kDa) or Tim13 (9.5 kDa). The amount of the Tim8 cross-linked product was consistently lower than the product obtained with Tim13. As suggested by the results of the coimmunoprecipitation experiments (Fig. 3C), this is consistent with the notion that Tim13 is found on the outer surface of the Tim8-Tim13 complex so that a higher proportion of this protein would be in contact with the Tom40 precursor. Cross-linking adducts were not seen when the immunoprecipitations were done with preimmune serum or when cross-linker was omitted from the reaction (Fig. 6B). Similarly, no cross-linked products were observed when Tom40 was imported into mitochondria from the tim8RIP or tim13RIP strains (Fig. 6A), effectively eliminating the possibility that the cross-linked product was a nonspecific immunoprecipitate of an adduct formed between Tom40 and one of the small TOM complex components Tom5, Tom6, or Tom7. When cross-linking was attempted after 15 min of importing the Tom40 precursor at 25 °C, little cross-linked product was seen (Fig. 6C). These observations suggest that contact between Tim8-Tim13 and the Tom40 precursor may occur at the initial stages of import.



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  		FIG. 6.
Tom40 precursor contacts the Tim8-Tim13 complex. A, radiolabeled Tom40 precursor was incubated with mitochondria isolated from wild type strain NCN251 (cont), the tim8RIP strain (8RIP), or the tim13RIP strain (13RIP) for the indicated times at either 0 or 25 °C in individual reactions containing 40 µg of mitochondrial protein. The import reactions were subjected to cross-linking with 300 µM DSG for 30 min on ice. Excess cross-linker was quenched, and the mitochondria were reisolated. In one reaction, the mitochondria were resuspended in cracking buffer and electrophoresed to indicate the total content of each reaction (Total). In other reactions, reisolated mitochondria were lysed in 1% Triton X-100 and subjected to immunoprecipitation (IP) with either Tim8 ({alpha}8) or Tim13 ({alpha}13) antiserum. Samples were subjected to SDS-PAGE, blotted to nitrocellulose, and images obtained by PhosphorImaging. Specific cross-linked adducts of the Tom40 precursor with Tim8 or Tim13 are indicated with arrows. B, radiolabeled Tom40 precursor was incubated with isolated mitochondria from a wild type strain (NCN251) at 0 °C for 15 min. The indicated reactions were then subjected to cross-linking with 300 µM DSG for 30 min on ice and processed as in A. Immunoprecipitation reactions were carried out with either Tim8 ({alpha}8) or Tim13 ({alpha}13) antiserum, preimmune serum from rabbits injected with Tim8 (8PIS) or Tim13 (13PIS) antigen, or protein A-Sepharose beads alone. Samples were analyzed as in A. The arrow indicates the position where adducts would be observed. C, radiolabeled Tom40 precursor was incubated with isolated mitochondria for 15 min at 25 °C. Cross-linking and immunoprecipitations were performed as in A. The arrow indicates the position where adducts would be observed. D, the precursor of a Tom40 assembly mutant stalls at a position where contact with the Tim8-Tim13 complex occurs. Radiolabeled Tom40 mutant precursor lacking residues 321–323 ({Delta}KLG) was incubated with mitochondria isolated from wild type strain NCN251 (cont), the tim8RIP strain (8RIP), or the tim13RIP strain (13RIP) for the indicated times at 25 °C in individual reactions containing 40 µg of mitochondrial protein. The import reaction was subjected to cross-linking with 300 µM DSG for 30 min on ice and processed as in A. Specific cross-linked adducts of the Tom40 mutant precursor with Tim8 or Tim13 are indicated with an arrow. In all panels the asterisk (*) indicates the position of the Tom40 precursor protein.

 
Additional evidence for the interaction of the Tim8-Tim13 complex with Tom40 at an early stage of import was obtained using an assembly mutant of the Tom40 precursor. When the Lys-Leu-Gly residues at positions 321–323 of the Tom40 protein are deleted, the mutant precursor ({Delta}KLG) is reduced in its ability to assemble into the 250-kDa intermediate and does not progress past this stage, even at 25 °C (38). When cross-linking was performed after import of the {Delta}KLG variant, adducts were detected between the mutant precursor and both Tim8 and Tim13 even after 15 min of import at 25 °C (Fig. 6D). Thus, the mutant precursor stalled at an early stage of assembly has a prolonged interaction with the Tim8-Tim13 complex.

To examine further the nature of the cross-linked intermediate, cross-linking was performed in mitochondria from a wild type strain after 15 min of import at 0 °C followed by treatment with proteinase K. The cross-linked product persisted after the proteinase K treatment demonstrating that the Tom40 precursor that cross-links to the Tim8-Tim13 complex is in a protease-inaccessible location (Fig. 7). It has been shown previously that Tom40 in an early assembly intermediate was inaccessible to externally added protease (45) in contrast to fully imported Tom40, which is cleaved into 26-kDa and 12-kDa fragments by proteinase K (Fig. 4G). Thus, these findings also support the view that Tom40 precursor interacts with the Tim8-Tim13 complex at an early stage of import.



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  		FIG. 7.
Cross-linked adducts of Tom40 are not accessible to externally added protease. Radiolabeled Tom40 precursor was incubated with mitochondria isolated from a wild type strain (NCN251) for 15 min at 0 °C, and the import reactions were subjected to cross-linking with 300 µM DSG as in Fig. 6. Samples were either untreated (-PK) or subjected to 0.1 mg/ml proteinase K digestion (+PK) for 15 min on ice before centrifugation to reisolate mitochondria. Lanes contain either the total sample (Total) or samples obtained after immunoprecipitation with Tim13 antiserum ({alpha}13). Samples were subjected to SDS-PAGE, transferred to nitrocellulose, and images obtained by PhosphorImaging.

 
Cross-linking to Tim23 Precursor—Previous studies in yeast have shown that the Tim8-Tim13 complex is involved in the import of Tim23 and that cross-links between the complex and the precursor can be detected (23, 24, 26). It was of interest to determine whether the N. crassa Tim8-Tim13 complex also cross-linked to Tim23. Tim23 was imported under conditions of depleted membrane potential to arrest the precursor as a translocation intermediate. Cross-linker was added, and the samples were subjected to immunoprecipitation using antiserum to Tim8 and Tim13. Both proteins were found to form adducts with Tim23 precursor (Fig. 8). It has also been reported that the Tim9-Tim10 complex contacts Tim23 precursor during import (24, 47). Therefore, after cross-linking, Tim23 import reactions were also examined using antiserum to Tim9. As shown in Fig. 8, cross-links between the Tim23 precursor and Tim9 were observed.



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  		FIG. 8.
Tim8, Tim13, and Tim9 contact the Tim23 precursor during import. Radiolabeled Tim23 precursor was incubated with mitochondria isolated from a control strain (NCN251), the tim13RIP strain (13RIP), or the tim8RIP strain (8RIP) for 20 min at 25 °C. In each case the membrane potential was dissipated with carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin to stall the import of Tim23 as a translocation intermediate. The import reactions were subjected to cross-linking with 300 µM DSG for 30 min on ice. Excess cross-linker was quenched, and mitochondria were reisolated. One sample was dissolved directly in Laemmli gel cracking buffer (Total). Additional samples were lysed in 1% Triton X-100 and subjected to immunoprecipitation (IP) with Tim8 antiserum ({alpha}8), Tim13 antiserum ({alpha}13), Tim9 antiserum ({alpha}9), Tim 13 preimmune serum (13PIS), or unbound protein A-Sepharose beads. Immunoprecipitates were subjected to SDS-PAGE, blotted to nitrocellulose, and images obtained by PhosphorImaging. The position of specific cross-linked adducts of the Tim23 precursor with Tim8, Tim9, or Tim13 is indicated by the arrow.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that the N. crassa mitochondrial intermembrane space contains an 80-kDa hetero-oligomeric complex consisting of the Tim8 and Tim13 proteins. Examination of the tim8RIP and tim13RIP strains revealed that the absence of either Tim8 or Tim13 severely reduced the level of the other protein, favoring the hypothesis that they exist in a hetero-oligomeric complex that stabilizes both proteins. No growth abnormalities were observed in either mutant strain, and we conclude that the presence or absence of the complex does not affect the growth of N. crassa under the conditions tested. As described previously for yeast and humans (23, 25), we detected a reduction in the import of the Tim23 precursor into mitochondria lacking Tim8-Tim13 when the membrane potential across the inner membrane was reduced.

We observed a reduction in import of the precursors for Tom40 and porin, both {beta}-barrel proteins of the outer mitochondrial membrane. For Tom40, cross-links were readily detected with Tim13, and to a lesser extent with Tim8, during import of the precursor protein, suggesting that the precursor is in close proximity to the Tim8-Tim13 complex during import. When considered together with the reduction in import of Tom40 seen in the tim8RIP and tim13RIP mutant strains, these data strongly suggest a direct involvement of the Tim8-Tim13 complex in Tom40 import. The role played by the complex appears to be in the early stages of Tom40 import because the ability to detect cross-links is reduced in samples where import has proceeded for longer times. Similarly, cross-links of Tom40 precursor to the assembly factor Mas37 are not seen after longer import times (46). At the time when the Tom40 precursor can be cross-linked to the Tim8-Tim13 complex, it is not accessible to externally added proteases. This is consistent with the previous observation that Tom40 in early intermediates exists in the intermembrane space and is not accessible to protease (45). The Tim8-Tim13 complex is not part of the 250-kDa Tom40 assembly intermediate which contains other proteins such as Tom5, Mas37, and Tob55/Sam50 (45, 46, 48, 49), because the intermediate does form in mitochondria lacking the Tim8-Tim13 complex. The lack of an observable assembly intermediate containing the Tom40 precursor and the Tim8-Tim13 complex suggests that it does not survive the conditions of BNGE, which has been used to characterize the other intermediates of the Tom40 assembly pathway, or it is relatively short lived. Because cross-links are only observed at early stages of import, it seems likely that interactions of Tom40 precursors with Tim8-Tim13 are lost as the precursor progresses along the assembly pathway. However, cross-links can be observed after prolonged periods of import with a mutant form of the Tom40 precursor ({Delta}KLG) that is reduced in its ability to progress into the early 250-kDa intermediate form (38). All of these data suggest a model in which the Tom40 precursor interacts with the Tim8-Tim13 complex at an early point in the assembly pathway, most likely as it emerges from the TOM complex and is exposed to the intermembrane space.

How can the observations of decreased in vitro import of Tom40 and porin in mitochondria lacking the Tim8-Tim13 complex be reconciled with the finding that mutant cells exhibit no growth phenotype and contain the affected proteins at normal levels? The function served by Tim8-Tim13 in the assembly of these proteins may be a redundant one. In vivo, the rate of assembly of the affected proteins in mutant cells must not be limiting for growth and must be sufficient to allow accumulation to normal steady-state levels. Assembly of {beta}-barrel proteins in Escherichia coli provides an analogy that is similar in at least some respects. The 17-kDa Skp protein resides in the periplasmic space and acts as a chaperone for the assembly of {beta}-barrel proteins into the bacterial outer membrane (50, 51). Skp was found to bind early folding intermediates of OmpA and to be required for release of outer membrane proteins from the plasma membrane (52). The protein has also been shown to interact with PhoE while portions of the precursor of this {beta}-barrel protein are still in the inner membrane translocation channel (53). Although deletion of the gene encoding Skp does result in reduced steady-state levels of outer membrane proteins (50), it does not affect the growth of cells (52). However, synthetic growth defects are observed when the Skp deletion is coupled with deletion of genes encoding other periplasmic chaperones, suggesting that redundant pathways employing these chaperones exist for {beta}-barrel assembly in E. coli (54).

In N. crassa, both the Tim8-Tim13 complex and the Tim9-Tim10 complex appear to be involved in the import of Tim23. We observed an effect on the import of the Tim23 protein in the tim8RIP and tim13RIP mutants under conditions where the membrane potential was reduced, and we also detected cross-links between an arrested Tim23 precursor and the Tim8-Tim13 complex. However, we also found cross-links between Tim9 and an arrested Tim23 translocation intermediate, and other studies have shown that the N. crassa Tim9-Tim10 complex binds to N. crassa Tim23 peptides (47). The involvement of the Tim9-Tim10 complex in Tim23 import in yeast is not yet resolved because cross-links between Tim23 precursor and the Tim9-Tim10 complex have been reported (24), but peptide scans of yeast Tim23 with yeast Tim9-Tim10 revealed no binding (16).

It has been suggested that the effects of Mohr-Tranebjaerg syndrome result from deficiencies in Tim23 assembly which eventually lead to a reduction in overall import efficiency of matrix proteins. Conceivably, reduced assembly of outer membrane {beta}-barrel protein could exacerbate such effects on mitochondrial protein import and overall mitochondrial function and could occur even if mitochondrial membrane potential was normal. However, studies on a patient suffering from the syndrome revealed no clear effects on mitochondrial structure or oxidative phosphorylation (34). Thus, the relationship of any import defect to the manifestation of the disease remains unknown.

We have shown that the Tim8-Tim13 complex is involved in the import and assembly of Tom40, porin, and Tim23. Thus, the complex interacts with two different classes of membrane-spanning protein: {beta}-barrel proteins and a multitopic {alpha}-helical membrane-spanning protein. The role suggested for the Tim8-Tim13 complex in the import of Tim23 was to trap the precursor and prevent backsliding out of the TOM complex (23). The complex could play a similar role with the outer membrane {beta}-barrel proteins by preventing backsliding until the precursors are locked into their respective assembly pathways. Alternatively, Tim8-Tim13 might act as a chaperone to prevent aggregation of {beta}-strands until they begin to associate with further chaperones/assembly factors or to integrate into the membrane. Our findings, together with the recent observations that both Mas37 and Tob55/Sam50 are involved in the assembly of {beta}-barrel proteins (46, 48, 49), point to an increasingly complex picture of {beta}-barrel protein assembly in the mitochondrial outer membrane.


   FOOTNOTES
 
* This work was supported by a grant from the Canadian Institutes of Health Research (to F. E. N.) and by a graduate scholarship from Alberta Ingenuity (to S. C. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 780-492-5375; Fax: 780-492-9234; E-mail: frank.nargang{at}ualberta.ca.

1 The abbreviations used are: TOM, translocase of the outer mitochondrial membrane; BNGE, blue native gel electrophoresis; DSG, disuccinimidylglutarate; MOPS, 4-morpholinepropanesulfonic acid; RIP, repeat induced point mutation; TIM, translocase of the inner mitochondrial membrane. Back


   ACKNOWLEDGMENTS
 
We are grateful to Nancy Go for excellent technical assistance and to Troy Locke for running the Sephacryl columns. We also thank B. A. Roe, D. Kupfer, H. Zhu, J. Gray, S. Clifton, R. Prade, J. Loros, J. Dunlap, and M. Nelson. This project was aided by data obtained from the Neurospora crassa cDNA Sequencing Project (supported by funds from the National Science Foundation-Experimental Program to Stimulate Competitive Research). Our work was also aided by the Neurospora genome sequencing project at the Whitehead Institute Center for Genome Research (supported by the National Science Foundation).



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