Characterization of rat TOM40, a central component of the preprotein translocase of the mitochondrial outer membrane.

We cloned a 38-kDa rat mitochondrial outer membrane protein (OM38) with structural homology to the central component of preprotein translocase of the fungal mitochondrial outer membrane, Tom40. Although it has no predictable alpha-helical transmembrane segments, OM38 is resistant to alkaline carbonate extraction and is inaccessible to proteases and polyclonal antibodies added from outside the mitochondria, suggesting that it is embedded in the membrane, probably in a beta-barrel structure, as has been similarly speculated for fungal Tom40. Immunoprecipitation demonstrated that OM38 is associated with the major import receptors rTOM20 and rTOM22, and several other unidentified components with molecular masses of 5-10 kDa in digitonin-solubilized membrane: OM10, OM7.5, and OM5. Blue native polyacrylamide gel electrophoresis revealed that OM38 is a component of a approximately 400-kDa complex, firmly associating with rTOM22 and loosely associating with rTOM20. The preprotein in transit to the matrix interacted with the TOM complex containing OM38, and immunodepletion of OM38 resulted in the loss of preprotein import activity of the detergent-solubilized and reconstituted outer membrane vesicles. Taken together, these results indicate that OM38 is a structural and functional homolog of fungal Tom40 and functions as a component of the preprotein import machinery of the rat mitochondrial outer membrane.

We cloned a 38-kDa rat mitochondrial outer membrane protein (OM38) with structural homology to the central component of preprotein translocase of the fungal mitochondrial outer membrane, Tom40. Although it has no predictable ␣-helical transmembrane segments, OM38 is resistant to alkaline carbonate extraction and is inaccessible to proteases and polyclonal antibodies added from outside the mitochondria, suggesting that it is embedded in the membrane, probably in a ␤-barrel structure, as has been similarly speculated for fungal Tom40. Immunoprecipitation demonstrated that OM38 is associated with the major import receptors rTOM20 and rTOM22, and several other unidentified components with molecular masses of 5-10 kDa in digitoninsolubilized membrane: OM10, OM7.5, and OM5. Blue native polyacrylamide gel electrophoresis revealed that OM38 is a component of a ϳ400-kDa complex, firmly associating with rTOM22 and loosely associating with rTOM20. The preprotein in transit to the matrix interacted with the TOM complex containing OM38, and immunodepletion of OM38 resulted in the loss of preprotein import activity of the detergent-solubilized and reconstituted outer membrane vesicles. Taken together, these results indicate that OM38 is a structural and functional homolog of fungal Tom40 and functions as a component of the preprotein import machinery of the rat mitochondrial outer membrane.
Most mitochondrial proteins are synthesized in the cytosol as preproteins, delivered to the mitochondrial surface by cytosolic factors such as hsp70 and mitochondrial import-stimulating factor, and transported to the intramitochondrial compartments by the preprotein import machinery of the outer and the inner membranes (the TOM 1 and TIM complexes, respectively) (1)(2)(3)(4). The Saccharomyces cerevisiae TOM complex is composed of at least nine proteins (Tom71, -70, -40, -37, -22, -20, -7, -6, and -5) (5,6). Tom40, the central component of the translocation channel, stably associates with the Tom22 receptor and small Tom components, Tom7, -6, and -5, and forms a ϳ400-kDa general insertion pore complex in yeast (5). Composition of the Neurospora crassa TOM complex is similar to that of S. cerevisiae, but Tom5 has yet to be identified in N. crassa. The mitochondrial inner membrane has two separate import machineries (7): the Tim23-Tim17 system and the Tim54-Tim22-Tim18 system. The Tim23-Tim17 system functions in the translocation of preproteins across the inner membrane in conjunction with Tim44, mhsp70, and GrpE (8 -10), whereas the Tim54-Tim22-Tim18 system functions in collaboration with the intermembrane space proteins Tim13, Tim12, Tim10, Tim9, and Tim8, in the import of proteins without a cleavable presequence, such as the phosphate carrier, the ADP/ATP carrier, and several Tim proteins (Tim23, Tim22, and Tim17) (10 -16).
Although the fundamental mechanisms of mitochondrial protein import seem to be conserved from lower eukaryotes to mammals, only limited information is available for higher eukaryotic systems. Several mammalian counterparts have been identified and their roles are being studied: TOM20 (17)(18)(19)(20), TOM22 (21), TIM17 (22), TIM23 (22), TIM44 (22), and DDP1, a homolog of yeast Tim8 (23). In addition, several novel components that are thought to function as import receptors have also been found in mammalian mitochondria. Human TOM34 was cloned using a degenerate tetratricopeptide repeat sequence present in Tom70 and Tom20 (24). Metaxin is the 35-kDa C-terminal tail-anchor protein of the mitochondrial outer membrane (25). The antibodies raised against a 37-kDa outer membrane protein of rat liver mitochondria (OM37), inhibited preprotein import into the rat mitochondria (26). OM37 is anchored to the outer membrane through the N-terminal signal-anchor sequence. 2 These results suggest that features of the preprotein import system are unique to mammalian mitochondria and require further characterization. In the present study, we isolated the rat homolog of Tom40 and characterized it as a component of the TOM complex of rat liver mitochondria.

Methods
cDNA Cloning of Rat Tom40 -A 641-bp cDNA sequence encoding part of mouse TOM40 was assembled from two partial mouse EST nucleotide sequences (dbEST numbers 631778 and 757235). Based on this assembled sequence, the following oligonucleotides were synthesized: TOM40-1, 5Ј-GATGAATTCCCAACCCGGGGACGTT-3Ј (coding strand); TOM40-2, 5Ј-CAGAAGCTTTGTGATGCTCTGGAGGTA-3Ј (anticoding strand). Underlining in TOM40-1 and TOM40-2 indicates the restriction sites of EcoRI and HindIII, respectively. A 458-bp cDNA fragment was amplified from rat liver poly(A) ϩ -RNA by reverse transcriptase-polymerase chain reaction using TOM40-1 and TOM40-2 as the primers. This cDNA fragment was used as a probe to screen the gt10 rat cDNA library for rTOM40, and a ϳ1.6-kilobase pair cDNA encoding the entire rTOM40 was obtained.
Preparation of Antibodies against rTOM40 -A 1086-bp cDNA fragment was amplified by polymerase chain reaction using rTOM40 cDNA as the template and the following oligonucleotides as the primers: TOM40-3, 5Ј-ATCATATGGGGAACGTGTTGGC-3Ј (coding strand); TOM40-4, 5Ј-GGAGGGGATCCCCGATGGTGAGGCCAA-3Ј (anticoding strand). Underlining in TOM40-3 and TOM40-4 indicates the restriction sites of NdeI and BamHI, respectively. The obtained fragment was subcloned into the pET28a vector (Novagen) to create pET28a-NHIS40, which tags (His) 6 to the N terminus of the expressed protein. His-tagged rTOM40 was expressed in BL21(DE3) cells as inclusion bodies, which were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and the Coomassie Brilliant Blue-stained band was excised from the gel and used to raise antibodies in rabbits using the Ribi Adjuvant system (RIBI Immunochem Research Inc.).
Subcellular and Submitochondrial Fractionations-Subcellular fractionation of rat liver was performed as described previously (32). Submitochondrial fractionation by sucrose density gradient centrifugation was performed as follows. Mitochondria were diluted into 10 mM HEPES-KOH buffer (pH 7.4) containing 1 mM EDTA and protease inhibitor mixture (5 g/ml each of leupeptin, antipain, chymostatin, and pepstatin) and incubated at 0°C for 30 min. The mixture was sonicated on ice 5 times for 30 s each time, and centrifuged at 5,000 ϫ g for 10 min to obtain the supernatant. This fraction was layered over a linear gradient (0.6 to 1.6 M) of sucrose in hypotonic buffer and centrifuged at 100,000 ϫ g for 15 h at 4°C.
Immunofluorescence Microscopy-Immunofluorescence microscopy was performed as described previously (22). Briefly, normal rat kidney cells grown on glass coverslips in culture dishes were incubated with MitoTracker Red CMX Ros (Molecular Probes) at 37°C for 20 min. The cells were fixed with 50% methanol, 50% acetone for 2 min at room temperature. The coverslips were incubated with IgGs against rat TOM40 at room temperature for 1 h, washed, and then incubated with fluorescein isothiocyanate-conjugated goat antibodies against rabbit IgG. The images were obtained and analyzed using the confocal microscope, Radiance 2000 (Bio-Rad).
Immunoprecipitation of the TOM Complex-The mitochondrial outer membranes were incubated with 10 mM HEPES-KOH buffer (pH 7.4) containing 2% (w/v) digitonin, 200 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10% (v/v) glycerol (solubilization buffer) at 0°C for 30 min, followed by centrifugation at 100,000 ϫ g for 15 min. The supernatant pretreated with Protein A-Sepharose was incubated with anti-rTom20 or rTom40 IgG-bound Protein A-Sepharose at 4°C for 3 h. The reaction mixtures were centrifuged at 5,000 rpm for 5 min and the precipitates were washed 3 times with the solubilization buffer and suspended in the SDS-PAGE loading buffer. The eluted proteins were separated by SDS-PAGE and the gels were analyzed by immunoblotting or Coomassie Brilliant Blue staining.
Blue Native PAGE-Blue native PAGE was performed essentially as described previously (34,35). Mitochondrial outer membranes (50 g) were solubilized in 50 l of solubilization buffer and insoluble material was removed by centrifugation for 15 min at 100,000 ϫ g. The supernatant was mixed with 5 l of sample buffer (5% Coomassie Brilliant Blue G-250, 100 mM bis-Tris (pH 7.0), 500 mM 6-aminocaproic acid), and electrophoresed through 5 to 16% polyacrylamide gradient gels. For the second dimensional gel analysis, each individual lane was excised from the first gel and subjected to Tricine/SDS-PAGE.
Protein Import into Mitochondria-The reaction mixtures containing 25 g of mitochondria and 125 I-pAd or 35 S-pAd-dihydrofolate reductase (DHFR) were incubated in 50 l of 10 mM HEPES-KOH buffer (pH 7.4) containing 1 mM ATP, 20 mM sodium succinate, 5 mM NADH, 1 mg/ml fatty acid-free bovine serum albumin, and protease inhibitor mixture at 30°C for 30 min. When the import of 125 I-pAd was assayed, 1/10 volume of rabbit reticulocyte lysate was added to the reaction mixture to provide cytoplasmic chaperones. After import, the reaction mixtures were centrifuged, the precipitates were resolved by SDS-PAGE, and the gels were analyzed with a Bioimage Analyzer FLA2000 (Fuji).
Immunoprecipitation of the Import-arrested pAd-DHFR- 35 S-pAd-DHFR synthesized in the reticulocyte lysate was incubated at 0°C for 10 min in the import buffer (50 l) in the presence of 10 M methotrexate plus 1 mM NADPH and then 50 g of mitochondria were added to the reaction mixture, which was incubated further at 30°C for 30 min. The mitochondria were isolated by centrifugation and solubilized with the hypotonic buffer containing 1% (w/v) digitonin, 100 mM NaCl, 1 mM PMSF, 10% (v/v) glycerol, and protease inhibitor mixture at 0°C for 30 min. After centrifugation, the supernatant was subjected to immunoprecipitation as described above.
Reconstitution of Outer Membrane Vesicles-The reconstituted outer membrane vesicle was prepared as described previously with a slight modification (32). Mitochondrial outer membranes were solubilized with 10 mM HEPES-KOH buffer (pH 7.4) containing 1.5% (w/v) heptyl-␤-thioglucoside (HTG), 150 mM NaCl, 1 mM PMSF, 10% (v/v) glycerol, and protease inhibitor mixture at 0°C for 30 min, followed by centrifugation at 100,000 ϫ g for 15 min. The supernatant was incubated with Bio-Beads SM-2 (Bio-Rad) at 4°C overnight to reconstitute the outer membrane vesicles. Immunodepletion was performed as follows. Aliquots of the HTG-solubilized supernatant fractions were incubated at 4°C for 6 h with preimmune IgG-, anti-rTOM20-, or anti-rTOM40-IgGbound Protein A-Sepharose. The Protein A-Sepharose beads were removed by centrifugation and the supernatant was subjected to the reconstitution of membrane vesicles.
Preparation of MPP-loaded Outer Membrane Vesicles-MPP was loaded in the mitochondrial outer membrane vesicles according to the method of Mayer et al. (36,37). Purified yeast MPP (1.5 mg/ml) was added to the outer membrane vesicles in 10 mM HEPES-KOH buffer (pH 6.5) and the mixture was immediately frozen in liquid nitrogen and then placed on ice to allow the mixture to thaw slowly. A one-fifth volume of 100 mM HEPES-KOH buffer (pH 7.4) was added to the mixture, and this was then incubated at 30°C for 5 min and layered over a discontinuous gradient of 0.3, 0.88, and 1.5 M sucrose in 10 mM HEPES-KOH buffer (pH 7.4) containing 1 mM EDTA and 150 mM NaCl, followed by centrifugation at 100,000 ϫ g for 60 min at 4°C. The MPP-loaded membrane vesicles were recovered from the 0.88 -1.5 M sucrose interphase.
Preprotein Import into the Reconstituted Outer Membrane Vesicles-The outer membrane vesicles (10 g) were suspended in 50 l of 10 mM HEPES-KOH buffer (pH 7.4) containing 0.22 M mannitol, 0.07 M sucrose, 50 mM NaCl, 0.2 mg/ml fatty acid-free bovine serum albumin, and 50 g/ml HSP70 and incubated with 125 I-pAd at 0 or 30°C for 30 min. After the binding reaction, the mixtures were diluted 3-fold with HEPES-KOH buffer (pH 7.4) containing 200 mM NaCl (final concentration 150 mM NaCl), and ultracentrifuged to separate the supernatant and the membrane fractions. Both fractions were resolved by SDS-PAGE and the gels were analyzed using a Bioimage Analyzer FLA2000 for pAd. When the processing of pAd by the MPP-loaded outer membrane vesicles was to be monitored, 2 mM MnCl 2 was added to the reaction mixture.

RESULTS
Isolation of Rat Tom40 cDNA-Two sequences that partially overlapped and that were predicted to encode the partial amino acid sequence of the mouse counterpart of fungal Tom40 (dbEST numbers 631778 and 757235) (38,39) were found in the EST data base. We screened the rat gt10 cDNA library for the rat counterpart using the assembled nucleotide sequence as the probe and obtained a cDNA clone of ϳ1.6-kilobase pair that carried a putative open reading frame of 1086 bp encoding a 37918-Da protein. We concluded that this clone encoded the entire region of the authentic protein (OM38), because (i) the nucleotide sequence around the putative initiator methionine fit well with the Kozak motif (ACCATGG for (A/T)XXATGG), and (ii) the in vitro translated protein and the authentic protein as detected by immunoblotting exhibited the same mobility on SDS-PAGE and the molecular size estimated from the mobility coincided well with that calculated from the predicted sequence.
The sequence identity of OM38 to N. crassa and S. cerevisiae Tom40 was 28.6 and 26.8%, respectively (Fig. 1A). The predicted sequence of OM38 exhibited a similar hydropathy profile as fungal Tom40, and also contained no obvious ␣-helical hydrophobic segments (Fig. 1B). Because OM38 appeared to be buried deeply in the mitochondrial outer membrane as described below, it was likely inserted into the membrane in a ␤-barrel structure, as has also been speculated for fungal Tom40. OM38, as demonstrated by the experiments described below, constitutes a structural and functional homolog of fungal Tom40 and, hereafter, we refer to this protein as rTOM40. While this study was in progress, the amino acid sequences of the predicted human and mouse homologs of Tom40 with 92 and 81% identity to the rat sequence, respectively, were deposited into the data base (GenBank accession numbers AF043250 and AF044249, respectively).
Subcellular and Submitochondrial Localization of rTom40 -The subfractionation of the rat liver demonstrated that rTOM40 cofractionated with the mitochondrial marker protein, monoamine oxidase, but not with either cytochrome P450 (M1) or cytochrome H450, the marker proteins of rat liver microsomes (27) and cytosol (28), respectively ( Fig. 2A). Immunofluorescence microscopy also revealed that rTOM40 colocalized with MitoTracker as a filamentous structure in normal rat kidney cells (Fig. 2B).
In order to examine the submitochondrial localization of rTOM40, mitochondria were sonicated under hypotonic conditions and then subjected to sucrose density gradient centrifugation. rTOM40 was co-fractionated with monoamine oxidase, but not with rTIM23, an intrinsic inner membrane protein (22) (Fig. 2C). rTOM40 was resistant to alkaline carbonate (pH 11.5) extraction and high salt treatment of the mitochondria, and was solubilized from the membrane only in the presence of detergent (Fig. 3A). The intrinsic inner membrane protein, rTIM23, behaved similarly to rTOM40 during these treatments, whereas HSP60, the soluble protein in the matrix, was easily solubilized by alkaline carbonate treatment. The topology of rTOM40 in the outer membrane was then probed by proteinase K treatment of the mitochondria. As shown in Fig. 3B, rTOM40 was resistant to the externally added proteinase K, whereas rTOM20, which is the major import receptor and is anchored to the outer membrane through the N-terminal transmembrane segment extruding the bulk portion to the cytosol (20), was completely digested by this treatment. rTOM40 was also resistant to the treatment of trypsin added from outside the mitochondria (data not shown). rTOM40 was completely digested by proteinase K, however, under hypotonic conditions. The integrity of the inner membrane was preserved under these conditions, because proteinase K degraded only the Nterminal segment of rTIM23 that extrudes into the intermem- FIG. 2. Subcellular localization of rTOM40 in rat liver cells. A, 50 g each of mitochondria (mit), microsomes (ms), and cytosol (cyt) proteins from the rat liver were subjected to SDS-PAGE and immunoblotting using antibodies against the indicated proteins. The immunoreacted bands were visualized by ECL (Amersham Pharmacia Biotech). B, immunofluorescence detection of rTOM40 in normal rat kidney cells. C, submitochondrial localization of rTOM40. Submitochondrial fractionation by sucrose density gradient centrifugation was performed as described under "Experimental Procedures." After centrifugation, the samples were fractionated from the top of the tubes. The fractions were subjected to SDS-PAGE followed by immunoblot analysis using antibodies against the indicated proteins. Inp, 20 g of the unfractionated sample.
brane space (22) and the matrix-localizing HSP60 was resistant to the treatment (Fig. 3B, lane 4). Thus, rTOM40 is an integral membrane protein embedded in the mitochondrial outer membrane in a conformation that is inaccessible to the protease from the outside, although it is accessible from the inside. Furthermore, polyclonal antibodies raised against SDSdenatured rTOM40 at a concentration as high as 300 g/ml did not inhibit the import of pAd into the mitochondrial matrix. In contrast, antibodies raised against rTOM20 inhibited the import almost completely at a lower concentration (Fig. 4), indicating that rTOM40 is inaccessible to polyclonal antibodies added from outside the mitochondria. Taken together, rTOM40 acquires a conformation in the outer membrane that does not allow access to either proteases or antibodies. Similar properties have been noted for N. crassa Tom40; i.e. it is not accessible to either trypsin or to antibodies, but is accessible to proteinase K (38). In contrast, S. cerevisiae Tom40 assumes a topology that allows access to both proteases and antibodies (39).
rTOM40 Participates in Preprotein Import into Mitochondria-To assess the function of rTOM40 in preprotein import, we examined whether the import-arrested preprotein actually interacted with rTOM40. The pAd-DHFR fusion was imported into the mitochondria in a ⌬-dependent manner and the mature portion was protected against externally added proteinase K (Fig. 5A, lane 4). In contrast, in the presence of methotrexate, which induces tight folding of DHFR, the mature form as well as the precursor was completely digested by proteinase K (lane 6), indicating that the mature form had not translocated the outer and inner membranes and remained stacked in the translocation machinery, exposing a significant portion outside the mitochondria. The pAd-DHFR-stacked mitochondria were solubilized with digitonin and subjected to immunoprecipitation. As shown in Fig. 5B (lane 3), antibodies against rTOM40 precipitated pAd-DHFR and the mature form. Since rTOM40 is contained in the ϳ400-kDa TOM complex in digitonin-solubilized membrane (see below), these results indicate that the preprotein in transit to the matrix is actually integrated into the TOM complex, although the direct interaction of the preprotein with rTOM40 has yet to be shown. In contrast, the

FIG. 3. Characterization of rTOM40 as an integral membrane protein.
A, mitochondria were treated with 100 mM Na 2 CO 3 (pH 11.5), 1 M NaCl, or 1% Triton X-100 at 0°C for 30 min. The reaction mixtures were centrifuged to separate the supernatant (S) and precipitate (P) fractions, and each fraction was subjected to SDS-PAGE followed by immunoblot analysis using antibodies against the indicated proteins. B, rat liver mitochondria were treated with or without 100 g/ml proteinase K at 0°C for 20 min under isotonic or hypotonic conditions. After addition of 1 mM PMSF, the reaction mixtures were resolved by SDS-PAGE and analyzed by immunoblotting with the antibodies against the indicated proteins.
FIG. 4. Polyclonal antibodies against rTOM40 did not inhibit mitochondrial preprotein import. Mitochondria were preincubated with preimmune IgG (PI), anti-rTOM40 IgG (␣40), or anti-rTOM20 IgG (␣20) at 0°C for 30 min, reisolated, and subjected to the import of 125 I-pAd. The reaction mixtures were resolved by SDS-PAGE and the gels were analyzed by FLA2000. The import efficiencies were calculated as relative amounts of mature adrenodoxin (mAd), setting the amount of mAd imported into the untreated mitochondria as 100%. Inp: 100% of 125 I-pAd used. ND, not determined.

FIG. 5. rTOM40 interacts with the import-arrested preprotein.
A, reticulocyte lysate-synthesized 35 S-pAd-DHFR was preincubated with (ϩ) or without (Ϫ) methotrexate plus 1 mM NADPH (Mtx) and subjected to the import reaction. After import, the reaction mixtures were treated with or without 100 g/ml proteinase K at 0°C for 20 min and were resolved by SDS-PAGE followed by PhosphorImager analysis. As a control, the import reaction was performed in the presence of 10 M carbonyl cyanide p-chlorophenylhydrazone (lane 2). Inp, 20% of 35 S-pAd-DHFR used. B, immunoprecipitation of the import-arrested precursor with anti-rTOM40 IgG. 35 S-pAd-DHFR was imported into mitochondria in the presence or absence of Mtx as in A. The mitochondria were isolated and solubilized with 1% digitonin. The supernatants were divided into two aliquots, which were subjected to immunoprecipitation with preimmune IgG (PI) or anti-rTOM40 IgG (␣40). mature form was not precipitated at all in the absence of methotrexate, indicating that the mature protein had already exited the TOM complex stage.
rTOM40 Is Contained in the ϳ400-kDa TOM Complex, and Associates with rTOM20, rTOM22, and Several Other Components-S. cerevisiae Tom40 forms ϳ400-kDa complex with Tom22, Tom7, Tom6, and Tom5 in the digitonin-solubilized membrane (5), whereas the majority of the import receptors were not found in this ϳ400-kDa complex. A similar complex was detected in N. crassa mitochondria, although Tom5 was not identified (40,41). In N. crassa, the TOM core complex composed of Tom40, Tom22, Tom7, and Tom6 was isolated after dodecyl maltoside solubilization of the outer membrane (41). We therefore examined the interaction of rTOM40 with other import components of the rat mitochondrial outer membrane using immunoprecipitation. The outer membrane from rat liver mitochondria was solubilized with 2% digitonin and subjected to immunoprecipitation using antibodies against rTOM20 or rTOM40, and then the import components of the outer membrane in the precipitates and the supernatants were analyzed by immunoblotting. As shown in Fig. 6A, antibodies against rTOM40 efficiently precipitated rTOM20 and rTOM22. In contrast, there was no significant interaction between rTOM40 and the other import components of the outer membrane, rTOM70, 3 metaxin, and OM37.
Upon SDS-PAGE, the immunoprecipitates produced Coomassie Brilliant Blue-stained bands of rTOM40, rTOM20, and rTOM22 (Fig. 6B), which were confirmed by immunoblotting (data not shown). In addition, several protein bands with apparent molecular masses of 10 (OM10), 7.5 (OM7.5), and 5 (OM5) kDa were detected. Because these bands were not detectable in the precipitates with preimmune IgG, they were considered to be the components of the preprotein import machinery of the rat mitochondrial outer membrane. We then determined the size of the TOM complex by subjecting the digitonin-lysed mitochondrial outer membrane to blue native PAGE, which allows separation of the protein complex under native conditions. As shown in Fig. 6C, the major fraction of rTOM40 migrated as a ϳ400-kDa complex and the small fraction migrated as a ϳ190-kDa complex. rTOM22 was contained in the ϳ400-kDa complex firmly associating with rTOM40, but not in the ϳ190-kDa complex. In contrast to the results obtained using immunoprecipitation (see Fig. 6A), rTOM20 was mostly dissociated from the ϳ400-kDa complex. A similar phenomenon was observed for the N. crassa TOM complex; following blue native PAGE, the import receptors, Tom70 and Tom20, were mostly dissociated from the holo complex composed of Tom70, Tim40, Tom22, Tom20, and two small Tom subunits (41). Negative charges provided to the complex by Coomassie Brilliant Blue are thought to have destabilized the complex (41).
Function of rTOM40 as Assessed by Reconstituted Outer Membrane Vesicles-The involvement of rTOM40 in mitochondrial preprotein import was further assessed using the reconstituted outer membrane vesicles. For this purpose, the pre-3 H. Suzuki and K. Mihara, unpublished observations. FIG. 6. rTOM40 is a component of the ϳ400-kDa complex associating with rTOM20, rTOM22, and several unidentified components. A, interaction of rTOM40 with the import components of the outer membrane as probed by co-immunoprecipitation. The outer membranes were solubilized with 2% digitonin and aliquots of the supernatant were subjected to immunoprecipitation using preimmune (PI), anti-rTOM20 or anti-rTOM40 IgGs. The immunoprecipitates (P) and the supernatants (S) were subjected to SDS-PAGE followed by immunoblotting with antibodies against the indicated proteins. rTOM22 was detected using polyclonal antibodies against human TOM22 (21). Inp, an aliquot of the supernatant fraction. B, protein components of the immunoprecipitated complex as revealed by SDS-PAGE. The mitochondrial outer membranes were solubilized with 2% digitonin and aliquots of the supernatants were subjected to immunoprecipitation with preimmune IgG (PI), anti-rTOM40 IgG (␣40), or anti-rTOM20 IgG (␣20). The immunoprecipitates were washed with the solubilization buffer and analyzed using SDS-PAGE and Coomassie Brilliant Blue staining. The protein bands of rTOM40, rTOM22, and rTOM20 were identified by immunoblotting. C, analysis of rTOM40 with blue native PAGE. Rat liver mitochondrial outer membranes were solubilized with 2% digitonin, and solubilized supernatants were subjected to blue native PAGE as described under "Experimental Procedures." Gel slots were excised and subjected to the second dimensional Tricine/SDS-PAGE. The gels were analyzed by immunoblotting with the indicated antibodies. Marker proteins used were serum albumin, 67 kDa; lactate dehydrogenase, 140 kDa; catalase, 232 kDa; apoferritin, 440 kDa; and thyroglobulin, 669 kDa. protein import activity of the isolated outer membrane vesicles was examined. When 125 I-labeled pAd was incubated with the outer membrane at 30°C, pAd was inserted into the vesicles in a time-and temperature-dependent fashion, becoming resistant to salt extraction (Fig. 7A). These results suggest that pAd was partly translocated from the salt-labile cis-site of the membrane to reach the trans-site to acquire salt resistance at 30°C (36,42). To confirm this further, outer membrane vesicles were prepared in which purified MPP was enclosed by freezing and thawing (37). After incubation with 125 I-labeled pAd, the vesicles were recovered by centrifugation and analyzed by SDS-PAGE. The vesicles prepared in the presence of MPP, but without freezing-thawing, bound to pAd in a temperature-dependent manner (Fig. 7B, lanes 2 and 3) without any processing of pAd. On the other hand, vesicles prepared by freezingthawing in the presence of MPP processed pAd in a temperaturedependent manner (lanes 5 and 6). The processed form of pAd was completely degraded by externally added proteinase K (lane 7). These results indicate that the presequence portion including the MPP-processing site was translocated across the outer membrane and processed by the MPP present in the lumen, whereas the mature portion remained untranslocated and was thus degraded by proteinase K, confirming a previous report (37). Of note, however, a substantial fraction of the precursor that had reached the salt-resistant trans-site remained unprocessed by MPP. We speculate that the mitochondrial matrix proteins may be required in the lumen of the vesicles to facilitate access of the cleavage site of partly translocated preprotein to MPP. We then examined the function of rTOM40 with the reconstituted outer membrane vesicles using salt-resistant binding of pAd to the membrane as the criterion. For this purpose, the 1.5% HTG-solubilized outer membranes were subjected to immunodepletion using antibodies against rTOM40 or rTOM20. Then the supernatants were treated with Bio-Beads to remove the HTG and to reconstitute the outer membrane vesicles (32). Antibodies against rTOM40 depleted rTOM40 almost completely, whereas rTOM20 was unaffected by the treatment, and vice versa (Fig. 8A, lanes 3 and 4). As shown in Fig. 8B, the reconstituted vesicles prepared from the rTOM40-or rTOM20-depleted extracts exhibited only marginal, if any, translocation activity for pAd as assessed by salt-resistant pAd binding (Fig. 8B, lanes 6 -9). The possibility that import components other than rTOM40 or rTOM20 were depleted from the reconstituted vesicles by these treatments was ruled out because 1.5% HTG dissociated the TOM complex FIG. 7. Import of the presequence portion of pAd into the mitochondrial outer membrane vesicles. A, temperature-and timedependent binding of pAd to the mitochondrial outer membrane vesicles. 125 I-pAd was incubated with the outer membrane vesicles at 0 or 30°C for the indicated times. The concentration of NaCl was then adjusted to 150 mM and the reaction mixtures were centrifuged to separate the membrane (P) and the supernatant (S) fractions. Both fractions were resolved by SDS-PAGE and the gels were analyzed with a PhosphorImager. Inp, 100% of 125 I-pAd used. B, translocation of presequence of pAd across the outer membrane as revealed by processing by MPP enclosed within the vesicles. 125 I-pAd was incubated with the MPP-loaded outer membrane vesicles at 0 or 30°C for 30 min under the same conditions as in A, except that 2 mM MnCl 2 was included in the reaction mixture. After the reaction, the reaction mixtures were treated with or without 50 g/ml proteinase K at 0°C for 20 min, 1 mM PMSF was added, and then the mixtures were centrifuged. The membrane vesicles were analyzed as in A. Inp, 100% of input 125 I-pAd.
FIG. 8. Function of rTOM40 as assessed by reconstituted outer membrane vesicles. A, characterization of the reconstituted outer membrane vesicles after immunodepletion of rTOM40 or rTOM20. The mitochondrial outer membranes were solubilized by 1.5% HTG-containing buffer. Aliquots of the solubilized material were either left untreated (ROM), or incubated with preimmune IgG (PI), anti-rTOM40 (␣40), or rTOM20 (␣20) IgGs, and were then served for reconstitution of the membrane vesicles as described under "Experimental Procedures." The reconstituted vesicles were analyzed by SDS-PAGE and immunoblotting with antibodies against rTOM40, rTOM20, and OM37. B, import of pAd into the reconstituted outer membrane vesicles. 125 I-pAd was imported to the reconstituted outer membrane vesicles at 0 or 30°C for 30 min. Other conditions are as described in the legend to Fig. 7 and under "Experimental Procedures." Inp, 100% of input 125 I-pAd. and rTOM40 migrated as a dimer in blue native PAGE (data not shown). Furthermore, immunoprecipitation with anti-rTOM40 IgG revealed that rTOM22 as well as rTOM20 was completely dissociated from rTOM40 (data not shown). Thus, participation of rTOM40 in the preprotein import reaction could be demonstrated. Reconstitution of the import activity using the HTG-solubilized and rTom40-depleted fraction and recombinant rTom40 was unsuccessful. DISCUSSION Although detailed findings have now been obtained regarding the import machinery of fungal mitochondria, very little is known about the import machinery of mammalian mitochondria. We have identified rat TOM40 as a component of the mammalian TOM complex. The amino acid sequence of rTOM40 was 28.6% identical with that of N. carassa Tom40. Despite this low identity, there are several common characteristics. (i) They are both deeply embedded into the outer membrane, since they assume a conformation that does not allow access for proteases or polyclonal antibodies added to the mitochondria from the outside. (ii) They have no predictable transmembrane segments with a potential ␣-helical structure. (iii) Rat TOM40, like its N. crassa counterpart, is associated with rTOM20 and rTOM22, and is present in the ϳ400-kDa complex in the digitonin-solubilized outer membrane together with several other components, OM10, OM7.5, and OM5. Furthermore, immunoprecipitation with the import-arrested preprotein as well as experiments with the rTOM40-depleted and reconstituted outer membrane vesicles demonstrated that rTOM40 functions as a component of the preprotein translocation machinery of the rat mitochondrial outer membrane. We thus conclude that rTOM40 is the major component of the TOM machinery of mammalian mitochondria and functions as the translocation channel, as is also the case for fungal Tom40. Our attempts at complementation of the function of ⌬tom40 yeast cells by rTOM40 were not successful.
Immunoprecipitation with IgGs against rTOM40 or rTOM20 as well as blue native PAGE revealed that the rat TOM complex contained several unidentified components, in addition to rTOM40, rTOM22, and rTOM20: OM10, OM7.5, and OM5. We speculate that these components might correspond to the small Tom proteins of the fungal TOM complex and we are currently characterizing them.
These results suggest that the TOM complex of mammalian mitochondria resembles the fungal Tom complex, but is distinct from the plant TOM system: the ϳ230-kDa complex containing Tom40, Tom20, and Tom7, but not Tom22 or Tom37 in a digitonin-solubilized state (43). Tom22 and Tom37 have not been identified in plant mitochondria (43). Further experiments are necessary to precisely define the structure and function of the mammalian TOM complex in order to clarify its similarity to and distinction from the TOM machinery of other organisms.