Analysis of the Functional Domain of the Rat Liver Mitochondrial Import Receptor Tom20*

Tom20 is an outer mitochondrial membrane protein and functions as a component of the import receptor complex for the cytoplasmically synthesized mitochondrial precursor proteins. It consists of the N-terminal membrane-anchor segment, the tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker segment between the membrane anchor and the TPR motif, and the C-terminal acidic amino acid cluster. To assess the functional significance of these segments in mammalian Tom20, we cloned rat Tom20 and expressed mutant rat Tom20 proteins in Δtom20 yeast cells and examined their ability to complement the defects of respiration-driven growth and mitochondrial protein import. Tom20N69, a mutant consisting of the membrane anchor and the linker segments, was targeted to mitochondria and complemented the growth and import defects as efficiently as wild-type Tom20, whereas a mutant lacking the linker segment did not. In vitro protein import into mitochondria isolated from the complemented yeast cells revealed that the precursor targeted to yeast Tom70 was efficiently imported into the mitochondria via rat Tom20N69. Thus the linker segment is essential for the function of rat Tom20, whereas the TPR motif and the C-terminal acidic amino acids are not.

Protein import into mitochondria depends on the import receptors of the outer membrane. These components are Tom70, Tom22, and Tom20 in fungi and yeast, plus an additional component, Tom37, in yeast (1). In yeast, these receptors function as the Tom70⅐Tom37 and Tom20⅐Tom22 subcomplexes (1,2). The precursors are targeted to Tom70⅐Tom37 through the action of an ATP-requiring cytoplasmic chaperone such as the mitochondrial import stimulation factor (MSF) 1 (2)(3)(4)(5), transferred to Tom20⅐Tom22 ATP dependently, and then translocated across the outer membrane (4). Urea-denatured precursors or those which can assume unfolded conformations by themselves or by the action of hsp70 bypass Tom70⅐Tom37, bind to Tom20⅐Tom22, and are then imported into mitochondria independently of the cytoplasmic ATP (4,5).
Relatively little is known about the import machinery of mammalian mitochondria. The 29-, 42-, and 52-kDa components of the rat liver outer mitochondrial membrane have been reported to be the components of the import machinery (6,7). However, the function of these proteins remains unknown. The outer mitochondrial membrane proteins, OM37 in rats and metaxin in mice, have been shown to function as the components of the receptor for the precursor-MSF complex (herein referred to as the MSF-receptor; see Refs. 8 and 9). Recently, homologues of Tom20 in humans and an inner membrane component Tim17 in humans and Drosophila melanogaster have been identified and characterized (10 -14).
In the present study, we have identified a rat homologue of Tom20 by functional assay, analyzed its role in targeting the precursor to mitochondria, and identified the domain of rat Tom20 responsible for the function of the import receptor. Rat Tom20 complemented both the growth and the mitochondrial import defects of ⌬tom20 yeast cells on a nonfermentable carbon source, which corresponded well with the correct targeting of the expressed rat Tom20 to yeast mitochondria. Taking advantage of this complementation, we analyzed the functional segment of rat Tom20 as the import receptor since Tom20 exhibits characteristic structural features that are shared by yeast, fungi, and mammals: an N-terminal hydrophobic segment, a putative tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker region between the hydrophobic segment and the TPR motif, and a cluster of negatively charged amino acid residues at the C terminus. The truncated cDNA coding for the N-terminal 69 amino acid residues (rat Tom20N69) containing the membrane anchor and the linker segments, but lacking the TPR motif and the acidic amino acid cluster, complemented the defects of both growth and mitochondrial import of ⌬tom20 yeast cells as efficiently as wild-type rat Tom20. Rat Tom20⌬25-69 and Tom20⌬2-18, the mutants lacking the linker domain and most of the transmembrane segment, respectively, did not complement these defects although they were expressed and localized to mitochondria in yeast cells. In vitro import experiments with the mitochondria isolated from rat Tom20N69-complemented yeast cells revealed that the precursor was targeted to yeast Tom70 MSF dependently, then transferred to rat Tom20N69, and finally imported into the mitochondria. These results indicate that the linker segment is essential, whereas the TPR, as well as the C-terminal acidic amino acids, is dispensable for the import receptor function of rat Tom20.
Subfractionation of Rat Liver Mitochondria by Sucrose Density Gradient Centrifugation-Rat liver mitochondria were isolated as described (3), except that HES buffer (10 mM HEPES-KOH, pH 7.5, containing 1 mM EDTA and 300 mM sucrose) was used as the homogenization buffer. Mitochondria were swollen in 10 mM HEPES-KOH, pH 7.5, containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.1 mM chymostatin on ice for 10 min and sonicated 6 times for 10 s with 30-sec intervals. The mixture was centrifuged at 10,000 ϫ g for 10 min, and the supernatant was centrifuged at 100,000 ϫ g for 1 h. The precipitate was suspended in 10 mM HEPES-KOH, pH 7.5, containing 1 mM PMSF and layered over a 12-ml linear gradient of 0.6 -1.6 M sucrose in 10 mM HEPES-KOH buffer, pH 7.5. After centrifugation at 100,000 ϫ g for 16 h, 1 ml-fractions were collected from the top of the tubes.
Isolation of Rat Tom20 cDNA-The coding region of human cDNA was amplified by polymerase chain reaction (PCR) using the cDNA clone of 3259 base pairs (GenBank TM accession number D13641) as the template and the following oligonucleotides as the primers: 5Ј-GTA-GAGACCATGGTGGGT-3Ј (coding strand) and 5Ј-TTTTAAGTGG-GATCCTATTAT-3Ј (anticoding strand). The DNA fragment thus obtained was used as the probe to screen the rat cDNA library in gt10 for Tom20 cDNA.

Expression of Wild Type and Mutant Rat Tom20 Proteins in Yeast-
The yeast expression plasmid of rat Tom20 cDNA, pD2R20, was constructed as follows. The entire coding region of rat Tom20 cDNA was amplified by PCR and inserted into the EcoRI site downstream of the ADH1 promoter of pD2 (TRP1, 2 m). Yeast expression vectors for the truncated forms of rat Tom20 were prepared as follows. pD2R20 was subjected to PCR using a sense primer (OR20S) corresponding to the N terminus of rat Tom20 and antisense primers, OR20A140, OR20A103, and OR20A69, to obtain cDNAs for Tom20N140, Tom20N103, and Tom20N69. The PCR products were inserted into the EcoRI site of pD2 to obtain pD2R20N140, pD2R20N103, and pD2R20N69, respectively. The nucleotide sequences of OR20S, OR20A140, OR20A103, and OR20A69 were as follows where underlines indicate EcoRI sites: OR20S, 5Ј-TATGAATTCATGGTGGGCCGG-3Ј; OR20A140, 5Ј-CACGA-ATTCCTAACCAAAGCTCTGAGCACTG-3Ј; OR20A103, 5Ј-CAGGAAT-TCCTAAGGCTGTCCACACACAGC-3Ј; and OR20A69, 5Ј-CTCGAAT-TCTTAGAATTTCTGAACAGCTTCAG-3Ј.
In Vitro Import of pAd into Mitochondria-pAd import into yeast mitochondria was performed as follows. Mitochondria (50 g) isolated from ⌬tom20 yeast cells expressing rat Tom20N69 were incubated with or without pre-immune IgGs (100 g) or IgGs against yeast Tom70 (ammonium sulfate-fractionated, 100 g) or rat Tom20 (affinity purified, 4 g) at 0°C for 30 min in 50 l of the import buffer consisting of 10 mM HEPES-KOH, pH 7.5, 250 mM sucrose, 1 mM ATP, 1 mM reduced glutathione, 5 mM magnesium acetate, 20 mM sodium succinate, and 60 mM potassium acetate, washed once, and subjected to the import at 30°C for 60 min using 125 I-pAd⅐MSF complex or 125 I-pAd⅐hsp70 complex as the substrate. pAd import into rat liver mitochondria was performed as follows. Rat liver mitochondria (100 g) were treated with 100 g of pre-immune IgGs or 50 g of anti-rat Tom20 IgGs at 0°C for 60 min in 50 l of the import buffer, washed once with the import buffer, and subjected to the import using pAd-hsp70 or pAd-MSF-hsp70 as the substrate in which all of the components were labeled with 125 I (20).
Other Methods-Succinate-cytochrome c reductase was assayed as described (21). Yeast cell fractionation was carried out according to the method of Daum et al. (22), except that Zymolyase 100T was used to prepare the spheroplasts. Yeast lysates were prepared according to Yamazaki et al. (23).

Isolation of Rat Tom20 cDNA-A search of the EBI Data
Bank revealed that a human cDNA with an open reading frame encoding a protein of 145 amino acid residues (DDBJ, accession number D13641) exhibited a significant homology to Tom20 of Saccharomyces cerevisiae and Neurospora crassa. Using the coding region of this cDNA as a probe, we isolated the cDNA from the rat liver cDNA library. The cDNA encodes a (Fig. 1 pick;f1;0) protein of 145 amino acid residues. The sequence of the predicted protein shows an overall identity of 32, 31, and 98% with Tom20 from S. cerevisiae, N. crassa (Fig. 1), and humans, respectively. The predicted sequence is characteristic in that it contains an N-terminal hydrophobic segment (residues 7-24) that seems to function as the membrane anchor, followed by a charged amino acids-rich segment of 45 amino acid residues (the "linker segment", 25-69) and the C-terminal cluster of acidic amino acids (Glu 141 -Glu 145 ). These features are highly conserved in Tom20 from mammals, yeast, and fungi.
Tom20 has been reported to contain the TPR motif downstream of the linker segment (24). TPR is a highly conserved 2 S-i. Nishikawa and T. Endo, unpublished data. stretch of tandemly repeated amphiphilic ␣-helices (domains A and B) and is thought to mediate diverse protein-protein interactions (25). Tom20 from S. cerevisiae and N. crassa contains a perfect consensus sequence of the B-domain of the TPR: X-X-A-X-X-X-F-X-X-A-X-X-X-X-P-X-X. In addition, Tom20 and Tom70 from yeast and Neurospora share the sequence motif F-X-K-A-L-X-(V/L), or its minor variations, which is present in the B-domain of the TPR motif (26). However, the corresponding region of Tom20 from rats and humans exhibited weak homology to the TPR as well as to the F-X-K-A-L-X-(V/L) motif, and the secondary structure prediction shows that the corresponding region of mammalian Tom20 potentially forms a ␤-sheet structure (data not shown), a structure which is undetectable in the TPR motif (25). Although its identity as the TPR thus remains somewhat uncertain, we tentatively refer to this region of rat Tom20 as the TPR in the present study.
Intracellular and Submitochondrial Localizations of Rat Tom20 -Subfractionation of the rat liver indicated that rat Tom20 was cofractionated with a marker enzyme of the outer mitochondrial membrane, MAO, but not with cytochrome P450(M1) or hemoprotein H450, the marker proteins of microsomes (27) and cytosol (18), respectively ( Fig. 2A). When the mitochondria were subjected to hypotonic treatment followed by sucrose density gradient centrifugation, rat Tom20 co-sedimented with MAO at around fraction 5 but not with succinatecytochrome c reductase, the marker enzyme of the inner membrane (Fig. 2B). Thus rat Tom20 is the protein of the outer mitochondrial membrane.
Effect of Anti-Tom20 IgGs on the Protein Import into Rat Liver Mitochondria-It has been reported in yeast mitochondria that the precursors complexed with MSF are docked onto Tom70⅐Tom37 first, then transferred to Tom20⅐Tom22, and finally translocated across the outer membrane (4). Urea-denatured precursors or precursors that are able to maintain the unfolded conformations by themselves or through the action of hsp70 bypass Tom70⅐Tom37 and are directly targeted to Tom20⅐Tom22. We examined this with rat liver mitochondria and assessed the function of rat Tom20 during the initial step of the precursor import. pAd, MSF, and hsp70 were 125 I-labeled and mixed to preform the pAd⅐hsp70 and pAd⅐MSF⅐hsp70 complexes, and then the import of pAd in these complexes into the antibody-treated mitochondria was examined (Fig. 3). Anti-rat Tom20 IgGs did not inhibit MSF-dependent binding of pAd but inhibited its import into the matrix (Fig. 3, lanes 5 and 6), whereas they inhibited both the binding and the import of pAd in the hsp70-dependent pathway (lanes 7 and 8). These results indicate that Tom20 functions at the junction of both import pathways. The pAd-MSF complex first docks onto the MSF receptor located upstream of Tom20, and pAd is then transported into the mitochondria via Tom20, whereas pAd in the hsp70 complex binds directly to Tom20 and is then imported into the matrix. As reported previously, MSF was released to the supernatant in an ATP-dependent manner, whereas hsp70 was spontaneously released to the supernatant during this import reaction (lanes 2, 4, 6, and 8) (20). A significant amount of pAd remained bound to the antibody-treated mitochondria (lane 7). This is probably because a fraction of pAd bypassed Tom20 and was targeted to the unidentified component, such as the mammalian homologue of Tom22 since Tom22 has been reported to cooperate with Tom20 to function as an import receptor in N. crassa (28).
The Functional Domain of Rat Tom20 as Analyzed by Complementation of the Growth Defect of ⌬tom20 Yeast Cells-FIG. 2. Localization of rat Tom20 to the outer mitochondrial membrane. A, 100 g each of mitochondrial, microsomal, and cytosolic proteins from rat liver were subjected to SDS-PAGE and immunoblotting using IgGs against rat Tom20, MAO, cytochrome P450(M-1), or hemoprotein H450. B, rat liver mitochondria were subjected to hypotonic treatment and then fractionated by sucrose density gradient centrifugation. Each fraction was assayed for the amounts of total protein, succinate-cytochrome c reductase, MAO, and rat Tom20.
FIG. 3. Anti-rat Tom20 IgGs discriminate between the MSFand hsp70-dependent import of pAd into mitochondria. Mitochondria were treated with pre-immune IgGs or anti-rat Tom20 IgGs, washed once, and then used for the import reaction. The pAd⅐hsp70 and pAd⅐MSF⅐hsp70 complexes in which all of the components were labeled with 125 I were subjected to the import. After the import, mitochondria (Mt) and supernatant (S) fractions were separated by centrifugation and subjected to SDS-PAGE. Radioactive proteins were visualized with a Fuji BioImage Analyzer (BAS2000). Positions of hsp70, large and small subunits of MSF, pAd, and mature adrenodoxin (mAd) are indicated in the figure.
Introduction of the vector carrying rat Tom20 cDNA (pD2R20) into the mutant cells complemented the growth defect of the cells on a nonfermentable carbon source although the growth rate was slightly slower than that of wild-type cells (Figs. 4A-C). Western blotting of the lysate of the complemented cells indicated that rat Tom20 was expressed in ⌬tom20 yeast cells (Fig. 5) and that the expressed protein was co-fractionated with mitochondrial porin, but not with the proteins of microsomal or cytosolic fractions, indicating that it was correctly targeted to the mitochondria in yeast cells (Fig. 6). It should be noted that rat Tom20 recovered to yeast mitochondria was easily proteolysed to form a 15-kDa fragment. Since this fragment was resistant to alkali extraction, the processing seemed to occur at the C-terminal region of rat Tom20 in the cytoplasmic side.
We took advantage of this complementation to analyze the functional domain of rat Tom20 in ⌬tom20 yeast cells. It is noted in this context that the importance of the TPR motif of yeast Tom20 in the physical interaction with Tom70 carrying seven TPR motifs has been reported (29). We constructed yeast expression vectors harboring cDNAs coding for 1-140 (pD2R20N140), 1-103 (pD2R20N103), and 1-69 (pD2R20N69) of rat Tom20 and the cDNA (pD2R20⌬25-69 or pD2R20⌬2-18) in which residues 25-69 (the linker region) or 2-18 (Ϸ70% of the membrane-anchor segment) of Tom20 had been deleted, and we examined their ability to complement the defect of the respiration-dependent growth of ⌬tom20 yeast cells. FIG. 4. Complementation of the defect of respiration-dependent growth of ⌬tom20 yeast cells by rat Tom20 proteins. A, wild-type yeast cells transformed with pD2 or ⌬tom20 yeast cells transformed with pD2, pD2R20, pD2R20N140, pD2R20N103, or pD2R20N69 were streaked onto synthetic medium plates containing 2% glucose or 3% glycerol, and the plates were incubated at 30°C for 2 or 4 days, respectively. B, ⌬tom20 yeast cells harboring pD2R20, pD2R20⌬25-69, or pD2R20⌬2-18 were grown on a glucose-containing or glycerol-containing plate as described in panel A. C, yeast cells grown overnight at 30°C in glucose-containing medium were diluted with 20 -50 volumes of glycerol-containing medium and cultured at 30°C. Cell growth was monitored by measuring the absorbance at 600 nm. FIG. 6. Subcellular localization of rat Tom20 proteins expressed in ⌬tom20 yeast cells. A, ⌬tom20 yeast cells harboring pD2R20 were fractionated into mitochondria, microsomes, and cytosol. 100-g fractions were subjected to SDS-PAGE and immunoblot analysis with the IgGs against porin, Kar2p, or Bmh1p to assess mitochondria, microsomes, and cytosol, respectively. Note that anti-Bmh1p IgGs cross-react with Bmh2p in the cytosol. B, ⌬tom20 cells harboring pD2R20 or pD2R20D25-69 were fractionated into mitochondria, microsomes, and the cytosol fractions, and each fraction was subjected to immunoblot analysis with anti-Tom20 IgGs. ⌬tom20 cells harboring plasmids for wild type or C-terminaltruncated rat Tom20 proteins were able to grow on the glycerolcontaining plate although their growth was slightly slower than that of wild-type cells (Fig. 4A). In contrast, ⌬tom20 cells transformed with pD2R20⌬25-69 or pD2R20⌬2-18 could not grow on the glycerol-containing medium (Fig. 4, B and C). ⌬tom20 cells harboring these plasmids expressed Tom20 proteins with the expected molecular sizes although the extent of expression differed between them (Fig. 5). It is worth noting that the expression of Tom20N69 and Tom20⌬25-69 was significantly lower than that of wild type or other mutant Tom20 proteins and was probably due to their instability in yeast cells, although the expression of Tom20⌬25-69 was Ϸ3-fold higher than that of Tom20N69 (Table I). Cell fractionation indicated that Tom20N69 (not shown) and Tom20⌬25-69 were both targeted to mitochondria (Fig. 6B). Nevertheless, they were distinct in their complementation of the growth defect of ⌬tom20 cells: Tom20N69 complemented the defect, whereas Tom20⌬25-69 did not. The membrane-anchor mutant Tom20⌬2-18 was expressed at about 30% of the level of wildtype Tom20 but was unable to complement the growth defect of ⌬tom20 yeast cells (Table I). Taken together, these results suggest that the linker domain and the membrane anchorsegment are essential for the function of rat Tom20, whereas the TPR motif as well as the C-terminal acidic amino acid cluster are not.
Tom20⌬25-69 Is Unable to Complement the Defect of Mito-chondrial Protein Import in ⌬tom20 Yeast Cells-Western blotting with monoclonal anti-hsp60 antibody revealed a significant accumulation of pre-hsp60 in ⌬tom20 cells harboring pD2 (Fig. 7). This import deficiency was complemented by the expression of wild-type rat Tom20 or rat Tom20N69. No accumulation of pre-hsp60 was observed in ⌬tom20 cells expressing Tom20N140 or Tom20N103 (data not shown). In marked contrast, pD2R20⌬25-69 was unable to complement the defect of mitochondrial import of pre-hsp60 in ⌬tom20 cells (Fig. 7). Thus the TPR motif and the C-terminal acidic amino acid cluster are dispensable for the complementation of the defect of mitochondrial protein import in ⌬tom20 yeast cells.
Rat Tom20N69 in ⌬tom20 Yeast Mitochondria Functions Normally as the Precursor Receptor in Vitro-To further confirm the results obtained above, we performed an in vitro import assay using mitochondria isolated from ⌬tom20 cells expressing rat Tom20N69. 125 I-labeled pAd was incubated with MSF or hsp70 to preform the complexes, and the import of pAd in the complexes into mitochondria was examined. As shown in Fig. 8, pAd was actively imported into the mitochondria MSF dependently, and this import was inhibited by IgGs against FIG. 7. Effect of the expression of wild type and mutant forms of rat Tom20 upon mitochondrial import of pre-hsp60 in ⌬tom20 yeast cells. ⌬tom20 cells expressing wild type and mutant rat Tom20 proteins were grown at 30°C overnight in glucose-containing medium. The yeast cell lysates were prepared and were subjected to SDS-PAGE followed by Western blotting with the monoclonal antibodies against hsp60. The positions of pre-hsp60 (p) and hsp60 (m) are indicated in the figure.
FIG. 8. Effect of IgGs against rat Tom20 or yeast Tom70 on the in vitro import of pAd into mitochondria isolated from ⌬tom20 yeast cells expressing rat Tom20N69. Mitochondria isolated from ⌬tom20 yeast cells expressing rat Tom20N69 were incubated with or without preimmune IgGs or IgGs against yeast Tom70 or rat Tom20, washed once, and subjected to the import reaction using 125 I-pAd⅐MSF complex or 125 I-pAd⅐hsp70 complex as the substrate. The reaction mixtures were treated with 100 g/ml proteinase K at 0°C for 30 min and analyzed by SDS-PAGE and autoradiography. Other conditions are described under "Experimental Procedures." Percent inhibitions of MSFdependent import were as follows: ␣-yeast Tom70, 72% and ␣-rat Tom20, 91%. Percent inhibitions of hsp70-dependent import were as follows: ␣-yeast Tom70, 0% and ␣-rat Tom20, 92%.

TABLE I
Summary of experiments on ⌬tom20 yeast cells expressing rat Tom20 constructs The N-terminal transmembrane segment (TM), the putative tetratricopeptide repeat (TPR) motif, and the C-terminal acidic amino acid cluster in rat Tom20 are indicated. The amount of the expressed proteins was determined by scanning the X-ray films in Western blotting analysis using a dual-wavelength scanner (Shimadzu CS-930).
Rat Tom20 constructs expressed in ⌬tom20 yeast cells Respirationdependent growth

Expression of rat Tom20s
Localization of rat Tom20s Recovery of in vivo import of hsp60 yeast Tom70 or rat Tom20 (Fig. 8, top panel). In marked contrast, the hsp70-dependent import was inhibited only by IgGs against rat Tom20 but not by those against yeast Tom70 (Fig.  8, bottom panel). These results clearly indicate that rat Tom20N69 in the complemented yeast mitochondria functions normally as the import receptor and that pAd docked onto yeast Tom70 is transferred to rat Tom20N69 and then imported into the mitochondria.

DISCUSSION
In this paper, we have characterized the function of rat Tom20 in precursor targeting to and import into the mitochondria in vitro and identified the region responsible for its function as the import receptor both in vivo and in vitro (summarized in Table I).
The antibodies against rat Tom20 strongly inhibited hsp70dependent binding as well as the import of the precursor into rat liver mitochondria. In contrast, they did not interfere with the MSF-dependent binding of the precursor to the mitochondria but did inhibit its import into mitochondria. These results are consistent with our previous results showing that the precursors that can maintain the unfolded conformations by themselves or by complexing with hsp70 are directly targeted to Tom20, that the precursors that are complexed with MSF first dock at the MSF receptor located upstream of Tom20, and that the ATP-hydrolysis induced transport of the precursors via Tom20 (20). Similar results have been reported with yeast mitochondria (4). Thus, the essential part of the import apparatus seems to be conserved among species.
Taking advantage of rat Tom20-induced suppression of the growth defect of ⌬tom20 cells on a nonfermentable carbon source, we analyzed the functional segment of rat Tom20. The respiration defect of ⌬tom20 cells and their adaptation within days to the loss of Tom20 have been reported to be correlated with the loss of Tom22 and with its restoration, respectively (30,31). However, ⌬tom20 cells used in the present study grew normally in the glucose-containing medium and maintained their respiration deficiency throughout the experiments. Furthermore, Western blotting revealed that yeast Tom20 was absent, and the amount of Tom22 did not alter to any appreciable extent in ⌬tom20 cells expressing rat Tom20 mutants (data not shown). Thus, Tom22 is not the limiting factor for the defects of ⌬tom20 yeast cells under the present experimental conditions. Unexpectedly, Tom20N69, which contains both the membrane anchor and the linker segments but lacks the TPR segment and the C-terminal acidic amino acid cluster, complemented the growth defect as well as the defect of mitochondrial import in ⌬tom20 yeast cells. In contrast, the mutant lacking the linker segment could not complement these defects at all although it was expressed to a higher extent than Tom20N69 and was targeted to mitochondria. This was also supported by the finding that the mitochondria isolated from ⌬tom20 yeast cells expressing Tom20N69 imported the precursor both in an hsp70-dependent and in a MSF-dependent manner; anti-rat Tom20 IgGs inhibited both pathways, whereas anti-yeast Tom70 IgGs inhibited only the latter. These results indicate that rat Tom20N69 functioned as the import receptor and that transfer of the precursor from yeast Tom70 to rat Tom20N69 occurred normally in the complemented yeast mitochondria. Thus, we conclude that the putative TPR segment of rat Tom20 is dispensable for this function, whereas the linker domain is not. In support of this notion, only the membrane anchor and the downstream linker segment of Tom20 exhibit pronounced sequence conservation among species. Since Tom20 has been reported to bind precursor proteins through electrostatic interactions with the positively charged presequences in S. cerevisiae and N. crassa (28,32), we speculate that the charged amino acid-rich linker segment (K/R ϭ 17 residues and D/E ϭ 8 residues in a segment of 45 residues) is important for the recognition of the presequence of the precursors.
Haucke et al. (29) have shown that the TPR motif of yeast Tom20 increases interaction of Tom20 with the Tom70⅐Tom37 complex, but its mutation does not inactivate the receptor function of Tom20. However, we could not detect the requirement of the corresponding segment of rat Tom20 in the precursor import process either in vivo or in vitro. A possible explanation for this difference could be that the decreased interaction between Tom70 and Tom20 caused by the TPRdeletion of Tom20 was suppressed by the overexpression of the Tom20 mutants. Another possibility is that different regions of rat and yeast Tom20s are responsible for the interaction with Tom70 since human Tom20 has been reported to lack homology to the A-domain of the typical TPR motif and shows only weak homology to the core motif of the TPR B-domain (10). Analyses of precursor-receptor interactions or precursor transfer between the receptors using the cytoplasmic domains of Tom20, Tom70, or their mutants are required to clarify these issues as well as to clarify the mechanisms by which the mitochondriatargeting signals are correctly recognized and translocated across the outer mitochondrial membrane.