(Received for publication, March 21, 1997, and in revised form, May 10, 1997)
From the Department of Molecular Biology, Graduate School of
Medical Science, Kyushu University, Fukuoka 812, Japan, the
Kazusa DNA Research Institute, Chiba 292, Japan, the
§ Department of Chemistry, Faculty of Science, Nagoya
University, Chikusa-ku, Nagoya 464-01, Japan
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 tom20 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 Tom70·Tom37 and Tom20·Tom22 subcomplexes (1, 2). The precursors are targeted to Tom70·Tom37 through the action of an ATP-requiring cytoplasmic chaperone such as the mitochondrial import stimulation factor (MSF)1 (2-5), transferred to Tom20·Tom22 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 Tom70·Tom37, bind to Tom20·Tom22, 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.
MSF and hsp70 were purified from rat liver cytosol according to Hachiya et al. (3) and Deshaies et al. (15), respectively. Recombinant pAd was purified in 7 M urea as described (16). Antibodies against rat Tom20 were prepared by immunizing a rabbit with the recombinant protein that had been separated by SDS-PAGE. Antibodies against rat monoamine oxidase (MAO), yeast Bmh1p, yeast Kar2p, and yeast Tom70 were generous gifts from Akio Ito (Kyushu University), Masao Sakaguchi (Kyushu University), Masao Tokunaga (Kagoshima University), and Gottfried Schatz (Basel Biocenter), respectively. Antibodies against rat liver microsomal cytochrome P450(M-1), rat liver cytoplasmic hemoprotein H450, and yeast mitochondrial porin were as described (17-19). Monoclonal antibody against hsp60 (SPA-807) was purchased from Stressgen Biotechnologies Corp.
Subfractionation of Rat Liver Mitochondria by Sucrose Density Gradient CentrifugationRat 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 × g for 10 min, and the supernatant was centrifuged at 100,000 × 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 × g for 16 h, 1 ml-fractions were collected from the top of the tubes.
Isolation of Rat Tom20 cDNAThe coding region of human
cDNA was amplified by polymerase chain reaction (PCR) using the
cDNA clone of 3259 base pairs (GenBankTM accession
number D13641) as the template and the following oligonucleotides as
the primers: 5-GTAGAGACCATGGTGGGT-3
(coding strand) and
5
-TTTTAAGTGGGATCCTATTAT-3
(anticoding strand). The DNA fragment thus
obtained was used as the probe to screen the rat cDNA library in
gt10 for Tom20 cDNA.
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 µm). 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, 5-TATGAATTCATGGTGGGCCGG-3
; OR20A140,
5
-CACGAATTCCTAACCAAAGCTCTGAGCACTG-3
; OR20A103,
5
-CAGGAATTCCTAAGGCTGTCCACACACAGC-3
; and OR20A69,
5
-CTCGAATTCTTAGAATTTCTGAACAGCTTCAG-3
.
To construct pD2R2025-69, pD2R20 was subjected to PCR using the
oligonucleotides OR20S and ODA24 or OR20A and ODS. Two PCR products
were digested with EcoRI and BglII and ligated
together to the EcoRI-digested pD2 to obtain
pD2R20
25-69. To construct pD2R20
2-18, pD2R20 was subjected to
PCR using the oligonucleotides OR20A and OS19. The PCR product was
ligated to the EcoRI site of pD2. These manipulations
artificially produced Arg25-Ser26 in the mutant Tom20. The sequences of
oligonucleotides used are as follows where EcoRI and
BglII sites are underlined: OR20A, 5
-TATGAATTCTCATTCCACATCATCCTCAC-3
; ODS,
5
-CAGAGATCTTTCCTTGAAGAGATACACCTT-3
; ODA24,
5
-TTCAGATCTAAAGTAGATGCAGTACCCTAT-3
; and OS19,
5
-ATAGAATTC-3
.
tom20 yeast cells (SNY1005)2
harboring these plasmids were grown on synthetic medium (
Trp) plates
containing 2% glucose and were streaked onto new synthetic medium
(
Trp) plates containing either 2% glucose or 3% glycerol and left
at 30 °C for 2-4 days.
pAd import into
yeast mitochondria was performed as follows. Mitochondria (50 µg)
isolated from tom20 yeast cells expressing rat Tom20N69
were incubated with or without pre-immune IgGs (100 µg) or IgGs
against yeast Tom70 (ammonium sulfate-fractionated, 100 µg) or rat
Tom20 (affinity purified, 4 µg) at 0 °C for 30 min in 50 µl 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 125I-pAd·MSF complex or
125I-pAd·hsp70 complex as the substrate. pAd import into
rat liver mitochondria was performed as follows. Rat liver mitochondria (100 µg) were treated with 100 µg of pre-immune IgGs or 50 µg of
anti-rat Tom20 IgGs at 0 °C for 60 min in 50 µl 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 125I (20).
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).
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
(Glu141-Glu145). 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 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.
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 succinate-cytochrome 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 Tom70·Tom37 first, then transferred to Tom20·Tom22, 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 Tom70·Tom37 and are directly targeted to
Tom20·Tom22. 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 125I-labeled and
mixed to preform the pAd·hsp70 and pAd·MSF·hsp70 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
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 (
70% 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.
tom20 cells harboring plasmids for wild type or
C-terminal-truncated rat Tom20 proteins were able to grow on the
glycerol-containing 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
3-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 wild-type 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 anchor-segment are essential for the function of rat Tom20, whereas the
TPR motif as well as the C-terminal acidic amino acid cluster are
not.
|
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
To further confirm the results
obtained above, we performed an in vitro import assay using
mitochondria isolated from tom20 cells expressing rat
Tom20N69. 125I-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 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.
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 hsp70-dependent 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 = 17 residues and D/E = 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 Tom70·Tom37 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 TPR-deletion 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 mitochondria-targeting signals are correctly recognized and translocated across the outer mitochondrial membrane.