Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan
* Author for correspondence (e-mail: mihara{at}cell.med.kyushu-u.ac.jp )
Accepted 9 January 2002
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Summary |
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Key words: Mitochondrial protein import, Import receptor, Preprotein translocase, Mitochondrial outer membrane, Precursor proteins
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Introduction |
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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. Recently, several mammalian
counterparts were identified, and their roles are being studied. These
mammalian proteins are TOM40 (Suzuki et
al., 2000), TOM22 (Saeki et
al., 2000
), TOM20 (Hanson et
al., 1996
; Goping et al.,
1995
; Seki et al.,
1995
), TIM17 (Ishihara and
Mihara, 1998
), TIM23 (Ishihara
and Mihara, 1998
), TIM44
(Ishihara and Mihara, 1998
)
and DDP1, a homologue of yeast Tim8
(Koehler et al., 1999
). A rat
gene homologous to fungal Tom70 has been identified by differential display
PCR as a thyroid-hormone-regulated gene that is located in specific brain
regions, although its function remained to be determined
(Alvarez-Dolado et al., 1999
).
In addition, several novel components that are thought to function as import
receptors are also found in mammalian mitochondria. Human TOM34 contains a TPR
sequence with a sequence similarity to those of fungal Tom70 and Tom20
(Nuttall et al., 1997
). It is
loosely associated with the outer membrane via its N-terminal hydrophobic
segment, and antibodies against TOM34 inhibit mitochondrial import of
matrix-targeted preproteins (Nuttall et
al., 1997
). Metaxin is a 35 kDa C-terminal membrane-anchor protein
that has 25% sequence identity with the N-terminal region of yeast Tom37, and
antibodies against metaxin also inhibit mitochondrial import of a
matrix-targeted precursor preadrenodoxin (pAd)
(Armstrong et al., 1997
).
Whether metaxin is the mammalian counterpart of yeast Tom37 is not known. The
antibodies against OM37, a 37 kDa outer membrane protein of rat liver
mitochondria, inhibited mitochondrial docking of the pAd-MSF complex and the
subsequent transport of pAd into mitochondria
(Komiya et al., 1996
;
Komiya and Mihara, 1996
). The
homologue of OM37 is not found in yeast. These findings might reflect features
of the preprotein import system unique to mammalian mitochondria and require
further characterization.
In the present study, a 70 kDa outer membrane protein of rat liver
mitochondria (OM70) was identified as the rat homologue of Tom70 and was
characterized as the receptor of the rat TOM complex for a subset of
preproteins that carry internal mitochondria-targeting signals. Furthermore,
we demonstrated that the N-terminal segment, consisting of the N-terminal
hydrophilic segment, the transmembrane domain (TMD), and the following three
basic amino acid residues, is sufficient to function as a
mitochondria-targeting signal. This structural feature of the
mitochondria-targeting signal is also observed in the N-terminal
membrane-anchor protein rTOM20 (Kanaji et
al., 2000). An in vitro assay revealed that rTOM70 was targeted
and inserted into the mitochondrial outer membrane independent of the import
receptors, and this insertion strictly depended upon the basic amino-acid
residues in the C-terminal flanking region of the TMD.
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Materials and Methods |
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cDNA cloning of rTOM70
A 927 bp cDNA sequence encoding part of a putative mouse homologue of
fungal Tom70 was assembled from five partial mouse EST nucleotide sequences
(dbEST IDs: 756557, 794014, 851708, 1188043, 851269). On the basis of this
assembled sequence, the following oligonucleotides were synthesized: TOM70-1:
5'-GATGAATTCGATGCTCAAGGCAAATACATG-3'; TOM70-2:
5'-CAGGGATCCTAGCCTTGCTGATAAGCTCC-3'.
Underlining in the TOM70-1 and TOM70-2 sequences denotes the restriction
sites of EcoRI and BamHI, respectively. A 650 bp cDNA
fragment was amplified from the rat liver poly(A)+-RNA by RT-PCR
using TOM70-1 and TOM70-2 as the primers. This cDNA fragment was used as a
probe to screen the gt10 rat cDNA library for rat OM70, and an
2.5 kbp cDNA encoding the entire OM70 was obtained.
Preparation of antibodies against OM70
An 1830 bp cDNA fragment was amplified by PCR using OM70 cDNA as the
template and the following oligonucleotides as the primers: TOM70-3:
5'-GATCATATGGCCGCCTCTAAGCCC-3'; TOM70-4:
5'-GCGGGATCCTATAATGTCGGTGGTT-3'.
Underlining in TOM70-3 and TOM70-4 denotes the restriction sites of NdeI and BamHI, respectively. The obtained fragment was subcloned into the pET28b vector (Novagen) to create pET28b-NHIS70, which tags (His)6 to the N-terminus of the expressed protein. His-tagged OM70 was expressed in BL21 (DE3) cells as inclusion bodies, which were separated by SDS-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.).
Construction of expression plasmids for rTOM fusion proteins
The expression vector for rTOM70-HA was constructed as follows. The coding
region of rTOM70 cDNA was amplified by PCR using pET28b-NHIS70 as the template
and the following oligonucleotides as the primers: TOM70-5:
5'-TGCGGTACCACCATGGCCGCCTCTAAGCCCGTAGAG-3'; TOM70-6:
5'-CGCGGATCCTAATGTCGGTGGTTTTAATCCGTA-3'.
Underlining in TOM70-5 and TOM70-6 denotes the restriction sites of KpnI and BamHI, respectively. The isolated fragment was subcloned into KpnI-BamHI-digested pcDNA3.1 in which HA-tag sequence had been inserted to create pcDNA-rTOM70HA. pcDNA-rTOM70S6HA was constructed by the Kunkel method using pcDNA-rTOM70HA as the template. rTOM70-GFP was constructed using pET28b-NHIS70 as the template and the following oligonucleotides as the primers: TOM70-7: 5'-TGCTCTAGAACCATGGCCGCCTCTAAGCCCGTAGAG-3'; TOM70-8: 5'-CGCTCTAGATAATGTCGGTGGTTTTAATCCGTA-3'.
Underlining in TOM70-7 and TOM70-8 denotes the restriction sites of
XbaI. The obtained PCR fragment was subcloned into the XbaI
site of pRcG (Kanaji et al.,
2000) to create pRc-rTOM70GFP. cDNAs coding rTOM70(1-69)GFP,
rTOM70(42-69)GFP, rTOM70(1-66)GFP and rTOM70(1-65)GFP were all constructed by
PCR using pRc-rTOM70GFP as the template and appropriate oligonucleotides as
the primers: the 5'-upstream primers containing HindIII site
and 3'-downstream primers containing XbaI site. Thus obtained
fragments were subcloned into HindIII-XbaI digested
pRcG.
Subcellular and submitochondrial fractionations
Subcellular fractionation of the rat liver was performed as described
previously (Ishihara and Mihara,
1998). 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 the protease inhibitor
cocktail (PIC: 5 µg/ml each of leupeptin, antipain, chymostatin, and
pepstatin) (`hypotonic buffer') and incubated at 0°C for 30 minutes. The
mixture was sonicated on ice 5 times for 30 seconds each time and centrifuged
at 5,000 g for 10 minutes to obtain the supernatant. This fraction
was layered over a linear gradient of sucrose from 0.6 to 1.6 M in hypotonic
buffer and centrifuged at 100,000 g for 15 hours at 4°C.
Protein import into mitochondria
The reaction mixtures containing 25 µg mitochondria and rabbit
reticulocyte-lysate-synthesized 35S-Su9-DHFR, ADP/ATP carrier (AAC)
or rTOM40 were incubated in 50 µl 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 PIC (`import buffer') at 30°C for 30
minutes. When the import of 125I- or 35S-labeled
recombinant pAd was assayed, 1/10 of the volume of rabbit reticulocyte lysate
or 20 µg/ml MSFS (Alam et al.,
1994) was added to the reaction mixture, respectively. To examine
the effect of antibodies against the import components on the preprotein
import, mitochondria were pretreated with the antibodies in the homogenization
buffer (Ishihara and Mihara,
1998
) at 0°C for 30 minutes, washed once with the
homogenization buffer, then subjected to the import reactions. The import of
35S-rTOM70 into isolated rat liver mitochondria was performed as
described above. After import, the reaction mixture was treated with 100 mM
Na2CO3 (pH 11.5) as described previously
(Kanaji et al., 2000
) and the
mitochondria and supernatant were subjected to SDS-PAGE and the gels analyzed
for rTOM70 using a Bioimage Analyzer FLA2000 (Fuji). The membrane-insertion of
35S-AAC and 35S-rTOM40 was assessed as follows. After
the import, the reaction mixtures were incubated with 100 µg/ml of
proteinase K at 0°C for 30 minutes under hypotonic (for AAC) or isotonic
(for rTOM40) conditions, followed by incubation with 1 mM PMSF at 0°C for
15 minutes. The reaction mixtures were centrifuged, the precipitates were
resolved by SDS-PAGE, followed by fluoroimage analysis.
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, 50 mM NaCl, 1 mM PMSF and 10%
(v/v) glycerol (`solubilization buffer') at 0°C for 30 minutes, followed
by centrifugation at 100,000 g for 15 minutes. The supernatant
was incubated first with Protein A-Sepharose, centrifuged and the supernatant
was incubated with anti-rTOM20, anti-rTOM40 or anti-rTOM70 IgG-bound Protein
A-Sepharose at 4°C for 3 hours. The reaction mixtures were centrifuged at
5,000 rpm for 5 minutes, and the precipitates were washed three 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.
Blue native PAGE
Blue native PAGE was performed as previously described
(Schägger and von Jagow,
1991; Schägger et al.,
1994
). Mitochondrial outer membranes (50 µg) were solubilized
in 50 µl solubilization buffer, and insoluble material was removed by
centrifugation for 15 minutes at 100,000 g. The supernatant
was mixed with 5 µl 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,
individual lanes were excised from the first dimensional gel and subjected to
Tricine SDS-PAGE.
Yeast two-hybrid assay
Maintenance and transformation of yeast cells, using the MATCHMAKER GAL4
Two-Hybrid System 2 (Clontech), were performed according to the manufacturer's
protocol. The cytosolic domain of rTOM20 (D25-E145), rTOM22 (M1-R82), rTOM70
(R64-L610) and OM37 (R41-D338) were amplified by PCR, and the PCR fragments
were inserted separately downstream of the GAL4 DNA-binding domain (BD) in the
pAS2-1 plasmid (TRP1) or into the GAL4-activating domain (AD) in the
pACT2 plasmid (LEU2). Two-hybrid interactions were assayed using
either the HIS3 reporter or the lacZ reporter systems.
Cotransformation of two hybrid vectors into S. cerevisiae Y190
(MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112,
gal4, gal80
, cyhr2, LYS2:: GAL1UAS-HIS3TATA-HIS3,
MEL1URA3:: GALUAS-GAL1TATA-lacZ) was performed according to the
manufacturer's instruction. The transformants were screened for their
potential to grow on synthetic complete medium lacking tryptophan, leucine and
histidine. The transformants were also assayed for ß-galactosidase
activity of the lacZ reporter gene according to Adams et al.
(Adams et al., 1997
).
Transfection of COS-7 cells and immunofluorescence microscopy
COS-7 cells were cultured on coverslips in 35-mm dishes in 2 ml of DMEM
supplemented with 10% fetal calf serum at 37°C overnight under an
atmosphere of 10% CO2 in air. Transfection was performed using
FuGene 6 reagent (Roche Molecular Biochemicals). The cells were incubated for
24 hours. When mitochondria were to be stained, 100 nM Mito Tracker Red CMX
Ros (Molecular Probes, Eugene, OR) was added to the medium and incubated for
20 minutes before fixation. The cells were fixed with 50% methanol/50% acetone
for 2 minutes at room temperature. The coverslips were incubated with rabbit
polyclonal (BAbCO) or mouse monoclonal (BAbCO) anti-HA epitope tag antibodies
at room temperature for 1 hour, washed and then incubated with FITC-conjugated
goat antibodies against rabbit IgGs. To monitor the effect of brefeldin A
(BFA) on intracellular localization, cells were treated with 5 µg/ml BFA
for 1 hour. To stain the endoplasmic reticulum (ER), the fixed cells on
coverslips were incubated with rabbit anti-rat Calnexin (StressGen) antibodies
for 1 hour and then with FITC-conjugated goat antibodies against rabbit IgGs.
The images were obtained and analyzed using a confocal microscope, Radiance
2000 (BioRad).
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Results |
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The sequence identity of OM70 with N. crassa and S. cerevisiae Tom70 was 28.1% and 20.1%, respectively (Fig. 1A). The predicted sequence of OM70 exhibited a similar hydropathy profile to N. crassa and S. cerevisiae Tom70 and also contained a TMD in the N-terminus (Fig. 1B). The cytoplasmic domain of OM70 contained 10 TPR motifs, and 7 and 9 TPR motifs in S. cerevisiae and N. crassa, respectively. OM70, as demonstrated by the experiments described below, constitutes a structural and functional homologue of fungal Tom70; thus, we refer to this protein as rTOM70 hereafter. While this study was in progress, the amino-acid sequences of the predicted human and Drosophila homologues of Tom70 with 92% and 40% identity to the rat sequence, respectively, were deposited in the database (GenBank accession numbers NM_014820 and AE003634, respectively). Partial DNA sequence of a rat homologue of the fungal Tom70 had also been deposited in the database (EMBL accession number: AJ243368).
Subcellular and submitochondrial localization of rTOM70
Subfractionation of the rat liver and subsequent immunoblotting indicated
that rTOM70 was cofractionated with the mitochondrial marker protein, rTIM23,
but not with either cytochrome P450 (M1) or cytochrome H450, the marker
proteins of rat liver microsomes
(Matsumoto et al., 1986) and
cytosol (Ishihara et al.,
1990
), respectively (Fig.
2B). Immunofluorescence microscopy also revealed that the
endogenous TOM70 colocalized with MitoTracker as a filamentous structure in
HeLa cells (Fig. 2C).
To examine the submitochondrial localization of rTOM70, mitochondria were
sonicated under hypotonic conditions and subjected to sucrose density gradient
centrifugation. As shown in Fig.
2D, rTOM70 co-fractionated with the marker protein of the outer
membrane, rTOM40, but not with rTIM23, an intrinsic inner membrane protein
(Ishihara and Mihara, 1998).
rTOM70 was resistant to alkaline carbonate (pH 11.5) extraction or high
salt-treatment of the mitochondria (Fig.
3A). The intrinsic inner membrane protein, rTOM40, behaved
similarly to rTOM70 in these treatments, whereas hsp60, the soluble protein in
the matrix, was easily solubilized by alkaline-carbonate treatment. The
topology of rTOM70 in the outer membrane was then probed by trypsin treatment
of the mitochondria. As shown in Fig.
3B, 25 µg/ml trypsin cleaved rTOM70, and a cytoplasmic fragment
of
60 kDa was released into the supernatant fraction, whereas rTOM20,
which is the major import receptor, with the N-terminal membrane anchor
extruding the bulk portion to the cytosol
(Iwahashi et al., 1997
), was
completely digested by this treatment. The matrix-localizing malate
dehydrogenase (MDH) as well as the intermembrane space-localizing cytochrome c
was also resistant to this treatment, indicating that both the outer and inner
mitochondrial membranes remained intact during these treatments.
|
These results and the predicted primary amino-acid sequence indicate that
rTOM70 is an integral membrane protein that is anchored to the outer membrane
through the N-terminal TMD, extruding the bulk TPR-containing portion to the
cytosol with a membrane topology similar to that of fungal Tom70
(Söllner et al.,
1990).
Antibodies against rTOM70 inhibit mitochondrial import of preproteins
with the internal targeting signal
We examined the effect of antibodies against rTOM70 on the preprotein
import into mitochondria. As shown in Fig.
4A, specific IgGs against rTOM70, at concentrations as high as 400
µg/ml, did not inhibit mitochondrial import of pre-Su9-DHFR and pAd, the
preproteins with the matrix-targeted and cleavable presequence, whereas
anti-rTOM20 IgGs at the same concentration inhibited their import
(Fig. 4A, left panels). We then
examined the effect of the antibodies on the mitochondrial import of the AAC,
the inner membrane protein with the internal targeting signal
(Pfanner et al., 1987;
Wiedemann et al., 2001
). The
import of AAC was assessed by measuring the formation of the fragment AACf,
which is produced from correctly inserted AAC by proteinase K digestion of the
mitoplasts (Wiedemann et al.,
2001
; Kübrich et al.,
1998
; Ishihara and Mihara,
1998
). IgGs against rTOM70 inhibited AAC import in a
dose-dependent manner (Fig. 4A,
right panel). Similarly, they inhibited insertion of rTOM40 into mitochondria
(Fig. 4A, right panel).
Anti-rTOM20 IgGs also inhibited both insertion reactions. These results were
consistent with the reported specificity of fungal Tom70, which functions as
the receptor of preproteins with an internal targeting signal, such as the
phosphate carrier, AAC, Tom40 (Keil et
al., 1993
) and Tim 54 (Kurz et
al., 1999
).
|
In rat liver mitochondria, antibodies against OM37 inhibit both
mitochondria binding of the pAd-MSF complex and ATP-induced subsequent
transport of pAd across the outer membrane via rTOM20, thus inhibiting the
overall reaction of pAd import into the mitochondria
(Komiya et al., 1996;
Komiya and Mihara, 1996
). To
probe the function of rTOM70 in the preprotein transport pathways of the rat
mitochondrial outer membrane, we examined the effects of anti-rTOM70 and
anti-OM37 IgGs on the MSF-assisted mitochondrial import of pAd. As shown in
Fig. 4B (right panel),
anti-OM37 IgGs and anti-TOM20 IgGs efficiently inhibited the MSF-supported
import of pAd, consistent with our previous report
(Komiya et al., 1996
).
Anti-rTOM70 IgGs did not inhibit the same import. The same results were
obtained with rabbit reticulocyte lysate to provide cytoplasmic chaperones
(Fig. 4B, left panel). Taken
together, these findings indicate that rTOM70 functioned as the import
receptor for preproteins with an internal targeting signal and did so probably
upstream of OM37 or OM37-independently, upstream of rTOM20.
rTOM70 is loosely associated with the 400 kDa TOM complex
containing rTOM40 and rTOM22
S. cerevisiae Tom40 forms an 400 kDa complex with Tom22,
Tom7, Tom6 and Tom5 in digitonin-solubilized membranes
(Dekker et al., 1998
), whereas
most of the import receptors were not observed in the complex. A similar
complex was detected in N. crassa mitochondria, although Tom5 was not
identified (Künkele et al.,
1998
; Ahting et al.,
1999
). We therefore examined the interaction of rTOM70 with the
other import components of rat mitochondrial outer membranes using
immunoprecipitation. Rat liver mitochondrial outer membranes were solubilized
using 2% digitonin-50 mM NaCl and subjected to immunoprecipitation using
antibodies against rTOM20, rTOM40 or rTOM70. The import components of the
outer membrane recovered in the precipitates were then analyzed by
immunoblotting. As shown in Fig.
5A, antibodies against rTOM20 and rTOM40 efficiently precipitated
the other import components, whereas the antibodies against rTOM70 only
inefficiently precipitated rTOM20, rTOM22 and rTOM40, indicating that rTOM70
loosely interacted with the rat TOM complex. Solubilization by 2%
digitonin-200 mM NaCl completely disrupted this weak interaction (data not
shown).
|
We then examined the interaction of rTOM70 with the TOM complex using blue
native PAGE, which allows separation of the protein complex under native
conditions. As shown in Fig.
5B, the major fraction of rTOM40 migrated as an 400 kDa
complex, and the small fraction of rTOM40 was detected in the
190 kDa
complex. rTOM22 was contained in the
400 kDa complex. In contrast to the
results obtained by immunoprecipitation, rTOM70 and rTOM20 were mostly
dissociated from the
400 kDa complex, probably because of destabilization
by the negative charge of the Coomassie brilliant blue
(Ahting et al., 1999
). We then
examined the interaction of rTOM70 with other import receptors using a yeast
two-hybrid assay. When assayed using BD-rTOM20 as bait, yeast cells expressing
either AD-rTOM22 or AD-rTOM70 grew on synthetic medium without histidine
(Fig. 5C, upper panel), and the
cell lysates exhibited ß-galactosidase activity
(Fig. 5C, lower panel). Neither
AD-OM37 nor the empty vector pACT2 gave positive interaction signals. When
BD-rTOM22 was used as bait, only the cells expressing AD-rTOM70 gave positive
interaction signals (Fig. 5C)
and neither rTOM20 nor OM37 exhibited positive signals. Thus, rTOM70 interacts
with rTOM20 and rTOM22 through the cytosolic domains in vivo. In yeast, Tom20
directly interacts through its TPR motif with Tom70
(Haucke et al., 1996
). The
involvement of the TPR sequences in rTOM70 in the interactions described
herein remains to be determined.
Characterization of the mitochondria-targeting signal of rTOM70
In yeast, amino acids at positions 1 through 10 of Tom70 comprise a
hydrophilic, positively charged segment, which functions as the
matrix-targeting signal and a segment with nonpolar amino-acid residues at
positions 11 through 29 functions as the stop-transfer sequence
(Hase et al., 1984). McBride
et al. (McBride et al., 1992
)
later demonstrated that the nonpolar segment (amino acids 11-29) functions as
the signal-anchor sequence, and the hydrophilic N-terminal segment increases
the efficiency of binding and insertion of Tom70. In contrast, however, the
hydrophilic N-terminal segments of rats or N. crassa Tom70 do not
resemble the matrix-targeting signal; the segment of rTOM70 comprises an
N-terminal hydrophilic stretch at amino-acid positions 1 through 41, the
hydrophobic TMD at positions 42 through 63 and six arginine residues. We
recently demonstrated for the N-terminal membrane-anchor protein rTOM20 that
the TMD hydrophobicity and at least one net positive charge at the C-terminal
flanking segment are critical for mitochondria targeting
(Kanaji et al., 2000
). We
examined whether the targeting signal of rTOM70 follows these criteria. For
this purpose, we constructed rTOM70HA in which the hemagglutinin A (HA)-tag
was fused to the C-terminus of rTOM70. When expressed in COS-7 cells, rTOM70HA
colocalized with MitoTracker in a dispersed filamentous structure
(Fig. 6A upper panel). In
contrast, a mutant in which six arginine residues in the C-terminal flanking
region of the TMD were changed to serine residues (rTOM70S6HA) localized to
the reticular structure throughout the cells as well as to the plasma
membranes; a structure that is distinct from mitochondria identified by
MitoTracker. In the presence of BFA, which blocks anterograde vesicular
transport from the ER to the Golgi compartment, rTOM70S6HA colocalized with
the ER marker Calnexin (Fig. 6A
lower panel), indicating that the fusion protein was localized in the
secretory pathway and transported back to the ER in the presence of BFA. The
GFP-fusion construct carrying the N-terminal segment at amino-acid positions 1
to 69 of rTOM70 [rTOM70(1-69)GFP] colocalized well with MitoTracker to the
mitochondria (Fig. 6A).
rTOM70(42-69)GFP, the construct in which the N-terminal hydrophilic segment
(residues 1-41) of TOM70(1-69)GFP was deleted also localized to mitochondria,
although the targeting fidelity was slightly lower and a small fraction
localized to the organelles of the secretory pathway
(Fig. 6A). This effect remains
to be investigated.
|
The above results were also confirmed in an in vitro import system. As
shown in Fig. 6B,
reticulocyte-lysate-synthesized rTOM70HA was targeted to and imported into the
mitochondrial membrane. The same result was obtained for rTOM70(1-69)GFP. In
contrast, rTOM70S6HA, which failed to be targeted to mitochondria in vivo,
also failed to bind and insert into the mitochondrial membrane in vitro. We
further found that rTOM70(1-66)GFP carrying three arginine residues in the
C-terminal vicinity of the TMD localized correctly to mitochondria, whereas
rTOM70(1-65)GFP carrying two arginine residues in the same flanking region did
not (Fig. 6A). Therefore, the
segment of amino-acid residues 1 through 66 were sufficient to function as a
mitochondria-targeting signal. These results also suggested that at least
three basic residues in the C-terminal flanking region of the TMD were
necessary. We then examined the requirements of the import receptors for the
insertion of rTOM70 into a mitochondrial outer membrane using an in vitro
import system and found that the membrane insertion of rTOM70 was not affected
by trypsin-treatment of mitochondria, which degraded rTOM20, rTOM22, OM37 and
rTOM70 but not rTOM40 (Fig.
6C). Thus, rTOM70 was targeted to and inserted independently of
the import receptors into the outer membrane, probably directly into rTOM40,
although involvement of the other integral components of the rat TOM core
complex (OM5, OM7.5, and OM10) (Suzuki et
al., 2000) or the OM37 fragment is not yet known.
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Discussion |
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We previously demonstrated that TMD hydrophobicity and a net positive
charge at the C-terminal flanking segment of TMD are critical for mitochondria
targeting in rTOM20 (Kanaji et al.,
2000). These structural features are conserved among several other
mitochondrial outer membrane proteins, including Tom20 from various species,
Tom70 from N. crassa and rats and OM37. Here we demonstrated that the
amino acids at positions 1 through 66 of rTOM70 are sufficient to function as
the mitochondria-targeting signal, and the basic amino-acid cluster in the
C-terminal flanking region of TMD is critical for this function. These
features are in marked contrast to S. cerevisiae Tom70. It has a
composite topogenic signal at the N-terminus: a hydrophilic, positively
charged 11 residue segment that functions as the matrix-targeting signal and a
19 residue TMD that functions as the stop-transfer sequence
(Hase et al., 1984
). The in
vitro studies demonstrated that the TMD functions as the signal-anchor
sequence and is both necessary and sufficient for targeting, and residues 1
through 10 enhanced the rate of import
(McBride et al., 1992
).
Therefore, targeting and insertion mechanisms of rTOM70 are quite different
from S. cerevisiae Tom70. Rather, the structural feature of the TMD
and the flanking regions of rTOM70 resemble those of N. crassa Tom70.
The rTOM70 mutant, in which the basic aminoacid residues at the C-terminal
flanking region of the TMD were replaced by serine, was unable to be targeted
to mitochondria in vivo and, instead, was mistargeted to the secretory
pathway. This was clearly demonstrated using an in vitro import assay;
rTOM70S6HA failed to bind to and insert into the mitochondria. Therefore, the
region covered by the N-terminal hydrophilic segment (residues 1-41) and the
following TMD does not function by itself as the matrix-targeting signal.
Targeting and insertion strictly required the downstream basic amino-acid
residues. We speculate that the TMD of rTOM70 is recognized by SRP as soon as
it emerges from the ribosome, but the basic amino-acid residues at the
C-terminal flanking segment interfere with its function and eventually target
the nascent rTOM70 to mitochondria, as is the case for rTOM20
(Kanaji et al., 2000
).
rTOM70 was efficiently inserted in vitro into the trypsin-treated
mitochondria in which the import receptors rTOM70, rTOM20, rTOM22 and OM37
were removed. Thus, as is the case for fungal Tom70
(Schlossmann and Neupert,
1995), rTOM70 bypasses the import receptors and inserts into the
TOM core complex, probably directly into rTOM40. This is in marked contrast to
the results demonstrated by McBride et al.
(McBride et al., 1992
).
Insertion into rat heart mitochondria of a hybrid protein pOMD29 in which the
N-terminal mitochondria-targeting signal (residues 1-29) of yeast Tom70 was
fused to dihydrofolate reductase is significantly reduced by pretreatment of
the mitochondria with trypsin (McBride et
al., 1992
). In contrast, import of N. crassa Tom70 is not
affected by trypsin pretreatment of the N. crassa mitochondria
(Schlossmann and Neupert,
1995
), again suggesting a similarity in the insertion mechanism of
Tom70 between rats and N. crassa.
We speculate that a cytoplasmic factor recognizes these structural features of the mitochondria-targeting signal of N-terminal anchored outer membrane proteins and directs them to the TOM complex. These possibilities as well as the mechanism of membrane integration require further analysis.
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