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INTRODUCTION |
Many mitochondrial preproteins are synthesized with amino-terminal
extensions, termed presequences, that function as targeting signals to
direct the preproteins into mitochondria (1-3). Upon import into the
mitochondrial matrix, the positively charged amphipathic presequences
are cleaved off by a specific peptidase. Studies with fusion proteins
consisting of a presequence and a non-mitochondrial passenger protein
demonstrated that a presequence contains sufficient information for
specifically directing the import of proteins into mitochondria (4-6).
Extensive studies with fusion proteins and mutagenesis of presequences
revealed a predominant role of presequences in targeting of cleavable
preproteins (summarized in Refs. 7-9), although a contribution of the
mature protein parts was also found with some preproteins (10-12).
Numerous mitochondrial preproteins, however, are synthesized as
mature-sized non-cleavable preproteins. In a few cases, such preproteins were shown to contain a positively charged amphipathic targeting signal in the amino-terminal region, comparable to a non-cleaved presequence (13-17), or at an internal position of the
protein (18). In many other cases, in particular membrane proteins, the
available information on the targeting signals is limited. The largest
family of mitochondrial membrane proteins is the inner membrane
metabolite carriers, including the ADP/ATP carrier and the phosphate
carrier (19-21). Each carrier protein is ~300 amino acids in length
and contains six membrane-spanning segments. Three homologous domains,
each with two membrane-spanning segments, constitute a carrier protein.
On the one hand, the amino-terminal third of a carrier was sufficient
for targeting to mitochondria, and a region in the second half of this
domain seemed to constitute at least part of an import signal (22-25).
On the other hand, the carboxyl-terminal two-thirds of a carrier were
shown to be specifically imported into mitochondria (23, 26),
suggesting the possible presence of two targeting signals in carrier preproteins.
Although a further analysis of internal targeting signals in the
carrier preproteins has not been reported in the past 10 years, major
progress was made in the identification and characterization of the
mitochondrial outer membrane proteins that recognize and translocate
preproteins. A multisubunit machinery containing at least eight
different subunits was identified and termed the translocase of the
outer membrane (designated Tom) (reviewed in Refs. 1-3 and 27). Three
Tom proteins, Tom20, Tom22, and Tom70, were shown to function as import
receptors in vivo and in organello. The expressed
cytosolic domains of the import receptors were able to specifically
bind mitochondrial preproteins in vitro (28-32). Tom22
binds cleavable preproteins; Tom70 preferentially binds non-cleavable
preproteins; and Tom20 binds both cleavable and non-cleavable
preproteins. Synthetic presequence peptides were able to interact with
the purified cytosolic domains of Tom20 and Tom22 as shown by their
competition of binding of full-length cleavable preproteins (29, 30),
suggesting that the linear peptides contained sufficient information
for specific recognition by the mitochondrial presequence receptors.
The presequence peptides, however, did not influence the binding of
a carrier preprotein to Tom70 or Tom20 (29).
For this study, we attempted to obtain information about the mechanism
of interaction of carrier preproteins with import receptors. In
particular, we asked if a carrier preprotein contained short binding
sequences for receptors. Because the current knowledge did not allow
the prediction of possible binding sequences in a carrier preprotein,
we decided to undertake a systematic approach with cellulose-bound
peptide scans (33, 34) and the expressed receptor domains. A peptide
scan derived from a cleavable preprotein was selected for comparison
and validated the specificity of the approach, i.e. Tom20
and Tom22 preferentially bound to peptides derived from the
amino-terminal region of a presequence-containing preprotein. In
contrast, the amino-terminal region of the phosphate carrier was found
to be devoid of linear binding sequences for import receptors, yet
multiple binding sequences for Tom70 and also Tom20 were observed
throughout the remainder of the protein. Thus, both cleavable and
non-cleavable preproteins contain linear binding sequences that are,
however, of striking difference in the selection of receptors and the
distribution over the preproteins. This study implies the existence of
different mechanisms for recognition of non-cleavable and
cleavable mitochondrial preproteins.
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MATERIALS AND METHODS |
Expression and Purification of the Cytosolic Tom Domains in
Escherichia coli--
Expression and purification were performed
essentially as described (29). Briefly, the E. coli strain
BL21(DE3) was transformed with plasmid
pET19b-yTom20cd-His10,
pET19b-yTom22cd-His10, or pET19b-yTom70cd-His10. Expression was induced
by the addition of isopropyl-
-D-thiogalactopyranoside.
The bacterial pellet was resuspended in 5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9, and 2 mM phenylmethylsulfonyl fluoride, and sonified. After a
clarifying spin, the supernatant was applied to Mobicol columns containing
Ni2+-NTA1-agarose
resin (QIAGEN Inc.). After five washes with 80 mM
imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH
7.9, the Tom domains were eluted with 1 M imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9, and
dialyzed against 10 mM MOPS, pH 7.2. One aliquot was taken
to test for purity by SDS-polyacrylamide gel electrophoresis and for
protein concentration by the Bradford assay or absorption at 280 nm.
Screening of Cellulose-bound Peptide Scans for Binding of Tom
Proteins--
The cellulose-bound peptide scans were prepared by
automated spot synthesis (33, 35-37). The peptides were C-terminally
linked to a cellulose membrane (Whatman) via a (
-Ala)2
spacer. The dry membranes were incubated once with methanol and three
times with wash buffer (100 mM KCl and 30 mM
Tris-HCl, pH 7.6) at room temperature for 10 min. For analysis of
protein binding activity, the membrane was incubated with 150 nM Tom protein (expressed cytosolic domains) in binding
buffer (100 mM KCl, 5% (w/v) sucrose, 0.05% (v/v) Tween 20, 0.05% (w/v) bovine serum albumin, and 30 mM Tris-HCl,
pH 7.6) for 60 min at 25 °C with gentle shaking. Nonspecifically
bound protein was removed by washing the membrane with wash buffer for 3 min at room temperature, and peptide-bound protein was
electrotransferred onto polyvinylidene difluoride membranes using a
semidry blotter. The polyvinylidene difluoride membrane and the
cellulose membrane were laid on top of filter papers soaked in cathode
buffer (75 mM Tris base, 120 mM 6-aminohexanoic
acid, and 0.01% SDS) and overlaid with filter papers soaked in anode
buffers AI and AII (AI = 90 mM Tris base; and AII = 300 mM Tris base) at 4 °C. Blotting was performed at a
constant power of 0.8 mA/cm2 of cellulose membrane. The Tom
protein transferred to the polyvinylidene difluoride membranes was
detected by specific antisera using a fluorescence blotting substrate
(ECF, Amersham Pharmacia Biotech) and a fluorescence scanning system
(Fuji). Quantitation was performed with the program TINA (Raytest),
including subtraction of the local background for each peptide spot.
The means of at least three independent experiments for each peptide
spot were used (average units). The values for different membranes were
adjusted by use of identical reference peptides on each membrane. To
correlate the peptide binding values with the amino acid sequence, the
average units obtained for each peptide covering a given amino acid
were added and divided by the number of peptides (typically four to five different peptides/residue), yielding normalized units.
Synthesis of the Phosphate Carrier and Binding to
Tom70cd and Tom20cd--
The preprotein of the
phosphate carrier was synthesized in rabbit reticulocyte lysate in the
presence of [35S]methionine/cysteine and incubated with
Ni2+-NTA resin-bound Tom70cd or
Tom20cd for 40 min at 30 °C as described (29). Where
indicated, synthetic peptides, blocked amino-terminally by acetylation
and carboxyl-terminally by amidation, were added. Bound proteins were
eluted with 1 M imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9, and analyzed by digital
autoradiography (phosphor storage imaging technology, Molecular
Dynamics, Inc.).
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RESULTS AND DISCUSSION |
The Cytosolic Domain of Tom20 Binds to Peptides Derived from Both
the Cleavable Preprotein Cytochrome c Oxidase Subunit IV and the
Non-cleavable Preprotein Phosphate Carrier--
The import receptor
Tom20 possesses a hydrophobic membrane anchor directly at the amino
terminus, whereas the remainder of the protein is exposed to the
cytosol (Fig. 1A) (38-40).
The membrane anchor of Saccharomyces cerevisiae Tom20 was
replaced by a His10 tag; the resulting cytosolic domain
(Tom20cd) was expressed in E. coli cells,
purified by Ni2+-NTA affinity chromatography (29), and used
in the following binding studies.

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Fig. 1.
Binding of the cytosolic domain of Tom20 to
peptide scans derived from cytochrome c oxidase
subunit IV and the phosphate carrier. A, the cytosolic
domain of Tom20 (amino acid (aa) 30-183). The hydrophobic
membrane anchor was replaced by an amino-terminal His10
tag. OM, outer membrane; IMS, intermembrane
space. B, binding of 150 nM Tom20cd
to a peptide scan consisting of 13-mers derived from the preprotein of
S. cerevisiae CoxIV. The first peptide comprises amino acids
1-13 of the preprotein, the second peptide residues 4-16, and the
third peptide residues 7-19, etc. The labeling on the left indicates
the first amino acid of the left-most peptide of each row. The labeling
on the right side indicates the number of the right-most peptide.
C, binding of Tom20cd to a 13-mer peptide scan
derived from the S. cerevisiae phosphate carrier
(PiC). Construction and labeling of the
peptide scan were performed as described for B.
D, quantitation of bound Tom20cd. The amount of
Tom20cd at each peptide spot was determined as described
under "Materials and Methods" (averages from at least three
independent experiments; average units).
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For a peptide scan derived from a cleavable mitochondrial preprotein,
we chose the preprotein of subunit IV of S. cerevisiae cytochrome c oxidase (CoxIV), which has been intensively
studied for in organello import studies (4, 6, 41-45). The
scans consisted of 13-mer peptides overlapping by 10 residues, which were covalently attached to the cellulose membrane. Thus, 49 peptides were synthesized for this 155-residue preprotein. Tom20cd
was incubated to equilibrium with the cellulose-bound peptides,
followed by electrotransfer and immunodetection of the receptor domain (Fig. 1B). Bovine serum albumin, in a 50-fold molar excess
over the receptor domains, was included in the binding assay to
minimize nonspecific binding of proteins to the membranes. A
quantitative analysis was performed using the TINA program, which
corrects for the local background around each individual peptide spot. The best binding efficiency of Tom20cd was observed with
peptides derived from the amino-terminal region of CoxIV (Fig.
1D, upper panel).
For a non-cleavable preprotein, the S. cerevisiae phosphate
carrier was selected, which was previously shown to bind to Tom70 and
Tom20 in organello and in vitro (29, 46). 101 peptides (13-mer), again overlapping by 10 residues, were prepared on
the cellulose for this 311-residue preprotein. Tom20cd
bound to multiple regions of the phosphate carrier peptide scan (Fig.
1C). By inclusion of reference peptides on each peptide scan
(data not shown), the results from the CoxIV and phosphate carrier
peptide scans were quantitatively comparable. The quantitation is
presented in Fig. 1D (lower panel). The binding
efficiency of Tom20cd with numerous peptides derived from
the phosphate carrier was at least as high as that with amino-terminal
CoxIV peptides. Interestingly, peptides derived from the amino-terminal
region of the phosphate carrier did not interact with
Tom20cd.
The Cytosolic Domain of Tom22 Preferentially Binds to Peptides
Derived from CoxIV--
The single membrane anchor of Tom22 is located
in the carboxyl-terminal half of the receptor. In contrast to Tom20 and
Tom70, Tom22 exposes its amino terminus to the cytosol (Fig.
2A) (47-49). The cytosolic
domain of S. cerevisiae Tom22 with a carboxyl-terminal His10 tag (29) was expressed in E. coli and
purified via Ni2+-NTA.

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Fig. 2.
Binding of the cytosolic domain of Tom22 to
peptide scans derived from CoxIV and the phosphate carrier.
A, the cytosolic domain of Tom22 (amino acids
(aa) 1-97) received a carboxyl-terminal His10
tag. OM, outer membrane; IMS, intermembrane
space. B and C, binding of 150 nM
Tom22cd to the CoxIV-derived peptide scan and the phosphate
carrier-derived peptide (PiC) scan,
respectively, was performed and analyzed as described in the legend of
Fig. 1B. D, shown is the quantitation of bound
Tom22cd.
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Tom22cd was incubated with the CoxIV peptide scan (Fig.
2B) and the phosphate carrier peptide scan (Fig.
2C), followed by quantitative analysis (Fig. 2D).
Tom22cd efficiently bound to peptide spots in the
amino-terminal region of CoxIV, i.e. within the first 10 peptides. The peak region included peptides P4-P9 (Fig. 2,
B and D, upper panel). In comparison,
the peak region for binding of Tom20cd to CoxIV peptides
included peptides P1-P6 (Fig. 1D). Thus, both
Tom20cd and Tom22cd show the best binding to
the amino-terminal region of CoxIV, yet the peak is shifted by
approximately three peptides, i.e. ~9 amino acids. The
remainder of the CoxIV sequence provided only few binding peptides for
Tom22cd (Fig. 2, B and D, upper
panel).
The phosphate carrier peptide scan revealed several binding sequences
for Tom22cd scattered over various regions of the
preprotein (Fig. 2, C and D, lower
panel). In comparison with Tom20cd, however, a
striking difference was observed. Although numerous and efficient binding peptides for Tom20cd were found (Fig.
1D, lower panel), both the number of peptides and
the intensity of interaction with Tom22cd were
significantly lower.
The Cytosolic Domain of Tom70 Efficiently Binds to Peptides Derived
from the Phosphate Carrier--
The single membrane anchor of Tom70 is
located at the amino terminus (50, 51). The membrane anchor of S. cerevisiae Tom70 was replaced by a His10 tag; the
resulting cytosolic domain was expressed in E. coli cells
and purified (29). In contrast to Tom20cd and
Tom22cd, binding of Tom70cd to the CoxIV
peptide scan was nearly absent (Fig.
3B). A few weak binding spots
were observed in the amino-terminal region (Fig. 3D,
upper panel). With the phosphate carrier-derived peptide
scan, however, several clusters of high efficient binding of
Tom70cd were found (Fig. 3, C and D,
lower panel). Interestingly, only weak binding of
Tom70cd was observed within the first 23 peptides,
comprising the amino-terminal ~70 residues of the phosphate carrier.
We conclude that the non-cleavable preprotein of the phosphate carrier,
but not the cleavable CoxIV preprotein, contains multiple linear
binding sequences for Tom70cd.

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Fig. 3.
The cytosolic domain of Tom70 preferentially
binds to peptides derived from the phosphate carrier, but not from
CoxIV. A, the cytosolic domain of Tom70 (amino acids
(aa) 38-617) was equipped with an amino-terminal
His10 tag instead of the membrane anchor. OM,
outer membrane; IMS, intermembrane space. B and
C, binding of 150 nM Tom70cd to the
CoxIV-derived peptide scan and the phosphate carrier-derived peptide
(PiC) scans, respectively, was performed
and analyzed as described in the legend of Fig. 1B.
D, shown is the quantitation of bound
Tom70cd.
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Comparative Analysis of the Binding Properties of Tom20, Tom22, and
Tom70 at the CoxIV and Phosphate Carrier Sequences--
Since the
peptides overlap such that a single amino acid is covered by four to
five consecutive peptides of the scan (except the penultimate
amino-terminal and carboxyl-terminal residues), the quantitation data
shown in Figs. 1D, 2D, and 3D did not
readily allow a correlation of the binding efficiencies with the amino acid sequences of the preproteins. We therefore developed a score to
assign the binding efficiencies for the receptor domains throughout the
linear sequences of CoxIV and the phosphate carrier. For each amino
acid, the binding units determined for the peptides covering this
residue (average units in Figs. 1D, 2D, and
3D) were added and normalized for the number of peptides.
The resulting normalized units can be directly aligned with the linear
sequences of CoxIV (Fig. 4) and the
phosphate carrier (Fig. 5). The binding
properties for the CoxIV peptide scan were analyzed first since a
comparison was possible to the in organello import studies
with various constructs of CoxIV (4, 6, 41, 42, 44, 45) as well as the
in vitro binding studies with full-length cleavable
preproteins and the expressed cytosolic receptor domains (29, 32).

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Fig. 4.
Distribution of binding sequences for Tom70,
Tom20, and Tom22 on the cleavable preprotein of CoxIV. The binding
intensities of the cytosolic Tom domains for the CoxIV-derived 13-mer
peptides determined in the experiments of Figs. 1-3 were converted to
sequence-specific normalized (norm.) units as described
under "Materials and Methods." The primary structure of CoxIV is
indicated in single-letter code. The presequence comprises residues
1-25. The threshold values for "binders" (60 normalized units) and
"strong binders" (100 normalized units) are indicated by
dashed lines.
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Fig. 5.
Distribution of binding sequences for Tom70,
Tom20, and Tom22 on the non-cleavable preprotein phosphate
carrier. The binding intensities of the cytosolic Tom domains for
the 13-mer peptides derived from the phosphate carrier were converted
to normalized (norm.) units (directly comparable to the
values for CoxIV in Fig. 4). Thresholds for binders (60 normalized
units) and strong binders (100 normalized units) are indicated by
dashed lines. The six predicted transmembrane segments
(TM) (19) are marked.
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The Cleavable Preprotein of CoxIV--
Tom20cd and
Tom22cd strongly bound to peptides from the amino-terminal
region of CoxIV, whereas Tom70cd bound weakly to a few
amino-terminal peptides only (Fig. 4). This agrees well with the import
studies in mitochondria. Import of cleavable preproteins is
preferentially directed by the presequences via the Tom20-Tom22 pathway
and does not require Tom70 (4, 5, 38, 40, 47, 49, 52, 53).
A comparison between receptor binding to short peptides that lack
higher order structure and binding to full-length proteins with folded
structure poses the problem that folding differences may influence the
exposure or formation of potential binding sites. With presequences,
however, this problem is minimized since several studies demonstrated
that the presequences of preproteins are in loosely folded
conformations (8, 54) and thus should be close to the structural
properties of short peptides. Indeed, synthetic peptides derived from
the presequence of CoxIV specifically competed with the binding of
full-length cleavable preproteins to Tom20cd and
Tom22cd (29). No interaction of presequences with
Tom70cd was observed in the in vitro binding
studies with various full-length preproteins. In particular, synthetic
peptides derived from the CoxIV presequence did not influence the
interaction of preproteins (containing internal targeting information)
with Tom70cd (29). This indicates that the weak binding of
Tom70cd to CoxIV peptides (up to 40 normalized units) (Fig.
4) is not of functional relevance. In contrast, the areas of the best
binding of Tom20cd or Tom22cd to CoxIV peptides
yielded binding scores of >100 normalized units (Fig. 4). Based on
these considerations and the profiles obtained for binding to the
phosphate carrier peptide scan (see below), we operationally divided
binding into three categories to allow a qualitative comparison of
binding intensities: "strong binding" (
100 normalized units),
"binding" (
60 normalized units), and "poor to non-binding"
(<60 normalized units).
Significant binding of Tom20cd was almost exclusively
confined to the presequence of CoxIV (Fig. 4), which is in good
agreement with the dominant role of Tom20 as a presequence receptor
(29, 38, 40, 52). The peak of best binding of Tom22cd was
shifted by ~9 amino acids toward the carboxyl terminus of CoxIV and
thus was observed at the carboxyl-terminal end of the presequence and in the amino-terminal region of the mature protein part (Fig. 4). This
supports the proposed role of Tom22 as a presequence receptor (29, 48,
49, 53), yet additionally explains the contributions of mature protein
parts, particularly in the amino-terminal region, to the targeting
efficiencies of some preproteins (10). A structural analysis of the
CoxIV presequence showed the formation of a helical structure in the
amino-terminal half, whereas the remainder of the presequence did not
adopt a regular secondary structure (55), suggesting that the helical
structure may be of particular importance for the interaction with
Tom20. In organello import studies, together with an
analysis of the structural composition of the Tom machinery, indicated
that preproteins are initially recognized by Tom20 and are subsequently
transferred to Tom22 before their insertion into the general import
pore (47, 49, 56-58). The peptide binding properties seen here support
such a model in that Tom20 initially recognizes the very first part of a preprotein. Tom22 neighboring the import pore binds further toward
the carboxyl terminus of the preprotein such that the amino terminus of
the presequence is free for interaction with the import pore. The
remainder of the CoxIV sequence yielded only few efficient binding
peptides for receptor domains, particularly a potential binding of
Tom22cd to regions in the carboxyl-terminal half of mature
CoxIV (Fig. 4, lower panel). Future studies will have to address the significance of these possible recognition sequences.
The Non-cleavable Preprotein of the Phosphate Carrier--
With
Tom70cd, at least five regions of the phosphate carrier
with strong binding properties were found (Fig. 5). A strong binding
region was found in the second half of each of the three carrier
domains in the area of the second transmembrane segment (TM2, TM4, and
TM6) and carboxyl-terminal to this. Interestingly, the remainder of the
amino-terminal domain, i.e. the amino-terminal 70 amino
acids of the phosphate carrier, was completely devoid of efficient
binding sequences for Tom70 (Fig. 5, upper panel). The
middle domain and the carboxyl-terminal domain of the phosphate carrier
contain an additional strong binding region each: between the two
transmembrane segments in the case of the middle domain (between TM3
and TM4) (Fig. 5, middle panel) and in the area of the first
transmembrane segment of the carboxyl-terminal domain (TM5) (Fig. 5,
lower panel). The binding properties of Tom70 for the
carrier-derived peptide scan agree with and extend the current information on targeting signals of carrier preproteins that were obtained by in organello import studies with constructs of
preproteins. Douglas and co-workers (22, 24, 25) showed that the
amino-terminal third of the ADP/ATP carrier contained sufficient
information for targeting to mitochondria, yet the amino-terminal 72 residues were dispensable, indicating the presence of targeting
information in a region between residues 73 and 111. However, the
carboxyl-terminal two-thirds of two carriers were imported into
mitochondria in the absence of the amino-terminal domains,
demonstrating the presence of at least two independent targeting
signals within these preproteins (23, 26). The presence of several
strong binding regions in the middle domain and the carboxyl-terminal
domain with a comparable affinity compared with the region in the
amino-terminal domain (Fig. 5) suggests the presence of multiple
receptor-binding signals in carriers. Since the carrier constructs
mentioned above were imported with lower efficiency than the
full-length carrier (26), multiple signals appear to increase the
efficiency of the import process. It has been speculated that the
receptor function of Tom70 may include chaperone-like activities in
preventing aggregation and misfolding of the membrane proteins (27).
The presence of multiple binding sequences in a preprotein would
facilitate such a chaperone function.
The overall binding efficiencies of Tom70 for peptides of the phosphate
carrier peptide scan were higher than those of Tom20 (Fig. 5). This is
in agreement with the predominant role of Tom70 in carrier import,
whereas Tom20 is involved in carrier import to a smaller, yet
significant degree (39, 40, 51, 59). Indeed, Tom20cd is
able to bind carrier preproteins (29) and interacts with several
regions of the phosphate carrier peptide scan (Fig. 5). The binding
regions correlate in part with those of Tom70cd; two of the
three strong binders coincide with strong binders of
Tom70cd in the carboxyl-terminal domain. Moreover, the
amino-terminal 40 residues are devoid of significant binding peptides
for Tom20cd. However, there are three regions of the phosphate carrier peptide scan where binding of Tom70cd is
absent, whereas Tom20cd shows significant binding: the
regions between the transmembrane segments of the amino-terminal domain
and the carboxyl-terminal domain and a segment around the beginning of the first transmembrane segment (TM3) of the middle domain,
representing the third strong binder (Fig. 5).
Conflicting results were reported for the role of Tom22 in the import
of carrier preproteins. Although translocation intermediates of carrier
preproteins were shown to be transported to the import pore of intact
mitochondria via Tom22 (47, 60), no binding of carrier preproteins to
the expressed cytosolic domain of Tom22 was observed (29). The binding
yields of Tom22cd with the phosphate carrier peptide scan
were, overall, clearly lower than those of Tom70cd or
Tom20cd, but a few regions with binding scores at or above
the lower threshold were observed (Fig. 5). Interestingly, the
Tom22-binding regions largely coincide with the Tom20-binding regions.
Since Tom22cd does not bind the full-length phosphate carrier synthesized in a cell-free system (29), it may be speculated that the Tom22-binding segments in the phosphate carrier are not exposed on the surface of the preprotein, but become accessible to the
receptor only by unfolding of the carrier during the translocation process. It is thus possible that segments that are bound by Tom20 or
Tom22, but not by Tom70, are not exposed on the surface of the
preprotein, in agreement with the view that Tom70 is the first mitochondrial import receptor that interacts with carrier preproteins.
The few peptide sequences of the phosphate carrier that bind
Tom22cd were all characterized by a positive net charge
(Fig. 5), as is the case with the presequences that efficiently bind to
Tom22cd. The cytosolic domain of Tom22 contains a large
abundance of negatively charged residues (47-49). Therefore, an ionic
interaction between binding peptides and Tom22cd is likely
(29).
Synthetic Peptides Derived from the Phosphate Carrier Peptide Scan
Compete with Binding of the Full-length Phosphate Carrier to Tom70 and
Tom20--
The predominant role of presequences in import of
preproteins into isolated organelles and the competition of preprotein
binding to Tom20cd and Tom22cd by synthetic
presequence peptides validate the main observations of the CoxIV
peptide scan and thus the use of this peptide scan for defining
targeting sequences in a cleavable preprotein. The information on
signals in carrier preproteins and binding to the Tom70 or Tom20
receptor is more limited, yet agrees with the observations made with
the phosphate carrier peptide scan. To directly test predictions made
from this peptide scan, we synthesized four 13-mer peptides in soluble
form (Fig. 6A). The addition
of short peptides to in organello import studies is
difficult to interpret since short peptides bind to and insert into the
mitochondrial membranes, thereby disturbing various mitochondrial functions (44, 45, 61, 62). We therefore tested the influence of the
synthetic peptides on the binding of the full-length phosphate carrier
to Tom70cd or Tom20cd.

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Fig. 6.
Linear sequences identified with peptide
libraries inhibit binding of the full-length phosphate carrier to Tom70
and Tom20. A, shown are synthetic peptides derived from
the S. cerevisiae phosphate carrier
(PiC) sequence. The peptides were blocked
amino-terminally by acetylation and carboxyl-terminally by amidation.
B, the preprotein of the phosphate carrier was synthesized
in rabbit reticulocyte lysates in the presence of
[35S]methionine/cysteine. Tom70cd was bound
to Ni2+-NTA resin and incubated with the phosphate carrier
preprotein in the presence of peptide P1, P2, or P3 (200 µM each) as described under "Materials and Methods"
(29). Bound phosphate carrier was quantified by digital
autoradiography. The amount of phosphate carrier bound to
Tom70cd in the absence of peptide was set to 100%
(control). C, the experiment was performed as described for
B, except that Tom20cd and peptides P2-P4 were
used (200 µM peptides P2 and P4 and 100 µM
peptide P3).
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Peptides P1-P3 were selected for the binding assay with Tom70 since
peptides P1 and P2 are derived from the strong Tom70-binding region of
the phosphate carrier peptide scan, whereas peptide P3 is from a
non-binding region. The preprotein of the phosphate carrier was
synthesized and radiolabeled in rabbit reticulocyte lysate, and the
interaction with Ni2+-NTA resin bound Tom70cd
was analyzed (29). Peptides P1 and P2 strongly inhibited the binding of
the phosphate carrier (Fig. 6B, columns 2 and
3), whereas peptide P3 did not show an effect (column
4), in full agreement with the binding studies on the cellulose membrane.
For the binding assay with Tom20, peptides P2-P4 were used since, on
the peptide scan, peptides P2 and P3 efficiently bound Tom20cd, whereas P4 had a binding score below 50 units.
Indeed, peptides P2 and P3 strongly inhibited the binding of the
phosphate carrier to Ni2+-NTA resin-bound
Tom20cd (Fig. 6C, columns 2 and
3), whereas peptide P4 did not (column 4).
We conclude that peptides P1 and P2 in the case of Tom70 and peptides
P2 and P3 in the case of Tom20 bind to sites of the receptors that are
crucial for the interaction with the full-length preprotein. In
contrast, peptide P3 (for Tom70) and peptide P4 (for Tom20) do not
influence the specific receptor activity. These results provide good
evidence for the validity of the phosphate carrier-derived peptide scan
and indicate that the specific binding site of each receptor is able to
recognize different peptides.
Positively charged residues and an amphipathic character are crucial
for the function of mitochondrial presequences (8, 63-65). Moreover,
Káldi et al. (66) reported that positively charged
internal segments in Tim proteins function as targeting signals.
However, Davis et al. (67) showed that targeting signals are
not located in these positively charged segments, but in at least two
of the predicted four hydrophobic segments of Tim23. Although an
assessment of the predicted hydrophobic character of the phosphate
carrier indicated a loose preference for more hydrophobic segments
among the strong Tom70 binders, the charge distribution of the
phosphate carrier peptides clearly does not account for the efficiency
of interaction with Tom70 or Tom20. With both receptors, an uncharged
peptide (P2) and a charged peptide (P1 for Tom70; P3 for Tom20; and
both with 2 positively charged residues and 1 negatively charged
residue) show specific binding properties. Interestingly, peptide P3 is
a binder for Tom20, but a non-binder for Tom70, supporting the view of
a different specificity of the binding sites of the two receptors.
Together with the finding that presequence peptides do not influence
the binding of the phosphate carrier to Tom70 or Tom20 (29), we
conclude that the mechanism of interaction of phosphate carrier
peptides with receptors is significantly different from that of
presequence peptides. These observations support the proposal that
Tom20 contains two distinct binding sites, one for presequence-carrying
preproteins and one for preproteins with internal targeting information
(29, 32, 68).
Conclusions--
We report the first systematic analysis of
receptor-binding sequences in mitochondrial preproteins. Short peptides
derived from cleavable or non-cleavable preproteins can function as
specific binding sequences for the import receptors Tom20, Tom22, and
Tom70. The use of peptide scans for identification of mitochondrial
targeting signals is validated by the good agreement of our conclusions with the in organello import studies and in vitro
binding studies using entire cleavable preproteins. The nature of
targeting signals in a carrier preprotein has been unknown. We show
that multiple recognition sequences for import receptors are present
and confirm this conclusion by a specific inhibition of the interaction
between import receptors and a full-length carrier by short peptides. It cannot be formally excluded that, in addition to linear recognition sequences, more complex, discontinuous targeting information is present
in preproteins. However, the binding specificities for different
receptors and preproteins determined with the peptide scans reflect
very well the specificities that are observed by importing
full-length preproteins into mitochondria.
In a typical cleavable preprotein, the binding sequences are largely
confined to the amino-terminal presequence and the adjacent portion of
the mature protein, whereas binding peptides derived from a carrier
preprotein are found in several clusters throughout the protein, except
for the lack of binding sequences in the amino-terminal region.
Cleavable preproteins enter mitochondria with their amino terminus
first (69, 70). It may be speculated that carrier preproteins are bound
by the import receptors at multiple binding sites and enter the import
machinery with non-amino-terminal portions first. Presequences are
characterized by an abundance of positively charged residues. With the
phosphate carrier peptide scan, charged amino acids are apparently not
the determinant of the targeting function of peptides that specifically
bind Tom70 or Tom20 since both charged and uncharged peptides can be
effective. These results suggest a fundamental difference in the
mechanism of targeting of cleavable preproteins and carrier preproteins.