(Received for publication, April 22, 1997, and in revised form, June 5, 1997)
From the Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Straße 7, D-79104 Freiburg, Germany
The preprotein translocase of the outer mitochondrial membrane (Tom) is a multi-subunit complex required for specific recognition and membrane translocation of nuclear-encoded preproteins. We have expressed and purified the cytosolic domains of three postulated import receptors, Tom20, Tom22, and Tom70. Each receptor domain is able to bind mitochondrial preproteins but with different specificity. Tom20 binds both preproteins with N-terminal presequences and preproteins with internal targeting signals; the binding is enhanced by the addition of salt. Tom22 selectively recognizes presequence-carrying preproteins in a salt-sensitive manner. Tom70 preferentially binds preproteins with internal targeting information. A chemically synthesized presequence peptide competes with preproteins for binding to Tom20 and Tom22 but not to Tom70. We conclude that each of the three import receptors binds preproteins independently and by a different mechanism. Both Tom20 and Tom22 function as presequence receptors.
Nuclear-encoded mitochondrial proteins are synthesized as preproteins in the cytosol. They are targeted to receptors on the mitochondrial surface and are subsequently translocated across the outer mitochondrial membrane by a general import pore (1-5). The import receptors and the general import pore assemble into a dynamic high molecular weight complex, termed the preprotein translocase of the outer mitochondrial membrane (Tom) (6). Nine Tom proteins have been identified to date. Five of them expose major portions on the cytosolic side of the outer membrane: Tom20-Tom22 and Tom70-Tom37 were suggested to function as heterodimeric import receptors for distinct classes of preproteins (see below) (7-10). Tom72 is homologous to Tom70, yet is expressed at a low level and does not play a significant role in mitochondrial biogenesis (11, 12). The other four Tom proteins are associated with the general import pore: Tom40 is thought to represent the pore-forming component (13, 14); Tom5 promotes preprotein entry into the pore (15); and Tom6 and Tom7 modulate the association of the import pore with the receptor subcomplexes (16, 17).
Protein import studies with isolated mitochondria indicated that
Tom20-Tom22 functions as receptor for the typical mitochondrial preproteins that carry N-terminal cleavable targeting sequences, termed
presequences, which form positively charged amphipathic -helices (9,
18-22). Tom70-Tom37 was reported to be required for import of
noncleavable preproteins, carrying (poorly characterized) targeting
information in the mature protein but also for cleavable preproteins
(8, 23-26). Little is known about the function and specificity of
individual receptor proteins. Only the cytosolic domain of Tom70 has
been purified so far and used for analyzing binding of preproteins.
Schlossmann et al. (27) showed that the cytosolic domain of
Tom70 was able to bind noncleavable preproteins but also to some
cleavable ones. Schleiff et al. (28) expressed Tom20 as part
of a fusion protein with glutathione S-transferase and
reported the binding of various preproteins to the fusion protein but
also a high degree of nonspecific binding to the beads used.
For this report, we expressed and purified the cytosolic domains of Tom20, Tom22, and Tom70 and directly compared the ability of the three receptors to bind preproteins. We observed that each receptor domain could recognize and bind mitochondrial preproteins independently and with a different specificity. Only with Tom20 and Tom22, a synthetic presequence peptide competed for the binding of preproteins. Our studies indicate that each of the receptors binds mitochondrial preproteins by a different mechanism and show that two distinct presequence receptors function on the mitochondrial surface.
(His)10-tagged cytosolic domains of
Tom20, Tom22 and Tom70 were amplified using the polymerase chain
reaction and Saccharomyces cerevisiae genomic DNA as
template. To amplify yTOM20cd-His10 (N-terminal
His10 tag), the primers
5-TATCATATGGACTATCAAAGAAGAAATAGCCGCCAATTC-3
and
5
-TATGGATCCTCAGTCATCGATATCGTTAGCTTCAGC-3
were used. To amplify TOM70cd-His10 (N-terminal His10
tag), the primers 5
-GTCGACATATGCGAGGAAAAAAGAACACGATC-3
and
5
-GGATCCCTCGAGTTACATTAAACCCTGTTCGCG-3
were used. The polymerase chain
reaction products were digested with NdeI and
BamHI or XhoI and cloned into pET19b. To amplify
yTOM22cd-His10 (C-terminal His10
tag), the primers 5
-TATCCATGGTCGAATTAACTGAAATTA-3
and 5
-TATGGATCCTTAATGATGATGATGATGATGATGATGATGGTGGTTTCCGGATTTTGTGAAAGC-3
were used. After digestion of the polymerase chain reaction
product with NcoI and BamHI, it was cloned into
pET19b. The correct sequences of the clones were confirmed by DNA
sequencing.
Escherichia coli strain BL21(DE3) was transformed with the
plasmids pET19b-yTom20cd-His10,
pET19b-yTom22cd-His10, or
pET19b-yTom70cd-His10. The cells were grown
overnight in LBA medium (0.5% (w/v) Bacto-yeast extract, 1% (w/v)
Bacto-peptone, 0.5% (w/v) NaCl, 100 mg/l ampicillin) at 35 °C.
10-ml cultures were diluted 200-fold into LBA medium and grown up to an
A600 of 0.7 in a 2-liter fermenter at pH 7.0. Then isopropyl--D(
)-thiogalactopyranoside was added to
the culture to a final concentration of 2 mM. The culture
was incubated for a further 5 h. Cells were harvested by
centrifugation, resuspended in 50 ml of resuspension buffer (10 mM MOPS/KOH,1 pH
7.2) and aliquoted. After centrifugation and removal of the supernatant, the bacterial pellets were frozen in liquid nitrogen and
stored at
80 °C.
The bacterial pellet was resuspended in binding buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris/HCl, pH 7.9, containing 2 mM phenylmethylsulfonyl fluoride) and sonified in a Branson sonifier at 50% duty and setting 5 with a microtip by 3 × 10 pulses of 1 s at 0 °C. The suspension was centrifuged at 16,000 × g and 2 °C. The supernatant was applied on 1-ml Mobicol columns (Mobitec) that had been loaded with 800 µl of Ni-NTA-agarose resin slurry (Qiagen) and equilibrated with binding buffer. After an immediate spin in a microcentrifuge, the columns were washed five times with 500 µl of washing buffer (80 mM imidazole, 500 mM NaCl, 20 mM Tris/HCl, pH 7.9). The protein-loaded resin was resuspended in washing buffer and aliquoted.
One aliquot was analyzed by SDS-PAGE to assay the purity and the amount of protein bound to the Ni-NTA-agarose. The identity of the purified proteins was confirmed by Western blotting with the corresponding antibodies.
Assay for Binding of PreproteinsThe protein-loaded Ni-NTA resin (final amount of 50 pmol of each Tom protein/100-µl assay) was equilibrated with assay buffer (20 mM imidazole, 0-200 mM KCl, 10 mM MOPS/KOH, pH 7.2, 1% (w/v) BSA) by mixing, centrifugation, and discarding the supernatant (three times). The resuspended resin was divided into equal volumes and transferred into 1-ml Mobicol columns that were closed at the bottom side. A mixture of 35S-labeled preproteins in assay buffer (maximum 7% (v/v) rabbit reticulocyte lysate) was added, and the resin was resuspended by vortexing. Where indicated, the synthetic presequence peptide CoxIV(1-23) or the control peptide SynB2, which were blocked N-terminally by acetylation and C-terminally by amidation, were included (to avoid peptide dimers, the cysteine at position 19 of CoxIV(1-23) was replaced by a serine). The total volume of the reaction was 100 µl. The columns were incubated under mixing at 30 °C for 40 min in a thermomixer (Eppendorf, setting 11). After centrifugation, the resin was washed three times with the corresponding assay buffer without BSA. Finally all bound proteins were eluted with elution buffer (1 M imidazole, 500 mM NaCl, 20 mM Tris/HCl, pH 7.9) and precipitated by adding 12% (w/v) trichloroacetic acid. After 20 min of incubation at 0 °C, the proteins were sedimented by centrifugation (40,000 × g) and washed with acetone. The protein complexes were analyzed by SDS-PAGE and digital autoradiography (phosphor storage imaging technology; Molecular Dynamics).
Miscellaneous ProceduresThe following procedures were carried out as described previously: synthesis of preproteins in rabbit reticulocyte lysate and labeling with [35S]methionine/[35S]cysteine (29, 30) and SDS-PAGE (31).
For expression of the cytosolic domains
of S. cerevisiae Tom20, Tom22 and Tom70, the predicted
single membrane anchor sequence of each protein was replaced by a
His10 tag. Tom20 and Tom70 carry the membrane anchor at the
N terminus and expose the C terminus on the cytosolic side (Fig.
1A) (19, 24, 25); therefore the His10 tag was placed at the N terminus of the cytosolic
domains. Tom22 exposes the N terminus on the cytosolic side and
contains a membrane anchor in the middle and a C-terminal segment on
the intermembrane space side (32). The cytosolic domain of Tom22 was
thus tagged with a C-terminal His10 tag. By use of the
plasmid pET19 and induction by
isopropyl--D(
)-thiogalactopyranoside, the cytosolic
domains were expressed in E. coli cells to ~1-5% of
total cellular protein (Fig. 1B, lanes 2-4). The
cytosolic domains were purified from the soluble fraction of the
E. coli cells by Ni-NTA affinity chromatography. The
proteins were more than 95% pure (Fig. 1C).
Equimolar amounts of the cytosolic domains of Tom20, Tom22, and Tom70
bound to the Ni-NTA resin were used for binding assays with
mitochondrial preproteins (Fig. 2,
lanes 1-3). The control sample contained Ni-NTA resin that
was subjected to a mock treatment with lysates from noninduced E. coli cells (Fig. 2, lane 4). The following preproteins
were synthesized in rabbit reticulocyte lysates in the presence of
[35S]methionine/[35S]cysteine: a fusion
protein between the presequence of Fo-ATPase subunit 9 and
entire dihydrofolate reductase (Su9-DHFR) (33); as control, the DHFR (a
cytosolic enzyme) alone; a fusion protein containing the presequence
plus 87 N-terminal amino acid residues of mature cytochrome
b2 and the DHFR (b2-DHFR; a
19-residue hydrophobic sorting segment of the 80-residue presequence
was deleted) (34); the authentic precursor of cytochrome
c1 that includes an N-terminal presequence with
a hydrophobic sorting signal and a membrane anchor segment in the
mature protein part; the phosphate carrier (PiC) that is
synthesized without a presequence but contains internal targeting and
membrane anchor sequences. The labeled preproteins were incubated with
the resin-bound cytosolic domains at 30 °C under continuous mixing.
After washing, bound proteins were eluted and analyzed by SDS-PAGE and
digital autoradiography (Fig. 2).
Each of the cytosolic domains interacted with a distinct subset of the
proteins added. Tom20cd bound all four mitochondrial preproteins (Fig. 2, lane 1). Tom22cd bound
preferentially b2-DHFR and cytochrome
c1 and bound only very weakly Su9-DHFR (Fig. 2, lane 2), whereas binding of PiC was in the
background range (Fig. 2, lanes 2 and 4).
Tom70cd bound cytochrome c1 and
PiC but not the DHFR fusion proteins (Fig. 2, lane
3). DHFR alone did not bind to any of the three cytosolic domains
(Fig. 2). To minimize unspecific binding of preproteins to the
resin-bound cytosolic domains, we included a 300-fold molar excess of
BSA (over the purified cytosolic domains) in the binding assays;
however, the omission of BSA did not affect the efficiency and
selectivity of preprotein binding (not shown). We conclude that the
cytosolic domains of Tom20, Tom22, and Tom70 selectively bind distinct
subsets of mitochondrial preproteins.
The cytosolic domain of Tom22 contains a large number of
negatively charged amino acid residues and was suggested to interact with the positively charged amphipathic presequences (7, 9, 10, 32). We
thus wondered why Su9-DHFR bound to Tom22cd only with such
a low efficiency. The binding assays reported in Fig. 2 were performed
in the presence of 100 mM KCl that could interfere with a
possible ionic interaction between the charged residues of presequences
and Tom22. At low salt, the binding of Su9-DHFR to Tom22cd
was indeed strongly enhanced (Fig. 3).
Also the binding of b2-DHFR to Tom22cd was
increased at low salt, whereas that of cytochrome
c1 was largely salt-insensitive, and
PiC did not bind to Tom22cd at any salt
concentration tested (Fig. 3).
Tom20 contains a segment with negatively charged residues and was
similarly suggested to bind preproteins by ionic interactions (7, 10,
21, 35). We asked if lowering of the salt concentration enhanced
preprotein binding to Tom20cd. Surprisingly, the binding of
Su9-DHFR and b2-DHFR as well as that of cytochrome
c1 and PiC to Tom20cd
were inhibited at low salt (Fig. 4,
lane 1). An increase in the concentration of KCl to 200 mM even enhanced binding of all four preproteins to
Tom20cd (Fig. 4, lane 3), whereas DHFR did not
bind at any salt concentration tested (Fig. 4, lanes 1-3). The binding of preproteins to Tom20cd and
Tom22cd therefore reveals an opposite dependence on the
salt concentration (Fig. 4, lanes 1-3 versus 4-6).
Binding of cytochrome c1 and PiC to
Tom70cd, however, were only slightly influenced by a
variation of the KCl concentration from 0-200 mM (Fig.
4, lanes 7-9). Moreover, Su9-DHFR and
b2-DHFR did not bind to Tom70cd at any salt
concentration.
We conclude that binding of preproteins to Tom20cd is stimulated by salt, whereas the binding of preproteins to Tom22cd is inhibited by salt. Binding of preproteins to Tom70cd is salt-insensitive. Only Tom20cd is able to bind significant amounts of all four mitochondrial preproteins tested.
A Chemically Synthesized Presequence Peptide Competes with Binding of Preproteins to Tom22 and Tom20To further determine the
specificity of preprotein binding to the cytosolic domains, we used two
chemically synthesized peptides (Fig.
5A). The 23 N-terminal
residues of the 25-residue presequence of subunit IV of cytochrome
c oxidase (CoxIV) function as a mitochondrial import signal;
they can form an amphipathic -helix with a positively charged side
and a hydrophobic side (36-39). The peptide SynB2 contains the same
number of positively charged residues but lacks the hydrophobic site
and does not function as mitochondrial import signal (36).
Binding of preproteins to Tom22cd was performed in the
presence of different concentrations of the presequence peptide or the
control peptide. The presequence peptide strongly inhibited the binding
of Su9-DHFR and b2-DHFR (Fig. 5B, lanes
2-7). Half-maximal inhibition of preprotein binding occurred at
~6 µM peptide in the case of Su9-DHFR and at ~10
µM peptide in the case of b2
-DHFR (Fig.
5C). In concentrations up to 20 µM, the
peptide SynB2 revealed only a slight inhibitory effect on binding of
these two preproteins to Tom22cd (Fig. 5, B,
lanes 8 and 9, and C); at 30 µM, it caused a partial inhibition of binding (Fig. 5,
B, lane 10, and C). The inhibitory
effect of the presequence peptide on binding of cytochrome c1 to Tom22cd was not as strong as
that on the binding of the two DHFR fusion proteins but still
significantly distinct from that of the peptide SynB2 (Fig. 5,
B and C).
Binding of the preproteins cytochrome c1 and
PiC to Tom70cd was inhibited neither by the
presequence peptide (Fig. 6, A
and B) nor by the peptide SynB2 (not shown), independently
of the salt concentration in the assay.
With Tom20cd the presequence peptide competed with the
binding of all three presequence-containing preproteins,
b2-DHFR, cytochrome c1, and
Su9-DHFR (Fig. 7A) in contrast
to the control peptide SynB2 (Fig. 7B). The half-maximal
inhibition of binding was obtained at a concentration of the
presequence peptide of ~4 µM in the case of Su9-DHFR
(Fig. 7A, lanes 13-27, and C) and ~10 µM in the case of b2
-DHFR (Fig.
7C). In the case of cytochrome c1,
inhibition was weaker (Fig. 7, A and C). Binding
of the noncleavable preprotein PiC to Tom20cd,
however, was not competed for by the presequence peptide (Fig. 7,
A and C).
We conclude that a chemically synthesized presequence peptide competes with the binding of cleavable preproteins to Tom20cd and Tom22cd. Binding of preproteins to Tom70cd is not affected by the presequence peptide.
We report that the purified cytosolic domains of yeast Tom20, Tom22, and Tom70 selectively bind distinct subsets of mitochondrial preproteins. The overall pattern of preprotein binding to the recombinant domains agrees well with the preprotein specificities that have been predicted for these Tom proteins by studying their function with mitochondria or outer membrane vesicles (summarized in Refs. 1, 3, and 40). Together with the lack of binding of a nonmitochondrial protein and the competition by a synthetic presequence peptide, this underscores the validity of the in vitro binding assays presented here.
The analysis in the purified system provides several new insights in the mechanism of action of mitochondrial import receptors. First, each cytosolic domain alone can specifically bind preproteins without a requirement for other Tom proteins. Second, the characteristics of binding of the same preprotein are different for each of the cytosolic domains, suggesting different functional mechanisms for the three import receptors. Although it has been expected that Tom70, which preferentially binds preproteins with internal targeting information (23-27), functions differently than Tom20 and Tom22, the latter two receptors were assumed to bind preproteins by a similar mechanism. Both were suggested to bind positively charged presequences in a salt-sensitive (ionic) manner (7, 9, 10, 21, 35). Moreover, Mayer et al. (10) proposed that Tom20 and Tom22 do not constitute independent binding sites for preproteins but function as a single heterodimeric receptor.
The findings reported here suggest the following characteristics of
mitochondrial import receptors. (i) Tom22: Binding to Tom22cd requires the presence of a cleavable presequence.
Binding of a preprotein, which consists only of an amphipathic
mitochondrial presequence and a cytosolic passenger protein, is highly
sensitive to the addition of salt and is strongly competed for by a
synthetic presequence peptide. This suggests that binding occurs solely via the presequence and that the positively charged surface of the
presequence interacts with the highly negatively charged
Tom22cd (32) in a ionic manner. (ii) Tom20: Cleavable and
noncleavable preproteins bind to yeast Tom20cd, indicating
a broad substrate specificity for this import receptor in agreement
with the binding properties of a fusion protein between glutathione
S-transferase and human Tom20 (28). The binding of
preproteins to yeast Tom20cd is stimulated by the addition
of salt, leading to the unexpected conclusion that preprotein
recognition by Tom20cd is not mediated by ionic forces but
probably by a hydrophobic type of interaction. Tom20cd
probably interacts with the hydrophobic surface of the amphipathic
presequences. Interestingly, a higher concentration of a control
peptide, which contains the same number of positive charges as the
presequence peptide but lacks the hydrophobic character partially
inhibits preprotein binding to Tom22cd but not to
Tom20cd. The opposite influence of salt supports the view
that Tom22cd and Tom20cd recognize distinct
surfaces of a presequence. It has been shown that synthetic
presequences form amphipathic -helices at amphiphilic surfaces such
as detergent micelles or lipid bilayers but not in water (37-39),
suggesting that the interaction of presequences with the binding
site/cleft of a receptor may induce the formation of an
-helical
structure. Because a synthetic presequence peptide competes with the
binding of cleavable preproteins but not with that of a noncleavable
one to Tom20cd, it is likely that Tom20 employs distinct
mechanisms for recognition of presequences and internal targeting
sequences. (iii) Tom70: Binding of preproteins to Tom70cd
is largely insensitive to the presence or the absence of salt and is
not competed for by the presequence peptide, clearly distinguishing the
binding properties from that of Tom22cd and Tom20cd. Tom70cd apparently does not recognize
mitochondrial presequences but targeting information contained in the
mature part of preproteins.
The cleavable preprotein cytochrome c1 behaves differently than other cleavable preproteins because it does not only bind to Tom20cd and Tom22cd but also to Tom70cd. This broad receptor dependence of cytochrome c1 is in agreement with import studies of this preprotein into mitochondria (26, 41). Moreover, the binding to Tom20cd and Tom22cd is only partially competed for by the synthetic presequence peptide, and the binding to Tom22cd is only slightly inhibited by increasing the salt concentration. This suggests that cytochrome c1 may not only carry targeting information within the amphipathic part of the presequence but also in additional protein parts. Although further work is needed to identify putative internal targeting signals of cytochrome c1, previous studies on the import of chimeric preproteins into mitochondria indeed suggested that some cleavable preproteins carry targeting information not only in the presequence but also within the mature protein (42-44).
The binding characteristics observed with different preproteins raise the speculation that Tom20cd as well as Tom22cd may contain more than a single binding site for preproteins each. (i) The major binding site of Tom22cd is obviously that for the positively charged surface of presequences. In addition, internal targeting information of a preprotein can stabilize the binding (cytochrome c1) but is not sufficient to promote binding by itself (PiC). A putative second binding site on Tom22cd would thus not function independently but would play an auxiliary role. (ii) Tom20cd may contain two binding sites, one for the hydrophobic surface of presequences and one for internal targeting information, each of which is apparently competent for recognition of preproteins. (iii) Tom70cd contains a binding site for internal targeting information that is not influenced by presequences.
Irrespective of these speculations, it is evident that each of the three import receptors Tom20, Tom22, and Tom70 can bind mitochondrial preproteins independently of other Tom proteins. In particular, this demonstrates that two presequence receptors operate at the mitochondrial outer membrane. The concentration of presequence peptide needed for half-maximal competition of binding (4-10 µM) suggests a relatively low affinity for the import receptors. The presence of two presequence receptors that recognize distinct properties of the same presequence should thus strongly enhance the specificity and fidelity of preprotein recognition at the mitochondrial surface.
We thank Dr. Bernard Guiard for plasmids and Dr. Michael Ryan for critical comments on the manuscript.