(Received for publication, January 10, 1997, and in revised form, March 18, 1997)
From the Department of Biochemistry, McGill University, Montreal H3G 1Y6, Canada
Tom20 is part of a multiple component, dynamic complex that functions to import specific cytosolic proteins into or through the outer membrane of the mitochondrion. To analyze the contribution of Tom20 to precursor protein recognition, the cytosolic domain of the human mitochondrial import receptor, hTom20, has been expressed as a fusion protein with glutathione S-transferase and conditions established to measure specific interactions of the receptor component with precursor proteins in vitro. Reconstitution of receptor binding from purified components revealed that a prototypic matrix-destined precursor protein, pODHFR, interacts with Tom20 by a mechanism that is dependent on an active matrix targeting signal but does not require cytosolic components or ATP. Binding was influenced by both salt concentration and detergent. The effect of salt or detergent, however, varied for different precursor proteins. In particular, detergent selectively enhanced binding of pODHFR to receptor, possibly because of induced changes in the structure of the signal sequence. Finally, mutations were introduced into hTom20 which had a dramatic effect on binding of some precursor proteins but not on others. Taken together, the results suggest that hTom20 recognizes and physically interacts with precursor proteins bearing a diverse array of topogenic sequences and that such pleiotropic specificity for these precursor proteins may involve different domains within the receptor molecule.
The majority of mitochondrial proteins are synthesized on cytoplasmic polysomes and subsequently imported to a specific subcompartment within the organelle (for review, see Refs. 1-4). In most cases, delivery to mitochondria requires correct interactions between the precursor protein and proteinaceous receptors on the mitochondrial surface. Extensive studies in yeast and Neurospora have identified this receptor as a hetero-oligomeric complex composed of Tom20, Tom22, Tom37, and Tom70/71-72. The proteins Tom20 and Tom22 have been postulated to recognize incoming precursor proteins that harbor NH2-terminal cleavable targeting signals (5, 6), whereas the Tom37/70 complex is believed to import proteins that are presented to mitochondria as a complex with MSF (mitochondrial import stimulation factor) (7-9).
Tom20 was initially identified as a potential receptor because antibodies against this protein blocked in vitro import of precursor proteins (10-12). Subsequent analysis by electron microscopy revealed an exclusive location of Tom20 to the outer membrane (10, 11), and antibody labeling and protease treatment studies of both in vivo and in vitro imported Tom20 demonstrated that although a segment of the protein is embedded in membrane, the bulk of the polypeptide faces the cytosol (10-12). Functional studies showed that interference of Tom20 action either by antibody inhibition in vitro (10-18) or by genetic ablation in vivo (11, 19-21) resulted in a decreased level of import of a large variety of mitochondrial precursor proteins. To date, however, biochemical analyses of direct interaction of precursor proteins with purified Tom20 have not been reported.
We have recently identified and partially characterized the human homolog of Tom20 and discovered several interesting features that may distinguish the receptor from its fungal counterparts. First, antibodies against hTom20 inhibit import of uncoupling protein (UCP)1 (15) whereas a close relative of UCP, ADP/ATP carrier, is refractory to such inhibition in the fungal context (10). Also, hTom20 exhibits quality control properties and guards the outer membrane import machinery against unscheduled bypass import mediated by cryptic import signals (22). To gain biochemical insight into these and other properties of hTom20, we have expressed its cytosolic domain as a fusion protein with GST and analyzed direct interactions between the immobilized hTom20 domain and various types of precursor proteins. Our results show that hTom20 interacts directly with a diverse array of precursor proteins, perhaps involving different domain requirements for different classes of precursor protein. Also, the lack of interaction of hTom20 with a precursor protein bearing a cryptic matrix targeting signal suggests that quality control by hTom20 may arise because hTom20 physically blocks the precursor from gaining access to downstream components of the import machinery rather than by a mechanism that involves direct physical interaction with the cryptic signal sequence.
Previous articles (23-25) describe the routine procedures used in this study for in vitro transcription and translation, SDS-PAGE, Western blot analysis, fluorography, and quantitation of radioactive bands.
Plasmid ConstructionThe plasmid pGST-30hTom20 was
constructed as follows. The two primers
5
-GGGGGGATCCAGTGACCCCAACTTCAAGAAC and
5
-CCGGAATTCTCATTCCACATCATCTTCAGCC were used in polymerase chain
reaction with a hTOM20 template plasmid (15) to generate a
369-base pair fragment that encodes the soluble domain of hTom20 (amino
acids 30-145). This domain is designated as
30hTom20. The
polymerase chain reaction product was digested with the restriction
enzymes BamHI and EcoRI (New England Biolabs) and
ligated into the BamHI/EcoRI-digested vector pGEX-2T (Pharmacia Biotech Inc.). The ligation mix was transformed into
competent XL1-Blue Epicurian coli cells. DNA from a clone that harbored the correct recombinant plasmid was then transformed into
competent TOPP2 E. coli cells (Stratagene). The nucleotide sequence of the hTom20 insert was verified by sequencing.
100-ml cultures of bacteria
containing the plasmid producing the proteins GST or GST-30hTom20
were grown in LB media containing 50 µg/ml ampicillin. At an
A600 of 0.6, isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 1 mM, and the cultures were grown
for another 90 min. The bacteria were sedimented, and the resultant
pellet was resuspended in 6 ml of 0.4 × KMH-G (10 mM Hepes, 32 mM KCl, 0.8 mM magnesium acetate,
15% glycerol, pH 7.5). Cells were lysed at 4 °C by 5 × 15-s
bursts at setting 6.0 from a small probe sonicator (Sonic Dismembrator,
Fisher Scientific), insoluble debris was removed by centrifugation for
15 min at 17,000 × g, and the supernatant was
incubated for 1 h at room temperature with 4 ml of a 50% slurry
of glutathione-Sepharose 4B (Pharmacia) that had been equilibrated in
0.4 × KMH-G. The Sepharose was washed twice with 40 ml of
0.4 × KMH-G and then resuspended in 6 ml of 0.4 × KMH-G.
GST fusion protein was eluted from an aliquot of the Sepharose by
incubation for 30 min at room temperature in 50 mM Tris, pH
8, containing 10 mM reduced glutathione, and the protein
concentration was determined using Bio-Rad reagent. Unbound Sepharose
4B was added to equalize the amount of protein/input Sepharose
matrix.
For the binding assays, 10 µl of GST- or GST-30hTom20-bound
Sepharose beads was incubated with 2 µl of in vitro
35S-labeled translation product in a total volume of
50 µl containing 38 µl of 0.4 × KMH-G. The mixture was
incubated for 20 min at room temperature with shaking. The Sepharose
beads were then washed twice in 0.4 × KMH-G, and the protein was
eluted with 20 µl of 50 mM Tris, pH 8, containing 10 mM reduced glutathione. The proteins (15 µl of elution)
were separated by 12% SDS-PAGE and stained with Coomassie Brilliant
Blue R-250 (Bio-Rad) to assess relative amounts of eluted GST and
GST-
30hTom20. The gel was then soaked in Amplify (Amersham) for 15 min, dried, and exposed to film to assess levels of
35S-labeled translation product. The amount of
radioactivity was quantitated by using a PhosphorImaging system (FUJIX
BAS 2000 system).
The fusion
protein was produced in bacteria and purified by affinity binding to
glutathione-Sepharose 4B (Fig. 1A). Assays were then developed to measure direct interactions of precursor proteins selectively with GST-30hTom20. This was accomplished by
comparing the amount of precursor protein that was released as a
complex with either GST or GST-
30hTom20 following incubation with
reduced glutathione, which competitively disrupts GST interaction with
glutathione-Sepharose 4B. For each assay, it must be determined that
similar amounts of GST and GST-
30hTom20 are released from the
Sepharose. This is easily determined by subsequent analysis of released
complexes by SDS-PAGE and protein staining. Comparison of circular
dichroism spectra of GST,
30hTom20, and GST-
30hTom20 are
consistent with a properly folded hTom20 domain within the context of
the fusion protein (data not shown).
In Fig. 1, GST- and GST-30hTom20-Sepharose were incubated with
reticulocyte lysate containing the transcription-translation product of
pODHFR, a reporter protein in which the matrix targeting signal (MTS)
of preornithine carbamyl transferase has been fused to dihydrofolate
reductase (26). Selective interaction of pODHFR was observed with
GST-
30hTom20 but not with GST alone, as revealed by selective
release with reduced glutathione (Fig. 1B, lanes 2 and 3). In contrast, the glutathione treatment
liberated similar amounts of both GST and GST-
30hTom20 (Fig.
1C, lanes 2 and 3). Specificity of
binding of pODHFR to GST-
30hTom20 was established by two criteria.
First, interactions were not observed with
-lactamase (Fig.
1B, lanes 5 and 6), a bacterial
precursor protein that is efficiently translocated into endoplasmic
reticulum microsomes in vitro. Second, interaction of
[35S]pODHFR transcription-translation product with
immobilized GST-
30hTom20 was competed by chemically pure unlabeled
pODHFR isolated from expressing bacteria (26) (Fig. 2).
These findings, together with the lack of interaction of pODHFR with
GST alone, strongly suggest that the in vitro binding assay
reflects bona fide precursor-receptor interactions.
The MTS of pODHFR Is Both Necessary and Sufficient for Binding of the Precursor Protein to hTom20
The binding reactions described
in Figs. 1 and 2 were conducted with pODHFR derived as a
transcription-translation product in reticulocyte lysate. To determine
if precursor-receptor interactions can be detected using purified
components, binding reactions were conducted using pODHFR isolated from
expressing bacteria (26). Following elution from
GST-30hTom20-Sepharose, the protein was detected by Western blotting
with anti-DHFR antibodies. As shown in Fig. 3, binding
of chemically pure pODHFR was recorded for GST-
30hTom20 but not for
GST (lanes 2 and 3). This interaction depended on
the presence of the signal sequence in pODHFR (compare lanes
3 and 8) and was unaffected by the addition of
translation-competent reticulocyte lysate to the binding reaction
(compare lanes 2 and 3 with lanes 4 and 5).
Effects of ATP, NEM, Salt, and Detergent
Import of the pODHFR
transcription-translation product into mitochondria in vitro
is dependent on ATP (27) and is sensitive to inhibition by NEM (26). As
shown in Fig. 4, however, depletion of ATP (lanes
3 and 4) or alkylation of lysate proteins with NEM following translation of pODHFR mRNA had little or no effect on subsequent binding to GST-30hTom20. These findings are consistent with the observation that cytosolic components are not required for
pODHFR interaction with GST-
30hTom20 (Fig. 3) and suggest that the
ATP dependence and NEM sensitivity of import in vitro are at
steps other than direct interaction of the precursor protein with
Tom20.
In contrast, however, interactions of pODHFR with GST-30hTom20
exhibited a striking sensitivity to salt and detergent (Fig. 5, A and B). Standard binding
reactions contained an ionic composition that was similar to that used
for in vitro import (32 mM KCl and 0.8 mM magnesium acetate) (28). Elevating the salt
concentration by including NaCl in the binding reaction resulted in a
progressive loss of interaction of pODHFR with GST-
30hTom20, such
that ~50% of binding was lost at 200 mM. At the highest
assayed level of NaCl concentration, the amount of binding was reduced
to the background binding of pODHFR to GST (Fig. 5A). The
low level of background binding of pODHFR to GST, on the other hand,
was unaffected by increasing the salt concentration to 1 M
NaCl. Inclusion of the nonionic detergents Triton X-100 or Tween 20 stimulated binding of pODHFR to GST-
30hTom20 by about 3-fold, and
this effect for both detergents was detected at concentrations of the
detergent which were immediately above the critical micelle
concentration (0.015% v/v for Triton X-100 and 0.007% v/v for Tween
20), with no further stimulation observed (Fig. 5B).
The effects of detergent and salt on hTom20-precursor interactions were
also analyzed for yTom70(1-29)DHFR and human VDAC. The former is a
hybrid protein bearing the NH2-terminal signal anchor
sequence of yeast Tom70p (amino acids 1-29) fused to DHFR (23), and
the latter is the mammalian equivalent of the -barrel protein, porin
(29). Both proteins are efficiently targeted and inserted into the
outer membrane of mammalian mitochondria in vitro by a
pathway that is inhibited by antibodies against Tom20 (18). As shown in
Fig. 6, Triton X-100 stimulated binding of pODHFR to
GST-
30hTom20, had no effect on binding of yTom70(1-29)DHFR, and
inhibited the interaction of VDAC with the receptor (Fig. 6,
compare lanes 4 and 5). Likewise, the effects of
elevated concentrations of NaCl also were variable depending on
the precursor examined (Fig. 6, lane 6).
Mutations in hTom20 Which Differentially Affect Its Ability to Recognize Precursor Proteins
Two regions of hTom20 were targeted
for mutagenesis. First, a cluster of acidic residues at the extreme
COOH terminus of the molecule, Glu-Asp-Asp-Val-Asp-COOH (residues
141-145), were deleted and the mutation designated 141-145.
Second, a predicted amphiphilic helix, spanning amino acids 104-114,
which contains a uniform Gln face (Fig. 7A)
was modified by introducing an Ala point substitution within the middle
of the Gln face at amino acid 108, and the mutation was designated
Q108A. The mutations were introduced into GST-
30hTom20, and their
effect on the ability of hTom20 to recognize various precursor proteins
was examined. As shown in Fig. 7B, neither mutation had a
significant effect on binding of pODHFR and yTom70(1-29)DHFR. However,
they both dramatically reduced binding of VDAC and UCP. The latter is a
polytopic integral protein of the inner membrane (30) which, like VDAC,
contains complex, multiple targeting regions within the molecule
(31).
A Cryptic MTS Does Not Interact with hTom20
Previous studies
have shown that the NH2-terminal 15 amino acids of yeast
Tom70 can function as a cryptic signal and direct import of an attached
reporter protein into the matrix compartment of mammalian mitochondria
in vitro, but only if hTom20 is physically removed from the
import machinery (22). To determine if this quality control property of
hTom20 correlates with its ability to recognize and interact with the
cryptic MTS, a fusion protein bearing the cryptic signal,
yTom70(1-15)DHFR, was examined for binding to GST-30hTom20.
Compared with pODHFR, such binding was negligible (Fig.
8). It is likely, therefore, that the ability of Tom20
to refuse entry of the cryptic signal into the import machinery does
not involve a direct physical interaction with the signal.
Tom20 is but one component of a multimeric receptor complex in the
mitochondrial outer membrane, which also includes Tom70/71-72, Tom37,
and Tom22 (1, 2, 6, 32-34). Nevertheless, extensive genetic and
biochemical analyses have revealed an important role for Tom20 in the
overall function of the receptor complex in mediating import of most
precursor proteins (10, 11, 13-22). To date, however, these studies
have been conducted on Tom20 as a native component of an intact import
machinery in mitochondria, and therefore contributions from other
components have been difficult to exclude. Here, we have examined
direct interactions between hTom20 and precursor proteins by expressing
the hTom20 cytosolic domain as a GST fusion protein in bacteria and
developing an assay that discriminates between specific and nonspecific
interactions in the binding reaction. By these criteria we established
that a fusion protein, pODHFR, which bears a prototypic cleavable MTS, interacts with hTom20 by a mechanism that is specific to mitochondrial precursor proteins and is dependent on the presence of a bona fide but not a cryptic MTS (Figs. 1 and 8). This interaction was competed by excess precursor protein (Fig. 2), did not require cytosolic components or ATP, and was observed for chemically pure pODHFR (Fig. 3). In addition, interactions with hTom20 were observed for several other proteins whose topogenic domains are very different from that of pODHFR: VDAC, a -barrel protein of the outer membrane which is the mammalian homolog of fungal porin (29); yTom70 (1-29)DHFR, a fusion protein bearing the 29-amino acid outer membrane signal anchor sequence of Tom70; and UCP, a polytopic integral protein
of the inner membrane which is a member of the ADP/ATP and
Pi carrier family (30) (Figs. 6 and 7). Comparisons of the characteristics of receptor interactions for these various proteins revealed several important features about hTom20 function.
Consistent with observations made with intact mitochondria (21), we found that precursor-hTom20 interactions were sensitive to salt concentration (100-1,000 mM) (Fig. 5), implying that electrostatic interactions are involved in the binding reaction. Of note, however, pODHFR and yTom70 (1-29)DHFR were more sensitive than VDAC to elevated concentrations of NaCl, suggesting that binding of VDAC to Tom20 may involve a different type of interaction. This conclusion was supported by the finding that mutations in Tom20 were identified which drastically curtailed the ability of the receptor to interact with VDAC and UCP but had negligible influences on binding of pODHFR and yTom70(1-29)DHFR. Not only may different precursor proteins interact with the receptor through different physical interactions, therefore, but these binding reactions may also involve different regions of the Tom20 molecule. In fact, the notion that Tom20 has more than one binding site for precursor proteins was first suggested to explain the correlation between a Tom20 proteolytic fragment (10) and the differing sensitivities of some precursor proteins to import into elastase pretreated mitochondria (35, 36).
Of particular interest was the finding that binding of pODHFR to hTom20 was enhanced by detergent and that this effect became dramatically apparent at or above the critical micelle concentration of the detergent (Fig. 5). Binding of yTom70(1-29)DHFR, in which the MTS of pODHFR has been replaced by a signal anchor sequence, showed no sensitivity to detergent, whereas VDAC binding was actually inhibited by detergent (Fig. 6). Although the enhancing effects of detergent on pODHFR interactions with hTom20 may involve several parameters, these findings are consistent with the observation that the pODHFR MTS is a lipophilic sequence that adopts an amphipathic helix upon binding to detergent micelles (37). In column chromatography, pODHFR, but not yTom70(1-29)DHFR, migrated with a higher hydrodynamic radius in the presence of detergent than in its absence,2 suggesting that detergent was bound to the amphipathic matrix targeting sequence (38, 39). Therefore, it may be that in vitro, such a detergent-induced helix is a preferred structure for recognition by the receptor. During import into intact mitochondria, such a preferred structure could be manifested as a result of interactions of the MTS with the surface of the membrane lipid bilayer prior to receptor binding (40).
Finally, we analyzed a cryptic MTS derived from the hydrophilic, positively charged NH2-terminal domain of the yeast Tom70 signal anchor sequence (amino acids 1-10). This sequence can direct attached passenger proteins into the matrix compartment of mammalian mitochondria only if Tom20 is removed from the receptor complex, and conversely, replacing yTom20 with hTom20 prevents import of the same protein into yeast mitochondria (22). These results suggest that hTom20 performs a quality control function that restricts bypass import into mammalian mitochondria (22). Two models were proposed to explain this observation. First, hTom20 might interact with both cryptic and active MTS sequences but reject the cryptic MTS and disallow it from proceeding to distal binding sites within the import machinery. Alternatively, hTom20 might physically block the translocation pore and occlude the cryptic signal from entering because the signal does not interact with the receptor. The findings presented here suggest that the quality control function of hTom20 occurs by a mechanism that does not involve a direct interaction with the cryptic MTS.