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Previous Article | Next Article 
Molecular and Cellular Biology, August 2003, p. 5217-5224, Vol. 23, No. 15
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.15.5217-5224.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Indian Institute of Chemical Biology, Calcutta 700032, India
Received 27 January 2003/ Returned for modification 4 April 2003/ Accepted 7 May 2003
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ABSTRACT |
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640 kDa) from the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania, consisting of 130-Å particles, was isolated. The complex, when incorporated into phospholipid vesicles, induced specific, ATP- and proton motive force-dependent transfer of Leishmania tRNATyr as well as of oligoribonucleotides containing the import signal YGGYAGAGC. Moreover, allosteric interactions between tRNATyr and tRNAIle were observed in the RNA import complex-reconstituted system, indicating the presence of primary and secondary tRNA binding sites within the complex. By a combination of antibody inhibition, photochemical cross-linking, and immunoprecipitation, it was shown that binding of tRNAIle to a 21-kDa component of the complex is dependent upon tRNATyr, while binding of tRNATyr to a 45-kDa component is inhibited by tRNAIle. This "ping-pong" mechanism may be an effective means to maintain a balanced tRNA pool for mitochondrial translation. |
INTRODUCTION |
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Using an in vitro evolution protocol, it was recently shown that Leishmania mitochondria recognize a number of short sequence motifs homologous to multiple domains in tRNAs, suggesting the presence of several import signals (3). Moreover, novel positive and negative allosteric interactions between these aptamers, as well as between intact tRNAs, at the inner membrane were described (3). The RNAs could be classified into two types: type I RNAs are efficiently transferred through the inner membrane but are inhibited by type II. In contrast, type II RNAs have poor inner membrane transfer efficiencies and are stimulated by type I. For example, tRNATyr(GUA) is a type I RNA containing the conserved motif UAGAGC in the D domain, while tRNAIle(UAU) is type II with the sequence UCGCGGGUU in the variable loop-T domain (V-T) region (3). The mechanism of these allosteric interactions is unknown, but there are several possibilities. A single conformationally flexible dimeric or multimeric receptor could bind to either a type I or a type II motif. Alternatively, distinct type I and type II receptors may interact directly or indirectly through a third subunit. A related issue is whether the effector and substrate binding subunits for either RNA are identical or different.
To begin to define the molecular components of the inner membrane import apparatus involved in such allosteric interactions, we have isolated a protein complex from mitochondrial inner membrane that is functional for import and probed the role of individual subunits of the complex using a combination of chemical cross-linking and immunochemical approaches. The results reveal specific interactions between distinct type I and type II receptors at the inner membrane.
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MATERIALS AND METHODS |
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Purification of RIC. Purified Leishmania mitochondria (20 mg of protein) were extracted with 320 µM digitonin as above, and mitoplasts were separated by centrifugation at 3,450 x g. Inner membranes were then prepared by three freeze-thaw cycles, suspended at 20 mg of protein/ml in sucrose-Tris-EDTA (STE) containing 0.5% sodium deoxycholate (DOC), 0.4 M KCl, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM EGTA, and incubated on ice for 30 min with gentle agitation. The DOC extract was clarified by centrifugation at 16,000 x g for 10 min. An RNA affinity column was prepared by immobilizing 500 pmol of RNA containing the D arm of tRNATyr (see Fig. 3) and a 3'-terminal poly(A) tail on 0.1 ml of oligo(dT) cellulose and equilibrated with buffer DB (1) containing 0.1 M KCl. The DOC extract was diluted to 0.1 M in KCl with DB and loaded on the column, which was then washed sequentially with DB containing 0.125% DOC and 0.1, 0.25, 0.5, and 1.0 M KCl. The 0.5 and 1.0 M KCl fractions were concentrated by dialysis against dry Sephadex (1) or by centrifugal ultrafiltration. RNA binding activity was assayed by incubating the fractions with 32P-labeled tRNATyr in binding buffer (1) for 30 min on ice and observing the electrophoretic mobility shift of the RNA-protein complex in 6% polyacrylamide gel (60:1) in the presence of 5 mM MgAc2. Polyclonal antibodies against individual bands resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were raised in BALB/c mice. The RIC21p gel band was trypsinized, and the digest was analyzed by liquid chromatography/mass spectrometry (MS) and MS/MS in a Finnigan LCQ ion trap mass spectrometer system. BLAST analysis was performed against the L. major sequence database (http://www.sanger.ac.uk/Projects/L_major/).
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36 µg of protein/ml).
Import substrates.
32P-labeled Leishmania tRNATyr(GUA), tRNAIle(UAU), tRNAGln(CUG) and D arm oligoribonucleotides were prepared by T7 RNA polymerase transcription (1-3, 12). tRNAIle [42-66], covering positions 42 to 66 of the V-T region, was transcribed from a clone containing the internal HpaII fragment of the tRNAIle(UAU) gene in plasmid pGCN8 (3). High- and low-specific-activity substrates and effectors for allosteric interaction assays were prepared as described previously (3). 5-Bromouridine (5-BrU)-labeled tRNA was synthesized in transcription reactions containing 10 µM [
-32P]UTP and 20 µM 5-BrU triphosphate.
Import assays.
Import into intact mitochondria or mitoplasts was assayed by incubating the vesicles (100 µg of protein) with 32P-labeled RNA (100 fmol) in 20 µl of import buffer (10 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 1 mM DTT, and 4 mM ATP) at 37°C for 15 min; then, RNases A and T1 were added to final concentrations of 2.5 µg/ml and 50 units/ml, respectively, and RNase digestion continued at 37°C for 15 min. The mitochondria were washed with STE by centrifugation, and imported RNA was isolated by guanidinium isothiocyanate extraction and ethanol precipitation (12). Proteoliposomes (0.7 µg of RIC) were incubated with 100 fmol of substrate RNA as above and treated with RNase; then, 3 ml of liposome washing buffer (5 mM HEPES-KOH [pH 7.5], 2 mM DTT, 5 mM EDTA) was added, and the vesicles were recovered by centrifugation at 120,000 x g for 30 min at 4°C for RNA analysis. For allosteric interaction assays, proteoliposomes were incubated with a mixture of high-specific-activity substrate (50 fmol) and low-specific-activity effector (5 fmol) as indicated.
Electrophoretic and sedimentation analyses. Native RIC was electrophoresed on 4 to 15% gradient polyacrylamide (40:1) containing 50 mM Tris-HCl [pH 8.9], with 25 mM Tris-glycine as running buffer, at 8 V/cm. For Western blots, the gel was equilibrated with Tris-glycine-SDS buffer before transfer to a polyvinylpyrrolidone membrane. The blots were probed with mouse polyclonal anti-subunit serum at 1:100 dilution and developed with horseradish peroxidase-conjugated secondary immunoglobulin G. Sedimentation analysis was performed in 15 to 30% glycerol gradients in buffer DB at 150,000 x g for 4.5 h at 4°C.
Electron microscopy.
Freshly purified RIC (nondialyzed 1 M KCl fraction) was diluted to 3 µg/ml with buffer DB, and 10 µl was applied on carbon-coated grids for 2 min; the grids were washed twice with deionized water and stained with 1% uranyl acetate for 2 min before observation at 30,000x. Photomicrographs were scanned on a Bio-Rad G710 densitometer. Particle size measurements were performed using Quantity One software.
Photochemical cross-linking and immunoprecipitation. Mitoplasts were incubated with 5-BrU-labeled tRNA (1 nM) for 30 min on ice, washed, and irradiated with 313-nm UV on a transilluminator for 30 min with cooling to form RNA-protein cross-links (20). SDS was then added to 0.2%, and the proteins were diluted 20-fold into 1x TETN buffer (250 mM Tris-HCl [pH 7.5], 5 mM EDTA, 250 mM NaCl, 1% Triton X-100, 2 mg of BSA/ml) containing a 1:50 dilution of polyclonal mouse antiserum. Immunoprecipitation was performed with protein A-Sepharose. Immune complexes were dissociated in SDS-PAGE sample buffer and resolved by 8 M urea-6% PAGE in the presence of 0.05% SDS. Antibody-inhibition experiments were performed on mitoplasts as described previously (1).
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RESULTS |
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620 kDa (Fig. 1). The profile, though complex, is reproducible, with correspondence between the Coomassie- and silver-stained bands (Fig. 1). A number of substoichiometric bands were also present; these could be contaminants or proteolytic degradation products. Native gel electrophoresis of the affinity-purified fraction revealed the presence of a single protein species migrating with an apparent molecular mass of 640 kDa (Fig. 2). Sedimentation analysis yielded a value of 620 kDa (Fig. 2). Glycerol gradient centrifugation resulted in a 50% increase of specific activity over the affinity fraction but a relatively low yield (Table 1), probably due to inactivation during sedimentation. The peak protein fraction in the glycerol gradient was sufficient to induce import, and no other fraction contained activity (data not shown), indicating the absence of smaller essential components; however, the possibility of multiple large complexes of identical size cannot be presently excluded. Western blot analyses demonstrated the presence of several of the subunits, including the 52- and the 21-kDa species, as well as one or more proteins binding specifically to tRNATyr, but not to tRNAGln(CUG), which is not imported, in the native 640-kDa complex (Fig. 2).
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Preliminary mass spectrometric peptide sequencing (data not shown) indicated that the 21-kDa band of RIC contains two major proteins, in keeping with the stoichiometry of two to three copies per RIC (Fig. 1). These include a homologue of assembly subunit 6 (UCR_14kDa) (7) of the ubiquinol-cytochrome c reductase complex (respiratory complex III) of the inner mitochondrial membrane and an unknown protein with no database matches. The present data are not sufficient to define a precise function of each of these proteins in import but may indicate a close relationship between RIC and the mitochondrial electron transport machinery.
The complex is necessary and sufficient for import. To examine whether the RIC is functional for RNA import, it was incorporated into phospholipid vesicles. RIC-containing vesicles were incubated with radiolabeled tRNATyr and then treated with RNase and reisolated. In the presence of ATP, protection of the tRNA from RNase was observed, indicating uptake into the vesicles (Fig. 3A). No protection was observed with RIC-free vesicles. Disruption of the vesicles with DOC after the incubation rendered the RNA nuclease-sensitive, showing that intactness of the membrane is essential for the protection and that the RIC itself does not protect the RNA. The apparent Km for RIC-mediated import of tRNATyr into phospholipid vesicles was 1.16 nM (Table 2). tRNAGln(CUG), which is not imported in vivo (9), was not translocated by RIC (Fig. 3A); the tRNA was recovered intact from the postincubation supernatant (data not shown), arguing against specific degradation of tRNAGln as the reason for the lack of its import.
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imido analogue of ATP was unable to substitute for ATP, indicating the requirement for ATP hydrolysis (Fig. 3A). The apparent Km for ATP was 1.35 mM; this is comparable to the value of 1.52 mM for import into intact mitochondria (2). It has been shown previously that in intact mitochondria or mitoplasts, transfer of tRNA to the matrix requires a transmembrane proton motive force probably generated by the oligomycin-sensitive F1F0-ATPase's pumping protons out of the matrix at the expense of ATP hydrolysis (13). In a similar manner, RIC-mediated transfer was sensitive to the protonophore uncoupler carbonylcyanide m-chlorophenylhydrazone, which dissipates proton gradients (Fig. 3A), indicating that both ATP hydrolytic and proton pumping activities are contained within the RIC. Signal recognition by RIC is sensitive to mutations within the conserved motif. In addition to intact tRNAs, Leishmania mitochondria import short oligoribonucleotides containing the import signal YGGYAGAGC in the D arm of tRNATyr (2, 3). RIC-dependent transfer of a 27-mer oligonucleotide containing the wild-type sequence was observed (Fig. 3B). The mutation G22:C in the conserved import motif abolished import, whereas restoration of the loop-closing base pair of the hairpin in the double mutant G22:C, C13:G restored import to about two-thirds of the wild-type level. The mutation A23:U in the next position of the motif also abolished importability, but restoration of the base pair in A23:U, U12:A failed to regenerate activity. These results indicate that the stability of the hairpin is critical for signal recognition by RIC and, further, that the identity of A23 is important. The behavior of these mutants in the RIC-reconstituted system is identical to that in mitoplasts and different from that in intact mitochondria (2). Thus, RIC retains the RNA recognition specificity of the inner membrane.
Allosteric interactions within RIC. A recently described property of inner membrane import is that of cooperative and antagonistic interactions between two types of RNA ligand (3). Type I ligands, of which tRNAs (including tRNATyr) containing the conserved D arm domain are an example, can be transferred independently of other ligands, whereas type II ligands (such as tRNAIle) are dependent on type I ligands for transfer. Moreover, type II ligands inhibit transfer of type I ligands. To examine whether such interactions occur within the isolated complex, RIC-reconstituted phospholipid vesicles were assayed for import of tRNATyr in the presence of low concentrations of tRNAIle and vice versa. Indeed, the uptake of tRNATyr was inhibited by tRNAIle, while tRNATyr stimulated uptake of tRNAIle (Fig. 3C). The 50% effective concentration for tRNATyr effector was 0.033 nM, while the 50% inhibitory concentration for tRNAIle was 0.016 nM (Table 2). Thus, RIC contains receptors for both tRNAs, namely, the primary receptor (that for tRNATyr), which binds directly to its ligand, and the secondary receptor (that for tRNAIle), whose binding to its ligand depends upon conformational changes transmitted within the native complex subsequent to loading of tRNATyr.
Regulated interaction of tRNATyr with RIC45p. To detect these allosteric interactions at the levels of individual components of RIC, subunit-specific antibodies were employed. A preliminary Northwestern blot analysis of RIC revealed the presence of several tRNA binding proteins. Polyclonal mouse antibodies against these bands were prepared, and their specificity was verified by Western blots (data not shown). Antibody against the 45-kDa subunit (RIC45p) (Fig. 1) inhibited binding as well as import of tRNATyr (Fig. 4A). Moreover, in the presence of the antibody, tRNATyr was unable to stimulate the import of tRNAIle, implying either that RIC45p directly binds tRNATyr or that it has some other role unrelated to tRNA binding.
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The type I receptor is expected to interact with the corresponding import signal present in the D domain of tRNATyr. To examine whether this is the case for RIC45p, a cross-linking experiment was carried out with 5-BrU-labeled D arm oligonucleotide. A specific RNA-protein complex immunoprecipitable with anti-RIC45p antibody was observed (Fig. 4B), showing direct contacts between RIC45p and the D arm signal.
tRNATyr-dependent interaction of tRNAIle with RIC21p. Information was obtained on type II interactions by similar immunochemical experiments. Antibody against the 21-kDa band (RIC21p) had no effect on the binding of tRNATyr to the inner membrane and had only a two- to threefold effect on its import (Fig. 4). In contrast, the antibody totally inhibited binding as well as import of tRNAIle in the presence of tRNATyr (Fig. 4). It also antagonized the inhibition of binding and import of tRNATyr by tRNAIle. These results indicate that either RIC21p is the binding site for tRNAIle or that it somehow relays the conformational changes between type I and type II receptors. With 5-BrU-labeled tRNAIle, a tRNA-RIC21p complex which migrates slower than the free RNA and which is sensitive to protease (data not shown) was recovered using anti-RIC21p, but not anti-RIC45p, antibody when tRNAIle was incubated with mitoplasts in the presence of tRNATyr, but not in its absence (Fig. 4C). Moreover, the 5-BrU-labeled V-T segment of tRNAIle, which contains a type II signal, was specifically cross-linked to form a complex immunoprecipitable by anti-RIC21p antibody (Fig. 4D). We conclude that a component of RIC21p has the properties of a type II receptor for tRNAIle.
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DISCUSSION |
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The complex appears to be composed of roughly spherical particles of uniform size (Fig. 2). A solid protein sphere of 130 Å diameter (as determined by electron microscopy) would have a mass of 950 kDa, assuming a partial specific volume of 0.73 cm3 g-1 (9). However, the subunit composition, native gel migration, and sedimentation properties support a mass of 620 to 640 kDa (Fig. 1 and 2). This difference can be accounted for by assuming a hollow sphere: if one assumes a cylindrical pore of 45 Å diameter, the calculated mass of 750 kDa comes to within 20% of the determined value. The reason for only 3% of the particles showing fluid-filled cavities may be that the channels are gated by tRNA. This is supported by the observation that in the presence of tRNATyr, there is an increase in the fraction of particles showing pores (Fig. 2). Thus, one consequence of the binding of tRNA to its receptor on the RIC would be to induce the opening of the import channel. Assembly of proton translocation channels on the endoplasmic reticulum membrane induced by ribosome "ligand" has been previously observed (6).
The diameter of the putative translocation channel (45 Å) is considerably larger than the 20-Å protein transport channels on the mitochondrial outer membrane (9) or endoplasmic reticulum (6), which can accommodate unfolded polypeptide chains. The canonical tRNA structure is L shaped, with two 20-Å helical arms at an angle of 90° and a distance of 80 Å between the two extremities of the L (16). This means that in order to pass through the 45-Å pore, the tRNA structure has to be distorted by reducing or increasing the hinge angle. It may not be a coincidence that both the type I (D arm) and type II (V-T region) are located at or close to the hinge; interaction with the cognate receptors could lead to conformational changes within the tRNA appropriate for translocation.
By antibody inhibition, photochemical cross-linking, and immunoprecipitation, two components of the import complex were identified as proximal contact sites for tRNATyr and tRNAIle, respectively (Fig. 4). The RIC45p-tRNATyr complex is inhibited by tRNAIle, whereas the RIC21p-tRNAIle complex is stimulated by tRNATyr, as expected of type I and type II receptors, respectively. These complexes are specific for their respective tRNAs, i.e., the receptors are distinct. Additionally, blocking of RIC21p by antibody is sufficient to prevent inhibition of tRNATyr binding by tRNAIle (Fig. 4), indicating that a separate tRNAIle binding protein is not required to negatively regulate the type I tRNA. These findings can be accommodated by a simple two-receptor model (Fig. 5). RIC45p binds tRNATyr to form a translocation-competent complex from which the tRNA can be transferred to the import pore. The tRNATyr-bound complex contacts RIC21p, activating the tRNAIle binding site of the latter. Subsequent loading of tRNAIle on RIC21p results in a second conformational change that is transmitted back to RIC45p, leading to the dissociation of tRNATyr, loss of contact between the two receptors, and formation of a translocation-competent RIC21p-tRNAIle complex. Such feed-forward and feedback interactions involving transient contacts between the respective receptors explain how the type I and type II tRNAs can be transported through the same import pore without directly competing with one another, while maintaining a properly balanced pool within the mitochondrial matrix.
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ACKNOWLEDGMENTS |
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We thank Nirmalendu Das for help with the liposome preparation and the W. M. Keck Biomedical Mass Spectrometry Laboratory, University of Virginia, for peptide analysis.
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FOOTNOTES |
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