INTRODUCTION

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Mitochondria are genetic parasites presumed to have evolved from endosymbiotic bacteria. The recent sequencing of a large number of mitochondrial genomes has revealed an unexpected diversity in size and gene content (5). The loss of most of the original bacterial protein-coding genes, or their transfer to the nucleus, has been explained in terms of a "big bang" radiation of the different eukaryotic lineages from a single (monophyletic) endosymbiont (5). What is not so easily explained is the loss of a variable number of tRNA genes apparently randomly in different protists, higher plants, and at least one invertebrate lineage (6). At least 24 different tRNA species are required to read the universal genetic code. In kinetoplastid protozoa, including leishmanias and trypanosomes, a complete set of tRNAs are apparently imported in order to compensate for the total lack of mitochondrial tRNA genes (7, 8, 11, 16, 21-23). The mitochondria of tetrahymena import about two-thirds of their tRNAs (2), while in budding yeast only a single tRNA^Lys species is imported (15). In higher plants, different species mitochondrially import different sets of tRNAs; for example, mitochondria from wheat but not maize import tRNA^His (9). Thus, there appears to be a species-specific selectivity, reflecting perhaps the presence of different mitochondrial receptors recognizing individual or groups of import signals on tRNAs.

To study the basis of the selectivity of tRNA import, we have developed an in organello system using leishmania mitochondria (1, 12, 14). Similar systems from trypanosomes have been recently reported (18, 25). Our experiments showed that tRNA import is selective, e.g., tRNA^Tyr is efficiently imported whereas tRNA^Gln(CUG) is not (1), and that a conserved sequence motif in the D arm of tRNA^Tyr is necessary and sufficient for import (13). The importance of the D loop of tRNA^Ile for import in vivo has also been demonstrated (10). Importable RNA interacts directly with the outer mitochondrial membrane (12), and a 15-kDa RNA binding protein, TAB, associated with the outer membrane (OM) was purified and shown to function as an import receptor (1). Using specific antibody, it was further shown that TAB interacts with tRNA^Tyr but not with tRNA^Gln (13). Thus, the TAB-tRNA interaction accounts for the selectivity of import in this system.

More recent studies have indicated that, in addition to sequence discrimination at the OM, a second level of selection possibly occurs at the inner membrane (IM). Analysis of the distribution of imported tRNA^Tyr and other transcripts in the different intramitochondrial compartments, i.e., OM, intermembrane space (IMS), IM, and matrix (MX), showed that import occurs in a stepwise fashion, with a distinct kinetic separation of the OM and IM transfer steps (17). While OM transfer requires ATP, IM transfer is driven by both the electrical and chemical components of the electromotive force generated at the IM of energized mitochondria (17). These results lead to the hypothesis that distinct receptors occur at the OM and IM which concertedly determine sequence selectivity for MX entry of tRNAs. However, nothing is known about the specificity of the interaction at the IM or the identity of any of the components of IM transport machinery.

Here we show that a short oligoribonucleotide containing the D arm of tRNA^Tyr(GUA) rapidly and efficiently transits to the mitochondrial MX in vitro. Site-specific mutagenesis of the D arm was employed to detect similarities and differences in the sequence and/or structural requirements for OM and IM transfer.

	MATERIALS AND METHODS

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Cell culture and preparation of mitochondria. Promastigotes of Leishmania tropica strain UR6 were cultured at 22°C on solid blood agar medium (3) supplemented with 150 µg of biopterin and 50 µg of adenine per ml. Mitochondria were purified from DNase I-treated lysates by Percoll gradient centrifugation and stored in a 50% glycerol storage buffer, as described previously (14). Before use, mitochondria were diluted with cold isotonic sucrose-Tris-EDTA (STE) buffer (14), washed by centrifugation, and resuspended in STE at a final protein concentration of 8 to 10 mg/ml.

Submitochondrial fractionation. Separation of submitochondrial compartments was performed as previously described (17). Briefly, mitochondria were treated with 320 µM digitonin in STE buffer for 15 min on ice to selectively solubilize the OM (19). Mitoplasts were separated from the soluble fraction (OM plus IMS) by centrifugation. To further fractionate the mitoplasts, they were suspended in a solution of 0.6 M sucrose, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA and subjected to three freeze-thaw cycles. Soluble MX and particulate IM fractions were subsequently separated by centrifugation.

Preparation of import substrates. ³²P-labeled tRNA^Tyr(GUA) transcripts were synthesized by runoff transcription of the plasmid pSKB1 (1), which contains a cloned genomic copy of the corresponding Leishmania gene, using T7 RNA polymerase and [-³²P]UTP, as described previously (4). Wild-type and mutant D arm minihelix RNAs were synthesized by runoff transcription of the corresponding double-stranded oligonucleotides containing a T7 RNA polymerase promoter. To prepare the templates, the promoter primer GGAATTCTAATACGACTCACTATAGGGACTGTAGCTC, containing an EcoRI linker, a T7 RNA polymerase promoter, and nucleotides 5 to 13 of tRNA^Tyr(GUA) (11) (see Fig. 1), was annealed to the following oligonucleotides, each containing sequences complementary to positions 5 to 27 of tRNA^Tyr(GUA): wild type, ATGCTCTACCAATTGAGCTACAGTC; U¹⁷A, ATGCTCTACCTATTGAGCTACAGTC; G¹⁸C, ATGCTCTACGAATTGAGCTACAGTC; A²¹C, ATGCTCGACCAATTGAGCTACAGTC; G²²C, ATGCTGTACCAATTGAGCTACAGTC; G²²C, C¹³G, ATGCTGTACCAATTCAGCTACAGTC; A²³U, ATGCACTACCAATTGAGCTACAGTC; A²³U, U¹²A, ATGCACTACCAATTGTGCTACAGTC; C¹¹A, U¹²C, ATGCTCTACCAATTGGTCTACAGTC. The resulting partially double-stranded molecule was end-filled with Moloney murine leukemia virus reverse transcriptase (13) and purified by ethanol precipitation. The sequences of the 27-mer transcripts were verified by two-dimensional oligonucleotide fingerprinting (24).

In organello import assays. Unless otherwise stated, mitochondria or mitoplasts (100 µg of protein) were incubated in 20-µl reaction mixtures containing 10 mM Tris-HCl (pH 8), 10 mM magnesium acetate, 2 mM dithiothreitol, and 4 mM ATP with 100 fmol of ³²P-labeled substrate for 15 min at 37°C (17). Then RNase A (2.5 µg/ml) and RNase T₁ (50 U/ml) were added, and incubation continued for an additional 15 min at 37°C. The vesicles were washed in cold STE buffer by centrifugation. To measure total uptake, RNase-treated mitochondria or mitoplasts were lysed in guanidium isothiocyanate, and ³²P-labeled RNA was recovered by isopropanol precipitation, as described previously (13). Alternatively, intramitochondrial RNAs were assayed by subjecting postimport RNase-treated mitochondria to digitonin treatment, followed by freeze-thaw lysis to separate the submitochondrial compartments (as described above) and recovery of ³²P-labeled RNA from each fraction. Antibody inhibition experiments were carried out with mitochondria or mitoplasts successively incubated with 4 mg of bovine serum albumin/ml in STE for 1 h at 0°C and then with 100 µg of normal or anti-TAB immunoglobulin G (IgG) per ml (1) for 30 min at 0°C and finally washed with STE. To study the effect of uncouplers, mitoplasts were preincubated with 50 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 10 min on ice before the import reaction. ³²P-labeled RNA obtained in each case was analyzed by urea-10% polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Quantitation was performed by liquid scintillation counting of the dried gel band and/or scanning in a Bio-Rad model GS 710 densitometer.

In vivo matrix localization assay. ³²P-labeled minihelix RNA (1 pmol) was added to 5 × 10⁸ promastigotes in 0.4 ml of HEPES-buffered saline (21 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.7 mM NaH₂PO₄; pH 7.4) and the cells were electroporated at 450 V and 500 µF in a Bio-Rad gene pulser. Electroporated cells were incubated on ice for 5 min, centrifuged, resuspended in 1 ml of medium M199 containing 10% fetal bovine serum, and incubated at 22°C for 10 min. The cells were returned to ice and 0.2-ml aliquots were added to 0.8 ml of phosphate-buffered saline. After pelleting by centrifugation, the cells were suspended in 1 ml of hypotonic lysis buffer (14), lysed by two syringe passages, and returned to isotonicity by sucrose addition, as described previously (14). The particulate fraction containing mitochondrial vesicles was suspended in 0.1 ml of STE containing 10 mM MgCl₂ and incubated with DNase I (50 U/ml), RNase A (2.5 to 5 µg/ml), and RNase T₁ (50 to 100 U/ml) for 15 min at 37°C. The mitochondria were then washed with STE, lysed in guanidinium isothiocyanate, and processed for RNA isolation (13). Quantitations were performed by densitometry.

Binding assay. Mitochondria or mitoplasts (50 µg of protein) were incubated with 10 fmol of ³²P-labeled RNA in 10 µl of binding buffer (10 mM Tris-HCl [pH 8], 10 mM magnesium acetate, 1 mM dithiothreitol, 0.1 M KCl) for 30 min on ice. The vesicles were then washed in 1 ml of cold STE, and bound RNA was recovered and analyzed as above. To obtain the dissociation constant (K_d) of the wild-type D arm-receptor complex, mitoplasts were titrated with increasing concentrations (t) of ³²P-labeled RNA, and the bound (b) and free (f = t  b) RNA concentrations were determined. Scatchard plots of b/f versus b yielded K_d = (1/slope) and the total receptor concentration, R₀, as the intercept on the x axis. For each mutant, K_d was then derived from the equation K_d = [(t  b) (R₀  b)]/b.

	RESULTS

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Stepwise import of the D arm minihelix. It was shown earlier (13, 17) that the tRNA^Tyr(GUA) molecule can be replaced by progressively smaller derivatives containing the D arm region, with concomitant increases in the efficiencies of transfer across the outer and inner mitochondrial membranes. Such derivatives apparently use the same import pathway as the intact molecule, as demonstrated by competition and antibody inhibition experiments. This observation raises the attractive possibility of using small synthetic oligonucleotides to probe the transport systems on mitochondrial membranes. Accordingly, a 27-nucleotide RNA hairpin (minihelix) (Fig. 1A) containing the wild-type D arm sequence of the tRNA^Tyr(GUA) was synthesized by T7 RNA polymerase-mediated transcription of the appropriate oligonucleotide template. The energy-minimized secondary structure of this molecule (Fig. 1A) contains the 4-bp stem and 8-base loop present in intact tRNA. Neither the 5' terminal extension, containing the GGGA initiation sequence for T7 RNA polymerase, nor the 3' tail forms any detectable base pair with the remainder of the molecule, and this is true for the wild type as well as for all the mutant sequences used in this study (see below).

View larger version (36K): [in this window] [in a new window]   FIG. 1.   Import of D arm minihelix in vitro. (A) Sequence of the wild-type minihelix. Numbers correspond to positions on the intact tRNATyr molecule (11). The secondary structure shown was derived by energy minimization using the FOLDRNA program (26). Bases in italics are derived from the T7 RNA polymerase initiation sequence on the template. The boxed region contains the conserved nonanucleotide motif found in importable RNAs (13). (B) Total uptake of the wild-type minihelix by intact mitochondria. Reaction mixtures were incubated without (lane 1) or with (lanes 2 through 7) 4 mM ATP. In lane 3, Triton X-100 (0.5%) was added after the import incubation. In lanes 4 through 7, unlabeled yeast tRNA (lanes 4 and 5) or low-specific-activity tRNATyr transcript (lanes 6 and 7) was added as the competitor at concentrations of 0.1 (lanes 4 and 6) or 1 (lanes 5 and 7) pmol. After RNase treatment, the total internalized RNA was analyzed. (C) Intramitochondrial distribution of D arm minihelix. Intact mitochondria were incubated with wild-type RNA and ATP at 37°C for the time intervals shown. After each incubation, the mitochondria were RNase treated and subfractionated, and the contents of RNA in the IM and MX fractions were analyzed. (D) Import of D arm minihelix into mitoplasts. Mitoplasts (100 µg of protein) were incubated with the wild-type minihelix in the absence (lane 1) or presence (lanes 2 through 6) of 4 mM ATP. In lane 3, Triton X-100 (0.5%) was added after the incubation. Reactions 4 and 5 contained 1 pmol of yeast tRNA or tRNATyr, respectively, as the competitor. In reaction 6, mitoplasts were preincubated with 50 µM CCCP before the import incubation. RNase-resistant RNA was recovered for analysis. The region of the major minihelix band in each lane was excised and counted; after background subtraction, femtomole values were computed from the specific activity.

Effect of point mutations in the D arm on transfer through OM and IM in organello. The D arm of tRNA^Tyr(GUA) contains the motif UGGUAGAGC (Fig. 1A), which is conserved in the corresponding region of tRNAs imported in leishmania and trypanosome mitochondria as well as in a synthetic transcript imported through the same pathway (13). To determine the role of the primary sequence and secondary structure in the import signal, point mutations were introduced within this region. Mutant RNAs synthesized by in vitro transcription of the corresponding oligonucleotide templates were assayed for intramitochondrial distribution after uptake in the presence of ATP. In this assay, total uptake (i.e., the amount of RNA in the IM-plus-MX fractions, whereas the OM-plus-IMS fractions contained negligible amounts of RNA [data not shown]), is a measure of OM transfer, while the fraction of the total internalized RNA present in the MX represents IM transfer. For those mutants which are deficient in OM transfer, it is difficult to accurately assess IM transfer using intact mitochondria, since the amount internalized is very low. Therefore, these mutants were directly assayed for their import into mitoplasts.

View larger version (50K): [in this window] [in a new window]   FIG. 2.   Effect of mutations on intramitochondrial location of D arm minihelix. (A) 32P-labeled wild-type or mutant minihelix (100 fmol) was incubated with mitochondria (100 µg of protein). After 15 min at 37°C, RNase was added, and the washed mitochondria were fractionated into IM and MX compartments. Lanes 1 through 10 and 11 through 20 show the results of two different experiments. The RNAs used were as follows. Lanes 1 and 2, 19 and 20, wild type; lanes 3 and 4, G18C; lanes 5 and 6, U17A; lanes 7 and 8, A21C; lanes 9 and 10, G22C; lanes 11 and 12, G22C, C13G; lanes 13 and 14, A23U; lanes 15 and 16, A23U, U12A; and lanes 17 and 18, C11A, U12C. (B) 32P-labeled wild-type (lane 1), G22C (lane 2), G22C, C13G (lane 3), A23U (lane 4), and A23U, U12A (lane 5) RNAs (100 fmol of each) were incubated with mitoplasts (100 µg of protein) in the presence of ATP for 15 min at 37°C, and the RNase-resistant RNA was analyzed. Band quantitation was performed as in Fig. 1; the smear at the bottom of lane 7 was disregarded. (C) Matrix targeting in vivo. (Upper panel) Promastigotes were transfected with 32P-labeled wild-type minihelix (1 pmol) in the absence (lanes 1 to 3) or presence of 10 µM CCCP (lane 4) or of 50 µM oligomycin (lane 5). Aliquots of the transfected cells were lysed, and mitochondrial fractions were treated with RNase and DNase in the absence (lanes 1, 4, and 5) or presence of 1% Triton X-100 (lane 2) or 320 µM digitonin (lane 3). (Lower panel) Promastigotes were transfected with the wild type (lane 1) or the G22C (lane 2), G22C, C13G (lane 3), A23U (lane 4), A23+U, U12A (lane 5), and C11A, U12C (lane 6) mutants, and the RNase-resistant RNA associated with the mitochondrial fraction was analyzed. Quantitation was performed by densitometry.

Targeting of the D arm minihelix to the mitochondrial MX in vivo. From the preceding in organello analysis, it is evident that mutations in the conserved D arm motif result in deficiency of transport into the mitochondrial MX, due to a defect in either OM or IM transfer. To examine whether this is also true in vivo, ³²P-labeled minihelices were introduced into leishmania promastigotes by electroporation. Transfected cells were lysed by syringe passage in hypotonic medium, the mitochondrial fraction was recovered and treated with DNase and RNase, and the nuclease-resistant RNA was analyzed. Hypotonic treatment results in leakage of the contents of the IMS; this assay therefore scores exclusively for MX-localized RNA and does not distinguish between OM and IM transfer.

Sequence-specific binding of D arm variants to OM and IM. To determine whether the above effects on OM and IM transfer reflect altered binding to membrane-bound receptors, ³²P-labeled minihelices were allowed to interact with intact mitochondria or mitoplasts under conditions favoring specific binding but discouraging translocation (i.e., in the presence of 0.1 M KCl at 0°C, in the absence of ATP) (12). The vesicles were then washed, and bound RNA was analyzed by gel electrophoresis. Control experiments (data not shown) demonstrated that the bound RNA was completely sensitive to RNase, i.e., it was associated with the membrane surface.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Binding of minihelices to the OM and IM. (A) The following 32P-labeled RNAs were incubated with mitochondria (lanes 1 to 5) or mitoplasts (lanes 6 to 10): lanes 1 and 6, wild type; lanes 2 and 7, G22C; lanes 3 and 8, G22C, C13G; lanes 4 and 9, A23U; and lanes 5 and 10, A23U, U12A. After washing with STE, the membrane-bound RNA was recovered and analyzed as in Fig. 1. The amounts of wild-type RNA (taken as 100%) bound to mitochondria (lane 1) and mitoplasts (lane 6) were 0.64 and 1.12 fmol, respectively. (B) Stability of inner membrane complexes of wild-type (lanes 1 to 4) and G22C, C13G mutant (lanes 5 to 8) minihelices. Binding reactions with mitoplasts were performed with the incubation intervals shown. Control reactions without incubation (lanes 1 and 5) yielded 1.80 and 1.01 fmol, respectively. Quantitations were performed by densitometric scanning of the major band in each lane.

Competition between D arm variants at OM and IM. From the above experiments, two mutants were identified which are transferred through the OM, but not IM, with good efficiency: the A²³U, U¹²A mutant and the C¹¹A, U¹²C mutant. Conversely, the G²²C, C¹³G mutant is transferred efficiently through the IM but not the OM. Only the wild-type sequence is transferred through both the membranes efficiently. To examine whether the different structures use the same or distinct receptors at either membrane, competition assays were performed in which one labeled oligonucleotide was challenged with an excess of another oligonucleotide (either unlabeled or labeled to low specific activity). OM transfer of both the A²³U, U¹²A and C¹¹A, U¹²C mutants was efficiently competed out by the wild-type sequence, although in the latter case competition was somewhat less effective than in the self-self situation (Fig. 4A). At the IM, the wild-type structure and the G²²C, C¹³G mutant also competed with each other, and again, cross competition was quantitatively less than self-competition (Fig. 4B). Competition for transfer was paralleled by competition for binding at either membrane (data not shown). These results are consistent with the binding of the different structures to the same or similar receptors at either membrane, albeit with different affinities (Table 3).

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Cross competition between wild-type and mutant minihelices for transfer across OM and IM. Mitochondria (A) or mitoplasts (B) were incubated with high-specific-activity 32P-labeled RNA (L) in the absence or presence of low-specific-activity competitor (C; C/L, ratio of competitor to substrate), and total uptake was assayed with RNase protection. Panel A, lanes 1 to 2, 3 to 4, and 5 to 6, contained high-specific-activity wild-type, A23U, U12A, and C11A, A12U RNA (100 fmol), respectively. Wild-type competitor (1 pmol) was included in reactions 2, 4, and 6. In panel B, high-specific-activity wild-type (lanes 1 to 5) or G12C, C13G (lanes 6 to 10) RNA (100 fmol) was incubated without competitor (lanes 1 and 6), with 0.5 pmol (lanes 2 and 9) or 5 pmol (lanes 3 and 10) of wild-type competitor, or with 0.5 pmol (lanes 4 and 7) or 5 pmol (lanes 5 and 8) of G22C, C13G competitor. Quantitations were performed by densitometric scanning of the major band in each lane.

Dependence of OM and IM transfer on TAB. TAB is an OM-associated RNA binding protein required for the uptake of tRNA^Tyr into leishmania mitochondria (1). To examine the role of TAB in the uptake and intramitochondrial distribution of D arm minihelices, antibody inhibition experiments were performed (Fig. 5A). Anti-TAB antibody, but not normal IgG (Fig. 5A) or antibody against total leishmania antigen (data not shown), inhibited OM transfer of the wild-type sequence as well as that of the A²³U, U¹²A, and C¹¹A, U¹²C mutants. Thus, the mutants, like the wild type, interact with TAB at the OM.

View larger version (41K): [in this window] [in a new window]   FIG. 5.   Effect of anti-TAB antibody on transfer of RNA across the OM and IM. (A) Intact mitochondria (100 µg of protein) preincubated with normal (n) or anti-TAB () IgG were incubated with 100 fmol of wild-type D arm minihelix (left), A23U, U12A mutant (center), or C11A, U12C mutant (right) in the presence of ATP. The amounts of total internalized RNA (IM + MX) are expressed as percentages of the control values obtained in the presence of normal IgG. (B) Mitoplasts were incubated with normal (n) or anti-TAB () IgG and then with 100 fmol of tRNATyr(GUA) transcript (left), wild-type D arm minihelix (center), or G22C, C13G mutant (right) for 15 min at 37°C and treated with RNase, and the internalized RNA was recovered. IM transfer is expressed as the percentage of the control value obtained with normal IgG. Quantitations were performed as for Fig. 4. In panel B, middle, both bands are of nearly equal intensity and yield similar values for extent of inhibition.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this report, a simple method for probing the structural basis of the selectivity of mitochondrial tRNA import is described. The use of synthetic oligonucleotides as import substrates is based on our observation that the D arm of tRNA^Tyr is necessary and sufficient for import in organello (13) as well as in vivo (Fig. 2), and this approach also greatly facilitates the generation of mutations for the study of structure-function relationships. Furthermore, fractionation of the submitochondrial compartments allows an assessment of the effect of an individual mutation on the precise location of the corresponding structure within the mitochondrion. By all the criteria examined, i.e., competition by intact tRNA (Fig. 1), effect of anti-TAB antibody on import into mitochondria or mitoplasts (Fig. 5), dependence on ATP and temperature (Table 1), and inhibition of transfer by inhibitors and uncouplers (Fig. 1 and Table 1), the D arm minihelix appears to be using the same pathway for import as the parental molecule, although the rate and extent of import of the minihelix are noticeably higher (Fig. 1), presumably due to its smaller size and/or the lack of inhibitory sequences.

Site-specific mutagenesis of the D arm minihelix revealed a number of distinct phenotypes (Fig. 2 and Table 1). In the first group of mutants (U¹⁷A, G¹⁸C; A²¹C, G²²C; and A²³U), both OM and IM transfer were reduced relative to the wild type. In the second group (A²³U, U¹²A and C¹¹A, U¹²C), OM transfer was reasonably efficient (about 60% of the wild type), but IM transfer was considerably reduced, with the RNA accumulating at the IM (Fig. 2). The third type of mutation, i.e., G²²C, C¹³G, had the opposite property: efficient IM but defective OM transfer (Fig. 2). These results imply that the RNA sequence and/or structural specificities at the two membranes are nonidentical.

The differences are particularly pronounced when bases in the stem of the D arm (Fig. 1A) are altered. Each of the single mutations (G²²C and A²³U), or the double mutation on the same side of the helix (C¹¹A, U¹²C), results in a drastic reduction of secondary structure stability (Table 3); the FOLDRNA program (26) predicts relatively unstable energy-minimized structures with the canonical 4-bp stem replaced by loops, bulges, and non-Watson-Crick pairs (data not shown). Restoration of the loop-closing pair by the second C¹³G mutation, or of the neighboring pair by the U¹²A mutation, results in restoration of the wild-type conformation. It would seem from this limited structure-function study that the loop-closing pair (G²²:C¹³) is critical at both membranes, but for different reasons: while the G base is specifically recognized at the OM, base pairing is more important at the IM, since restoration of the pair in reverse results in reappearance of IM transfer (Fig. 2) and an increase in binding affinity for the IM receptor (Table 3). Conversely, the A²³ residue critically interacts with the IM receptor(s) but may be replaced by U at the OM. This would explain the lack of reversion for IM transfer in the A²³U, U¹²A mutant (Fig. 2); in fact, the binding affinity of this mutant for the IM is further lowered (Table 3). The importance of base pairing for IM transfer is further illustrated by the effects of the double mutation of C¹¹ and U¹² in the stem, which results in considerable weakening of the secondary structure (Table 3), while leaving the critical A²³ and other residues in the conserved region intact. This mutation, however, only marginally affects OM transfer (Fig. 2) or OM binding (Table 3), suggesting recognition of primary sequence, rather than secondary structure, by the OM receptor TAB. In fact, synthetic transcripts containing the nonanucleotide conserved motif but lacking significant secondary structure are transferred efficiently through the OM by the TAB-dependent pathway (13, 14). Regardless of the precise nature of the interactions, it is evident from these experiments that distinct receptors exist for OM and IM transfer. This conclusion was reinforced by the resistance of IM transfer to antibody against TAB (Fig. 5).

Almost nothing is currently known about the nature of tRNA import factors. A major problem in systems such as leishmania, ciliates, and higher plants is the nonavailability of mutants with defective mitochondrial function. The use of oligonucleotide probes, coupled with submitochondrial fractionation, constitutes an alternative and general approach for the identification of the components of the membrane transport machinery. Previous experiments using intact tRNA molecules indicated the occurrence of nonproductive binding to mitochondrial membranes, with only about 10% of RNA bound to the OM being internalized (12); moreover, tRNA^Gln binds efficiently to the OM in a TAB-independent manner but is poorly imported (13). This occurrence of nonproductive binding makes it difficult to decide whether a particular tRNA binding membrane protein is an import factor or is unrelated to import. In contrast, binding of D arm oligonucleotides to the IM shows a strict correlation with importability. The most striking example of this correlation is the loss of binding as well as import in the G²²C mutant, and their simultaneous restoration by the second mutation, C¹³G (Fig. 2 and 3 and Table 3). This should allow the identification of putative IM receptors by photochemical cross-linking and other methods.

The observed differential effects of the same mutation on OM and IM transfer support and refine the stepwise transport model (17) of mitochondrial tRNA import (Fig. 6). In this model, a tRNA species containing the conserved D arm import signal is proposed to bind to TAB at the OM and to be transferred in an ATP-dependent manner into the IMS, where it interacts with a different receptor or receptor complex on the IM. Subsequent translocation into the MX is driven by both the electrical () and chemical (pH) components of the proton motive force across the IM. In the in vitro system, the proton motive force is generated by F₁F₀ ATPase-catalyzed ATP hydrolysis, with concomitant translocation of protons across the IM (Fig. 6).

View larger version (31K): [in this window] [in a new window]   FIG. 6.   Stepwise transfer of tRNA through the two mitochondrial membranes. For details see the text. TAB, outer membrane receptor; IMRC, inner membrane receptor complex components (two components are shown); , membrane potential; pH, proton gradient; F1-F0 ATPase, oligomycin-sensitive proton pump.

Mitochondrial translation is dependent on the presence of an adequately balanced pool of different tRNA species in the MX. Independent evolution of OM and IM receptor specificities may be a means of ensuring this balance. Since the MX concentration of an individual species is dependent on the efficiencies of OM and IM transfer, a low efficiency of OM transfer would be compensated for by a high efficiency of IM transfer, and vice versa. There is also evidence that other domains of the tRNA molecule besides the D arm contain import signals. In tetrahymena, the anticodon of tRNA^Gln(UUG) functions as an import signal (20). Moreover, a tRNA^Ile derivative containing a nonfunctional D arm sequence from tRNA^Gln is nonetheless imported into leishmania mitochondria in vivo (10), indicating the presence of a signal elsewhere in the molecule, possibly in the anticodon arm, in addition to the one in the D arm. The presence of distinct OM and IM transport machineries would allow the sequential use of different signals on the same molecule for MX entry.