Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (154K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (10)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Brubacher-Kauffmann, S.
Right arrow Articles by Duchene, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brubacher-Kauffmann, S.
Right arrow Articles by Duchene, A. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research Pages 2037-2042  

Differential import of nuclear-encoded tRNAGly isoacceptors into Solanum tuberosum mitochondria
Introduction
Materials And Methods
   Isolation of mitochondria
   Transfer RNA extraction
   Northern blot analysis of tRNAs
   Transfer RNA purification and sequencing
Results
   Northern blot analysis revealed that some cytosolic tRNAGly probes hybridized to S.tuberosum mitochondrial tRNAs
   Isolation and sequencing of a new plant cytosolic tRNAGly, with a UCC anticodon
   Determination of partial cytosolic tRNAGly(GCC) and tRNAGly(CCC) sequences in S.tuberosum
   Further northern blot analyses supported an import of cytosolic tRNAGly(UCC) and cytosolic tRNAGly(CCC) into S.tuberosum mitochondria
   Isolation and identification of the mitochondrialtRNAs hybridizing with the cytosolic tRNAGly(UCC)and tRNAGly(CCC) probes: confirmation of an importof some nuclear-encoded tRNAsGly into mitochondria
Discussion
References

Differential import of nuclear-encoded tRNA<sup>Gly</sup> isoacceptors into Solanum tuberosum mitochondria

Differential import of nuclear-encoded tRNAGly isoacceptors into Solanum tuberosum mitochondria

Sabine Brubacher-Kauffmann, Laurence Maréchal-Drouard, Anne Cosset, André Dietrich and Anne-Marie Duchêne*

Institut de Biologie Moléculaire des Plantes du CNRS (UPR 0406), Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France

Received December 23, 1998; Revised and Accepted March 16, 1999

DDBJ/EMBL/GenBank accession no. AJ012213

ABSTRACT

In potato (Solanum tuberosum) mitochondria, about two-thirds of the tRNAs are encoded by the mitochondrial genome and one-third is imported from the cytosol. In the case of tRNAGly isoacceptors, a mitochondrial-encoded tRNAGly(GCC) was found in potato mitochondria, but this is likely to be insufficient to decode the four GGN glycine codons. In this work, we identified a cytosolic tRNAGly(UCC), which was found to be present in S.tuberosum mitochondria. The cytosolic tRNAGly(CCC) was also present in mitochondria, but to a lesser extent. By contrast, the cytosolic tRNAGly(GCC) could not be detected in mitochondria. This selective import of tRNAGly isoacceptors into S.tuberosum mitochondria raises further questions about the mechanism under-lying the specificity of the import process.

INTRODUCTION

In plant cells, translation occurs in three compartments: the cytosol, the mitochondria and the chloroplasts, and all tRNAs necessary for the transfer of the 20 amino acids to the elongating polypeptide chains have to be present in these three compartments. Despite its large size, the plant mitochondrial genome lacks a number of tRNA genes, and some of the tRNAs involved in translation of mitochondrial mRNAs are nuclear-encoded and imported from the cytosol (1,2). The number of imported tRNAs and their identities vary from one plant species to another. In Marchantia polymorpha, almost all the tRNA genes needed are present in the mitochondrial genome (3). In Arabidopsis thaliana, about one-third of the genes are missing (4) and the corresponding tRNAs have to be imported from the cytosol into the mitochondria. Concerning the mitochondrial-encoded tRNAs, these are of two types: the ‘authentic’ mitochondrial-encoded tRNAs called ‘native’ tRNAs, and the ‘chloroplast-like’ tRNAs whose genes originate from chloroplasts and were inserted into the mitochondrial genome during evolution.

Most of the time, simple sequence comparisons allow us to determine to which category a tRNA belongs (5,6). Nuclear-encoded tRNAs are usually very different from mitochondrial- and chloroplast-encoded tRNAs (<60% identity). Mitochondrial ‘native’ tRNAs show 70-75% identity with chloroplast and prokaryotic tRNAs. Mitochondrial ‘chloroplast-like’ tRNAs are identical or nearly identical to their chloroplast counterparts (95-100% identity). Whereas isoacceptor tRNA sequences may vary to a large extent from one cellular compartment to another, there is an impressive conservation of the tRNA sequences with a given genetic origin (nuclear, plastidic or mitochondrial) among higher plants.

The number of mitochondrial tRNAGly genes and the genetic origin of mitochondrial tRNAsGly change from one plant to another. Two genes coding for native tRNAGly isoacceptors (with GCC and UCC anticodons, respectively) have been identified in the mitochondrial genome of the bryophyte M.polymorpha (3). Only a native tRNAGly(GCC) gene was found in the mitochondrial genome of the dicotyledon A.thaliana (4). In wheat (monocotyledon), maize (monocotyledon) and larch (gymnosperm), a nuclear-encoded tRNAGly(GCC) was detected in mitochondria (7,8). In the dicotyledon Solanum tuberosum (potato), only a mitochondrial-encoded tRNAGly(GCC) has been identified so far in mitochondria (1). According to the ‘two out of three’ and the ‘wobble’ translation rules (9), at least one other tRNAGly, in addition to the already known organelle- or nuclear-encoded tRNAGly(GCC), would be necessary in the mitochondria of higher plants to decode the four GGN glycine codons. Here we show that in S.tuberosum mitochondria, the needs for glycine codon decoding are likely to be fulfilled thanks to a selective import of cytosolic tRNAGly(UCC) and tRNAGly(CCC), whereas cytosolic tRNAGly(GCC), which has a mitochondrial-encoded counterpart, is not imported.

MATERIALS AND METHODS

Isolation of mitochondria

In order to obtain potato mitochondria free from cytosolic nucleic acid contaminants, the crude mitochondrial pellet (10) was purified by centrifugation successively on a continuous Percoll/PVP gradient [0.3 M sucrose, 10 mM potassium phosphate pH 7.5, 1 mM EDTA, 0.1% (w/v) BSA, 0-30% (v/v) Percoll, 0-10% (w/v) polyvinylpyrrolidone K25 (PVP)] and on a 13.5/21/45% (v/v) discontinuous Percoll gradient (11). Purification on two gradients reduced cytosolic contamination of mitochondrial tRNAs 5-10 times compared with purification on a single continuous Percoll/PVP gradient.

Transfer RNA extraction

Total and mitochondrial tRNAs were extracted from bean (Phaseolus vulgaris) hypocotyls or potato (S.tuberosum) tubers according to Maréchal-Drouard et al. (12). The proportion of mitochondrial tRNAs in total tRNA preparations from such plant material does not usually exceed 0.5% (13).

Northern blot analysis of tRNAs

Transfer RNAs were separated on a 15% (w/v) polyacrylamide gel, electro-transferred onto Hybond-N nylon membranes (Amersham) and hybridized to oligonucleotide probes at 55°C (for cytosolic tRNAGly probes except AM40 and AM42), 45°C (for non-cytosolic tRNAGly probes) or 40°C (for AM40 and AM42) in 6× SSC, 0.5% (w/v) SDS. Washes were performed at the hybridization temperature in 2× SSC, 0.1% SDS. The oligonucleotides listed below were used as probes:

A.thaliana cytosolic 5S RNA (accession no. M65137), 5[prime]-GGAGGTCACCCATCCTAGTACTAC-3[prime]; A.thaliana cytosolic tRNALys(CUU) (accession no. U67679), 5[prime]-CGCCCACCGTGGGGCTCGAACCC-3[prime]; P.vulgaris cytosolic tRNALeu(C*AA)(6), 5[prime]-TGTCAGAAGTGGGATTTGAACCCA-3[prime]; A.thaliana cytosolic tRNAGlu(UUC) (accession no. AC000106), 5[prime]-CTCCTGGGTGAAAGCCAGATA-3[prime]; Lupinus luteus mitochondrial tRNAGly(GCC) (6), AM15 5[prime]-AGCGGAAGGAGGGACTTGAACCCTCA-3[prime]; A.thaliana cytosolic tRNAGly(GCC) (accession no. AB010700), AM23 5[prime]-TGCACCAGCCGGGAATCGAAC-3[prime], AM40 5[prime]-GCAGGGTACTATTCT-3[prime]; A.thalianacytosolic tRNAGly(CCC) (accession no. AC005309), AM29 5[prime]-TGCGCATCCAGGGAATCGAAC-3[prime], AM42 5[prime]-GGAGGGTACTATGAT-3[prime]; Rattus norvegicus cytosolic tRNAGly(UCC)(accession no. K03130), AM27 5[prime]-TGCGTTGGCCGGGAATTGAACCCGGGG-3[prime]; P.vulgaris cytosolic tRNAGly(UCC) (Fig. 2; accession no. AJ012213), AM30 5[prime]-TTGCTTGGUAGGCAATTATCC-3[prime], AM31 5[prime]-CCGGGTTCGACTCCCGG-3[prime], AM32 5[prime]-GCGTCTGTAGTCCAACGGTTAG-3[prime], AM33 5[prime]-TGCGTCTGCCGGGAGTCGAAC-3[prime].

Transfer RNA purification and sequencing

Separation of tRNAs by 2D-polyacrylamide gel electrophoresis (2D-PAGE) and dot blotting were performed according to Maréchal-Drouard et al. (12). Aminoacylations were assayed with 10-4 M [3H]glycine and with either a bean cytosolic enzymatic extract or a potato mitochondrial enzymatic extract (12).

Determination of the tRNA sequence was performed using the technique of Stanley and Vassilenko (14). For analysis by homochromatography (15), the tRNA was 3[prime] end-labeled with [32P]pCp (16) and statistically hydrolyzed by heating in water. Reverse transcription, tRNA circularization with T4 RNA ligase, and PCR were performed according to Yokobori and Pääbo (17).

RESULTS

Northern blot analysis revealed that some cytosolic tRNAGly probes hybridized to S.tuberosum mitochondrial tRNAs

As mentioned above, only a native tRNAGly(GCC) has been identified so far in mitochondria of S.tuberosum (1), although at least one additional tRNAGly isoacceptor would be necessary to decode the four glycine codons in mitochondria. The hypothesis of an import of some nuclear-encoded tRNAsGly into the mitochondria of S.tuberosum was therefore emitted. To test this hypothesis, total and mitochondrial tRNAs from S.tuberosum were run on 15% polyacrylamide gels and northern blot hybridizations were performed. Oligonucleotides complementary to different higher plant tRNA sequences were used as probes, taking advantage of the fact that only very few differences are observed from one higher plant to another in the sequence of tRNAs having the same genetic origin, that is nuclear-encoded tRNAs, mitochondrial ‘native’ tRNAs, or chloroplast-encoded tRNAs and mitochondrial-encoded ‘chloroplast-like’ tRNAs. Transfer RNALeu(C*AA), which is a known imported tRNA (1), was taken as an import reference. Cytosolic 5S RNA and cytosolic tRNALys(CUU) probes allowed the evaluation of cytosolic contamination. Cytosolic tRNAGly(GCC) and tRNAGly(CCC) probes (AM23 and AM29, respectively) were derived from A.thaliana sequences. For cytosolic tRNAGly(UCC), no sequence had at that time been identified in plants, and an oligonucleotide (AM27) complementary to the rat (R.norvegicus) cytosolic tRNAGly(UCC) was used as a probe. Until now, no eukaryotic cytosolic tRNAGly(ACC) has ever been identified.


Figure 1. Northern blots with S.tuberosum total (T) and mitochondrial (M) tRNAs. The oligonucleotide probes are given in Materials and Methods; mito. and cyto. indicate mitochondrial and cytosolic, respectively. Hybridization signals were quantified, and the ratio between the mitochondrial (M) signal and the total (T) signal is indicated. (A) The hybridizations obtained with the probes used as a first approach. (B) The results with the probes designed after analysis of the S.tuberosum tRNAGly sequences.

As expected, the mitochondrial tRNAGly(GCC) probe hybridized with S.tuberosum mitochondrial tRNAs (1), and the three cytosolic tRNAGly probes gave a signal with S.tuberosum total tRNAs (Fig. 1A). Hybridizations of cytosolic tRNAGly probes with mitochondrial tRNAs showed different results, depending on the probe. Indeed, no cytosolic tRNAGly(GCC) could be detected in mitochondria, but a strong signal was obtained with the rat cytosolic tRNAGly(UCC) probe (AM27) and a weaker one with the cytosolic tRNAGly(CCC) probe (AM29) (Fig. 1A). The tRNA hybridizing with AM27 was assumed to be a nuclear-encoded tRNAGly(UCC), but this had to be confirmed (see below).


Figure 2. Secondary structure of P.vulgaris tRNAGly(UCC). The nucleotides in bold correspond to those identified by direct sequencing of the tRNA. Oligonucleotides AM30-AM33 are indicated around the sequence. Open arrows point to the positions detailed in Table 1. D, dihydrouridine; [Psi], pseudouridine; A*, methyladenosine; ?U, unknown modified uridine.

These results suggested that cytosolic tRNAGly(UCC) and tRNAGly(CCC), but not cytosolic tRNAGly(GCC), were imported into S.tuberosum mitochondria, and that the level of import was dependent on the tRNA (Fig. 1A): strong import of the tRNA hybridizing with AM27, little import of tRNAGly(CCC), and no detectable import of tRNAGly(GCC).

Isolation and sequencing of a new plant cytosolic tRNAGly, with a UCC anticodon

In order to unambiguously identify the tRNA hybridizing with AM27, bean total tRNAs were separated by 2D-PAGE. Bean was preferred to potato for preparing total plant tRNA because of much higher yields. The material in each spot resolved by 2D-PAGE was eluted, hybridized with AM27 and tested by aminoacylation with [3H]glycine. The positive samples, which corresponded to confluent spots on the gel, were pooled and further purified by another 2D-PAGE, followed by a 15% polyacrylamide denaturing gel. Only one tRNA band yielding a hybridization signal with AM27 was thus obtained.

This purified tRNA was directly sequenced according to Stanley and Vassilenko (14). Nucleotides from positions 13 to 65 could be identified by this method (Fig. 2), yielding a partial sequence corresponding to a tRNAGly with an NCC anticodon. A few bases were modified: dihydrouridine was found at positions 20 and 20a, pseudouridine at position 55, and methyladenosine at position 58. An unmodified U was present at position 54 instead of the usual T54. An unknown modified nucleotide was found at the wobble position in the anticodon. Analysis by 2D-thin layer chromatography (1) (data not shown) suggested that this nucleotide, which was highly hydrophilic, was a modified U (G.Keith, personal communication).

In order to determine the whole sequence, RNA circularization was performed on the purified tRNA. This ligation product was used in a reverse transcription reaction with AM30 as a primer (Fig. 2). A PCR reaction was performed with the reverse transcription product in the presence of oligonucleotides AM30 and AM31 (Fig. 2). Analysis of the PCR product allowed the determination of the sequence of the 5[prime] and 3[prime] ends of the tRNA.

To confirm the data obtained by direct sequencing with the Stanley and Vassilenko method, a PCR reaction was performed with oligonucleotides AM32 and AM33 (Fig. 2), using as a template the product of a reverse transcription performed with the purified bean tRNAGly. This confirmed that the new tRNAGly had a UCC anticodon, and the obtained sequence corresponded exactly to that established by direct sequencing. The complete bean tRNAGly(UCC) sequence can be found under DDBJ/EMBL/GenBank accession no. AJ012213.

Before using this new plant tRNAGly(UCC) sequence for further confirmation of the mitochondrial tRNAGly pattern in potato, we verified that there were no differences between the potato and the bean tRNAGly(UCC) sequences. A PCR reaction was therefore performed with oligonucleotides AM32 and AM33 and with S.tuberosum total DNA as a template. The partial sequence thus obtained (nucleotides 22-53) was identical to that of the bean tRNAGly(UCC).

More recently, two sequences were obtained from the A.thaliana genome sequencing program (accession nos AB12245 and AC005315), which were identical to the P.vulgaris AJ012213 sequence. The P.vulgaris or A.thaliana tRNAGly(UCC) sequence presented 71% identity with the A.thaliana tRNAGly(GCC) sequence (accession no. AB010700), and 58% identity with the A.thaliana tRNAGly(CCC) sequence (accession no. AC005309).

Determination of partial cytosolic tRNAGly(GCC) and tRNAGly(CCC) sequences in S.tuberosum

Still to prevent misinterpretation of the northern blots, partial sequences of the S.tuberosum cytosolic tRNAGly(GCC) and tRNAGly(CCC) were determined. This was performed by PCR reactions with S.tuberosum total DNA as a template, and oligonucleotides specific for the 5[prime] and 3[prime] regions of either tRNAGly(GCC) or tRNAGly(CCC).

The cytosolic tRNAGly(GCC) sequence was previously determined in A.thaliana (accession no. AB010700), L.luteus (accession no. Z49255), Oriza sativa (accession no. X14039), Eleusine coracana (accession no. U02636), Sorghum bicolor (accession no. X0695) and Triticum aestivum (accession no. M28427). Exactly the same sequence was found in all cases. The partial S.tuberosum tRNAGly(GCC) sequence we obtained (nucleotides 23-52) was also identical to these previously established sequences.

The A.thaliana tRNAGly(CCC) sequence was the only plant tRNAGly(CCC) sequence known so far. In the partial S.tuberosum tRNAGly(CCC) sequence we obtained (nucleotides 22-52), only one divergence was found compared to the A.thaliana sequence: a G at position 50 of the tRNA instead of an A in A.thaliana.


Figure 3. Solanum tuberosum mitochondrial tRNAs fractionated by 2D-PAGE as described in (1). The gel was stained with methylene blue. Arrows indicate the spots hybridizing with tRNAGly probes as listed below the picture.

Further northern blot analyses supported an import of cytosolic tRNAGly(UCC) and cytosolic tRNAGly(CCC) into S.tuberosum mitochondria

To confirm the first observations (Fig. 1A), further northern blot hybridizations were performed with S.tuberosum total and mitochondrial tRNAs, using cytosolic tRNAGly probes which took into account the above sequence studies and which were 100% identical to S.tuberosum cytosolic tRNAGly sequences: oligonucleotides AM30 for tRNAGly(UCC), AM40 for RNAGly(GCC), and AM42 for tRNAGly(CCC) (Fig. 1B). As already shown in Figure 1A, the probes specific for cytosolic tRNAGly(UCC) and tRNAGly(CCC) hybridized to mitochondrial tRNAs, and no signal was obtained in the mitochondrial tRNA lane with the probe specific for cytosolic tRNAGly(GCC). Because A.thaliana cytosolic tRNAGlu(UUC) presented strong similarities with P.vulgaris or A.thaliana cytosolic tRNAGly(UCC), a futher control was performed with a cytosolic tRNAGlu(UUC) probe, which showed that tRNAGlu(UUC) was only located in the cytosol (Fig. 1B). Altogether, these results further supported the idea that cytosolic tRNAGly(UCC) and tRNAGly(CCC) were both imported into S.tuberosum mitochondria, whereas cytosolic tRNAGly(GCC) was not.

Isolation and identification of the mitochondrialtRNAs hybridizing with the cytosolic tRNAGly(UCC)and tRNAGly(CCC) probes: confirmation of an importof some nuclear-encoded tRNAsGly into mitochondria

To definitely identify the mitochondrial tRNAs hybridizing with the cytosolic tRNAGly(UCC) and tRNAGly(CCC) probes, S.tuberosum mitochondrial tRNAs fractionated by 2D-PAGE (1) (Fig. 3) were hybridized with different tRNAGly probes. As already shown, the tRNA eluted from spot 29 hybridized with the mitochondrial tRNAGly(GCC) probe (oligonucleotide AM15) (1). The material from the minor spot 44 hybridized with a cytosolic tRNAGly(CCC) probe (oligonucleotide AM29), whereas spots 48 and 52 contained a tRNA hybridizing with a cytosolic tRNAGly(UCC) probe (oligonucleotide AM30). As a complementary control, the same probes were also hybridized to Southern blots of restriction endonuclease-digested S.tuberosum mitochondrial DNA (data not shown). As expected, the mitochondrial tRNAGly(GCC) probe hybridized to the mitochondrial DNA, but no signal was detected with the tRNAGly(UCC) and the tRNAGly(CCC) probes, confirming that the mitochondrial tRNAs hybridizing with these probes had to be imported into mitochondria.


Figure 4. Homochromatography 3[prime]-end sequencing of the major tRNA present in spot 48 of the 2D-PAGE fractionation of S.tuberosum mitochondrial tRNAs shown in Figure 3. The obtained sequence is identical to that of the P.vulgaris cytosolic tRNAGly(UCC) from positions 61 to 75 (see Fig. 2).

Aminoacylation with [3H]glycine was obtained in the presence of a S.tuberosum mitochondrial enzymatic extract with the tRNAs eluted from the four spots mentioned above (29, 44, 48 and 52). This showed that each of these four spots contained a tRNAGly. Finally, spot 48, which is supposed to correspond to the cytosolic tRNAGly(UCC), appeared to contain several tRNAs which migrated as three bands on a 15% polyacrylamide gel. The tRNA eluted from the major band was 3[prime] end-labeled with [32P]pCp, statistically hydrolyzed in water, and analyzed by homochromatography (Fig. 4). The obtained 3[prime] end sequence, from nucleotides 61 to 75, was 100% identical to the previously determined sequence of the cytosolic tRNAGly(UCC), thus confirming that the major tRNA species in spot 48 corresponded to this nuclear-encoded tRNA. Spot 44 was a minor spot and contained little material, so that a similar sequencing experiment could not be performed in this case. Altogether, these results confirmed the presence of the cytosolic tRNAGly(UCC) and tRNAGly(CCC) in S.tuberosum mitochondria.

DISCUSSION

In this study, we show that nuclear-encoded and mitochondrial-encoded tRNAsGly coexist in S.tuberosum mitochondria. A similar result was obtained with P.vulgaris: northern blots were prepared with P.vulgaris total and mitochondrial tRNAs, and hybridization allowed the detection of both the mitochondrial-encoded tRNAGly(GCC) and the nuclear-encoded tRNAGly(UCC) in the mitochondrial tRNA population (data not shown). Coexistence of imported and organelle-encoded isoacceptors in mitochondria has been previously implied for tRNAIle in higher plants (18), and for tRNAIle, tRNAThr and tRNaVal in M.polymorpha (19). In some of these cases, tRNA isoacceptors might be redundant for decoding certain codons. Taking into account the ‘two out of three’ and the ‘wobble’ translation rules, two tRNAGly isoacceptors, with UCC and GCC anticodons, respectively, would a priori be sufficientto decode the four GGN glycine codons. Indeed, bothtRNAGly(UCC) and tRNAGly(GCC) genes were found in M.polymorpha, Pinus thunbergiana, Nicotiana tabacum or O.sativa chloroplast genomes and in M.polymorpha mitochondrial genome [refer to The Organelle Genome Database (GOBASE); http://megasun.bch.umontreal.ca/gobase ]. Translation rules in non-plant mitochondria allow an unmodified U at the first position of the anticodon to pair with all four bases at the third position of the codon for sets of four synonymous codons (9). This is the case for non-plant mitochondrial tRNAsGly: only a tRNAGly(UCC) gene is found in the mitochondrial genome of Caenorhabditis elegans, Homo sapiens or Saccharomyces cerevisiae (refer to GOBASE), and there is no mitochondrial import of cytosolic tRNAsGly in these organisms. According to our results, the situation is likely to be different in S.tuberosum mitochondria, where the organelle-encoded tRNAGly(GCC) probably decodes the GGC and GGU codons. Direct sequencing showed that, at least in bean, the U at the first position of the anticodon in the cytosolic tRNAGly(UCC) is modified, which may limit the decoding capacity of this tRNA to the GGA codon. In that case, the presence of the tRNAGly(CCC) may also be necessary in mitochondria to read the GGG codons. Our observations fit this analysis, as cytosolic tRNAGly(UCC) and tRNAGly(CCC) were found to be present in S.tuberosum mitochondria. It therefore seems likely that the set of tRNAsGly we identified fulfills the requirements for glycine codon recog-nition in S.tuberosum mitochondria and that no decoding overlap occurs.

Although one cannot rule out a difference in the stability of these isoacceptors in the organelles, we bring here strong evidence for a selective import of only two out of the three cytosolic tRNAsGly into S.tuberosum mitochondria. This is the first clearly documented case of a selective import of cytosolic isoacceptors into plant mitochondria, and only very few examples are known in other organisms: in yeast only one out of two cytosolic tRNAsLys is imported into mitochondria (20,21), and selective import of cytosolic tRNAGln isoacceptors into mitochondria has been observed in the protozoans Tetrahymena thermophila (22) and Leishmania tarentolae (23).

Two different mechanisms have been proposed for mitochondrial tRNA import. The first one is a direct import of the tRNAs through the mitochondrial membranes, and in Leishmania tropica the UGGYAGAG sequence present in the D-loop of the tRNAs was proposed to be essential and sufficient for in vitro import (24). The second mechanism is a co-import of the tRNAs with protein factors, including the corresponding aminoacyl-tRNA synthetases: in yeast, co-import of the cytosolic tRNALys(CUU) with the precursor of the mitochondrial lysyl-tRNA synthetase was proposed (25). In plants, little is known so far about the tRNA import mechanism, but it was shown that a point mutation in a normally imported tRNA, tRNAAla, blocked both the amino-acylation of this tRNA by alanyl-tRNA synthetase and its import into mitochondria (26). In S.tuberosum, preliminary studies showed that a mitochondrial enzymatic extract was able to aminoacylate both the mitochondrial-encoded tRNAGly(GCC) and the three cytosolic tRNAsGly, including the non-imported cytosolic tRNAGly(GCC). This suggests that recognition of the tRNAsGly aminoacylation identity is not sufficient to promote specific import. Therefore, if co-import of tRNAsGly with a mitochondrial glycyl-tRNA synthetase occurs, this may require another factor for specificity, to explain the presence or absence of the different cytosolic tRNAGly isoacceptors in mitochondria.

Whatever the mechanism of import, the sequence of the tRNA was found to be essential. In T.thermophila, substitution of a unique nucleotide in the anticodon abolished the import of a normally imported tRNAGln, or permitted the import of a normally non-imported tRNAGln (27). In L.tarentolae, the replacement of the D-stem of a non-imported tRNAGln by the D-stem of an imported tRNA (tRNAIle) allowed the import of the mutated tRNAGln (23). In yeast, in vitro and in vivo import of mutated tRNAsLys underlined the role of the anticodon region and of the acceptor stem in import (28). In transgenic tobacco, the substitution of U70 into C70 in tRNAAla abolished the import of this tRNA (26).

Comparison of the cytosolic tRNAGly sequences should point out the differences between the non-imported tRNAGly(GCC) isoacceptor and the imported tRNAGly isoacceptors [tRNAGly(UCC) and tRNAGly(CCC)]. As a first attempt to run this kind of alignment, we took into account the partial S.tuberosum tRNAGly sequences we established and, betting on the very high conservation of the cytosolic tRNA sequences from one plant to the other, we used the known A.thaliana tRNAGly sequences for the missing 5[prime] and 3[prime] end regions. On such a basis, the tRNAGly(GCC) sequence presents 71% identity with the tRNAGly(UCC) sequence and 83% identity with the tRNAGly(CCC) sequence. Only six nucleotides in tRNAGly(GCC) are changed both in tRNAGly(UCC) and in tRNAGly(CCC) (Table 1). Four of these are located in the acceptor stem. The A3-U70 base-pair should be pointed out, as the C3-G70 pair is an identity element for aminoacylation in yeast tRNAsGly (29). In the D-stem, A23, which theoretically pairs with C12, should somehow destabilize the tRNAGly(GCC) structure. The last difference is the wobble position in the anticodon. Some of these six positions may be essential to avoid or to promote import into S.tuberosum mitochondria. Expression of chimeric tRNAsGly in transgenic potato upon exchange of these nucleotides would be an approach to analyzing the sequence dependence of the import process and trying to understand why the cytosolic tRNAGly(GCC) is not imported. However, with a broader view the problem looks obviously more complex. Although the sequence of the cytosolic tRNAGly(GCC) is likely to be the same in the two plant species, this tRNA is not imported into the mitochondria of the dicotyledonous species S.tuberosum and is imported into the mitochondria of the monocotyledonous species Triticum aestivum (7). Such variations illustrate the flexibility of the import process.

Table 1. Comparison of plant cytosolic tRNAGly sequences
Nucleotides in tRNAGly(GCC) Nucleotides in tRNAGly(UCC) and tRNAGly(CCC)
A3-U70 G3-C70
A6-U67 U6-A67
A23 G23: in tRNAGly(UCC)
(C12-A23) (C12-G23)
C23: in tRNAGly(CCC)
(G12-C23)
G34 U34: in tRNAGly(UCC)
C34: in tRNAGly(CCC)
Only six nucleotides in tRNAGly(GCC) are changed both in tRNAGly(UCC) and tRNAGly(CCC). The positions of these nucleotides are also indicated in Figure 2.

REFERENCES

1. Maréchal-Drouard,L., Guillemaut,P., Cosset,A., Arbogast,M., Weber,F., Weil,J.H. and Dietrich,A. (1990) Nucleic Acids Res., 18, 3689-3696. MEDLINE Abstract

2. Dietrich,A., Weil,J.H. and Maréchal-Drouard,L. (1992) Annu. Rev. Cell. Biol., 8, 115-131. MEDLINE Abstract

3. Oda,K., Yamato,K., Ohta,E., Nakamura,Y., Takemura,M., Nozato,N., Akashi,K. and Ohyama,K. (1992) J. Mol. Biol., 223, 1-7. MEDLINE Abstract

4. Unseld,M., Marienfeld,J.R., Brandt,P. and Brennicke,A. (1997) Nature Genet., 15, 57-61. MEDLINE Abstract

5. Maréchal-Drouard,L., Weil,J.H. and Dietrich,A. (1993) Annu. Rev. Plant Physiol. Plant Mol. Biol., 44, 13-32.

6. Sprinzl,M., Horn,C., Brown,M., Ioudovitch,A. and Steinberg,S. (1998) Nucleic Acids Res., 26, 148-153. MEDLINE Abstract

7. Joyce,P. and Gray,M. (1989) Nucleic Acids Res., 17, 5461-5476. MEDLINE Abstract

8. Kumar,R., Maréchal-Drouard,L., Akama,K. and Small,I. (1996)Mol. Gen. Genet., 252, 404-411. MEDLINE Abstract

9. Hatfield,D., Lee,B. and Pirtle,R. (eds) (1992) Transfer RNA in Protein Synthesis. CRC Press, Boca Raton, FL, pp. 113-140.

10. Neuburger,M., Journet,E.P., Bligny,R., Carde,J.P. and Douce,R. (1982) Arch. Biochem. Biophys., 217, 312-323. MEDLINE Abstract

11. Jackson,C., Dench,J., Hall,D. and Moore,A. (1979) Plant Physiol., 64, 150-153.

12. Maréchal-Drouard,L., Small,I., Weil,J.H. and Dietrich,A. (1995) Methods Enzymol., 260, 310-327. MEDLINE Abstract

13. Guillemaut,P. and Weil,J.H. (1990) In Cherayil,J.D. (ed.), Transfer RNAs and Other Soluble RNAs. CRC Press, Boca Raton, FL, pp. 55-67.

14. Stanley,J. and Vassilenko,S. (1978) Nature, 274, 87-89. MEDLINE Abstract

15. Maréchal-Drouard,L., Ramamonjisoa,D., Cosset,A., Weil,J.H. and Dietrich,A. (1993) Nucleic Acids Res., 21, 4909-4914. MEDLINE Abstract

16. Maréchal,L., Guillemaut,P., Grienenberger,J.M., Jeannin,G. and Weil,J.H. (1985) FEBS, 184, 289-293.

17. Yokobori,S.I. and Pääbo,S. (1995) Proc. Natl Acad. Sci. USA, 92, 10432-10435. MEDLINE Abstract

18. Dietrich,A., Small,I., Cosset,A., Weil,J.H. and Maréchal-Drouard,L. (1996) Biochimie, 78, 518-529. MEDLINE Abstract

19. Akashi,K., Tazkenaka,M., Yamaoka,S., Suyama,Y., Fukuzama,H. and Ohyama,K. (1998) Nucleic Acids Res., 26, 2168-2172. MEDLINE Abstract

20. Martin,R., Schneller,J.M., Stahl,A. and Dirheimer,G. (1979) Biochemistry, 18, 4600-4605. MEDLINE Abstract

21. Tarassov,I. and Entelis,N. (1992) Nucleic Acids Res., 20, 1277-1281. MEDLINE Abstract

22. Rusconi,C. and Cech,T. (1996) EMBO J., 15, 3286-3295. MEDLINE Abstract

23. Nabholz,C., Hauser,R. and Schneider,A. (1997) Proc. Natl Acad. Sci. USA, 94, 7903-7908. MEDLINE Abstract

24. Mahapatra,S., Ghosh,S., Kanti Bera,S., Ghosh,T., Das,A. and Adhya,S. (1998) Nucleic Acids Res., 26, 2037-2041. MEDLINE Abstract

25. Tarassov,I. and Martin,R. (1996) Biochimie, 78, 502-510. MEDLINE Abstract

26. Dietrich,A., Maréchal-Drouard,L., Carneiro,V., Cosset,A. and Small,I. (1996) Plant J., 10, 913-918. MEDLINE Abstract

27. Rusconi,C. and Cech,T. (1996) Genes Dev., 10, 2870-2880. MEDLINE Abstract

28. Entelis,N., Kieffer,S., Kolesnikova,O., Martin,R. and Tarassov,I. (1998) Proc. Natl Acad. Sci. USA, 95, 2838-2843. MEDLINE Abstract

29. Nameki,N., Tamura,K., Asahara,H. and Hasegawa,T. (1997) J. Mol. Biol., 268, 640-647. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +33 3 88 41 72 29; Fax: +33 3 88 61 44 42; Email: anne-marie.duchene{at}ibmp-ulp.u-strasbg.fr

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification: 10 Apr 1999
Copyright©Oxford University Press, 1999.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
M. A. T. Rubio, J. J. Rinehart, B. Krett, S. Duvezin-Caubet, A. S. Reichert, D. Soll, and J. D. Alfonzo
Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import
PNAS, July 8, 2008; 105(27): 9186 - 9191.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
C. Pujol, M. Bailly, D. Kern, L. Marechal-Drouard, H. Becker, and A.-M. Duchene
Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants
PNAS, April 29, 2008; 105(17): 6481 - 6485.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
L. Delage, A. Dietrich, A. Cosset, and L. Marechal-Drouard
In Vitro Import of a Nuclearly Encoded tRNA into Mitochondria of Solanum tuberosum
Mol. Cell. Biol., June 1, 2003; 23(11): 4000 - 4012.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A.-M. Duchene, N. Peeters, A. Dietrich, A. Cosset, I. D. Small, and H. Wintz
Overlapping Destinations for Two Dual Targeted Glycyl-tRNA Synthetases in Arabidopsis thaliana and Phaseolus vulgaris
J. Biol. Chem., April 27, 2001; 276(18): 15275 - 15283.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (154K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (10)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Brubacher-Kauffmann, S.
Right arrow Articles by Duchene, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brubacher-Kauffmann, S.
Right arrow Articles by Duchene, A. M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?