The anticodon and the D‐domain sequences are essential determinants for plant cytosolic tRNA^Val import into mitochondria

Abstract

SummaryIn higher plants, one-third to one-half of the mitochondrial tRNAs are encoded in the nucleus and are imported into mitochondria. This process appears to be highly speci®c for some tRNAs, but the factors that interact with tRNAs before and/or during import, as well as the signals present on the tRNAs, still need to be identi®ed. The rare experiments performed so far suggest that, besides the probable implication of aminoacyl-tRNA synthetases, at least one additional import factor and/or structural features shared by imported tRNAs must be involved in plant mitochondrial tRNA import. To look for determinants that direct tRNA import into higher plant mitochondria, we have transformed BY2 tobacco cells with Arabidopsis thaliana cytosolic tRNA Val (AAC) carrying various mutations. The nucleotide replacements introduced in this naturally imported tRNA correspond to the anticodon and/or D-domain of the non-imported cytosolic tRNA Met±e . Unlike the wild-type tRNA Val (AAC), a mutant tRNA Val carrying a methionine CAU anticodon that switches the aminoacylation of this tRNA from valine to methionine is not present in the mitochondrial fraction. Furthermore, mutant tRNAs Val carrying the D-domain of the tRNA Met±e , although still ef®ciently recognized by the valyl-tRNA synthetase, are not imported any more into mitochondria. These data demonstrate that in plants, besides identity elements required for the recognition by the cognate aminoacyl-tRNA synthetase, tRNA molecules contain other determinants that are essential for mitochondrial import selectivity. Indeed, this suggests that the tRNA import mechanism occurring in plant mitochondria may be different from what has been described so far in yeast or in protozoa.

The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria

“…Altogether, these data suggest that some specificity factors have been lost in the in vitro import assays. For instance, aminoacyl-tRNA synthetases, although likely implicated in the import process (35), are not present in our in vitro system, which might partially explain the reduced specificity. We also have to explain why a cytosolic tRNA, with a priori the same sequence, is imported in one plant but not in the other (34).…”

“…Import of 35 Slabeled fusion protein GluRS-GFP into purified potato mitochondria was performed as described (16). F1␤ presequence was isolated as described (21).…”

Dual targeting is the rule for organellar aminoacyl-tRNA synthetases in Arabidopsis thaliana

“…Because all four pairs of aaRSs correspond to tRNAs imported into mitochondria from the cytosol, an attractive hypothesis is that some of these enzymes are needed for import of cytosolic tRNAs into mitochondria. Coimport of tRNA with the corresponding aminoacyl-tRNA synthetase has been described in yeast (26), and in plants, point mutations in tRNA Ala and tRNA Val blocked both the aminoacylation of these tRNAs and their import into mitochondria (27,28). In this context, it is interesting to note that although GlyRS-1 is efficiently and incontrovertibly imported into mitochondria, it has no apparent aminoacylation activity within the organelle, this activity being assured by GlyRS-2 (5).…”

On the Evolution and Expression of Chlamydomonas reinhardtii Nucleus-Encoded Transfer RNA Genes

“…Oligonucleotides (see supplemental Table 2) were 59-end labeled using T4 polynucleotide kinase and ½g-32 PATP under standard conditions. Hybridization and washing were performed as described in Delage et al (2003).…”