Overlapping Destinations for Two Dual Targeted Glycyl-tRNA Synthetases in Arabidopsis thaliana and Phaseolus vulgaris

Anne-Marie Duchêne,Nemo Peeters,André Dietrich,Anne Cosset,Ian D Small,Henri Wintz

doi:10.1074/jbc.m011525200

Abstract

In plant mitochondria, some of the tRNAs are encoded by the mitochondrial genome and resemble their prokaryotic counterparts, whereas the remaining tRNAs are encoded by the nuclear genome and imported from the cytosol. Generally, mitochondrial isoacceptor tRNAs all have the same genetic origin. One known exception to this rule is the group of tRNA(Gly) isoacceptors in dicotyledonous plants. A mitochondrion-encoded tRNA(Gly) and at least one nucleus-encoded tRNA(Gly) coexist in the mitochondria of these plants, and both are required to allow translation of all four GGN glycine codons. We have taken advantage of this atypical situation to address the problem of tRNA/aminoacyl-tRNA synthetase coevolution in plants. In this work, we show that two different nucleus-encoded glycyl-tRNA synthetases (GlyRSs) are imported into Arabidopsis thaliana and Phaseolus vulgaris mitochondria. The first one, GlyRS-1, is similar to human or yeast glycyl-tRNA synthetase, whereas the second, GlyRS-2, is similar to Escherichia coli glycyl-tRNA synthetase. Both enzymes are dual targeted, GlyRS-1 to mitochondria and to the cytosol and GlyRS-2 to mitochondria and chloroplasts. Unexpectedly, GlyRS-1 seems to be active in the cytosol but inactive in mitochondrial fractions, whereas GlyRS-2 is likely to glycylate both the organelle-encoded tRNA(Gly) and the imported tRNA(Gly) present in mitochondria.

Highlights

Southern blot analysis [17] of total A. thaliana DNA digested with PstI, BglII, or EcoRI suggested that the glycyl-tRNA synthetases (GlyRSs)-1 gene is a single copy gene, which was confirmed with the availability of the complete A. thaliana genomic sequence ((32); see the Arabidopsis Genome Initiative web site)
We show that two different GlyRSs are imported into A. thaliana and P. vulgaris mitochondria
GlyRS-1 is similar to eukaryotic enzymes, whereas GlyRS-2 presents similarities with E. coli GlyRS

Summary

Introduction

GlyRS-1 seems to be active in the cytosol but inactive in mitochondrial fractions, whereas GlyRS-2 is likely to glycylate both the organelle-encoded tRNAGly and the imported tRNAGly present in mitochondria. GlyRS-1 was shown to be active in the bean cytosol but not in mitochondria, whereas GlyRS-2 was able to aminoacylate either nuclearly or mitochondrially encoded tRNAsGly. Inverse PCR—A. thaliana total DNA was extracted from whole plants according to Dellaporta et al [16], digested with BglII, selfligated, and used as a template for PCR amplification [17] with divergent oligonucleotides AM10 (5Ј-GCGGGATCCGGATCGGAGCGATGGAGATTGGG-3Ј; the BamHI site for cloning is underlined) and AM12 (5Ј-CGCTCTAGAGGCTTCCCGTGCAGCTAAACCAG-3Ј; the XbaI site for cloning is underlined) (see Fig. 1A).

Results

Conclusion