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

Abstract

In plants, protein synthesis occurs in the cytosol, mitochondria, and plastids. Each compartment requires a full set of tRNAs and aminoacyl-tRNA synthetases. We have undertaken a systematic analysis of the targeting of organellar aminoacyl-tRNA synthetases in the model plant Arabidopsis thaliana. Dual targeting appeared to be a general rule. Among the 24 identified organellar aminoacyl-tRNA synthetases (aaRSs), 15 (and probably 17) are shared between mitochondria and plastids, and 5 are shared between cytosol and mitochondria (one of these aaRSs being present also in chloroplasts). Only two were shown to be uniquely chloroplastic and none to be uniquely mitochondrial. Moreover, there are no examples where the three aaRS genes originating from the three ancestral genomes still coexist. These results indicate that extensive exchange of aaRSs has occurred during evolution and that many are now shared between two or even three compartments. The findings have important implications for studies of the translation machinery in plants and on protein targeting and gene transfer in general.

Fig. 1.

Mitochondrial and/or chloroplast import of targeting sequence GFP or RFP fusions in tobacco protoplasts. The images are false-color maximum projections from a confocal microscope. The red channel (Left) shows chlorophyll autofluorescence, the green channel (Middle) shows GFP or RFP fluorescence (RFP for GluRS and ThrRS, GFP for all others); in Right, the two channels are superimposed. Several of the images include untransformed protoplasts in the field of view to demonstrate the lack of background fluorescence under the conditions used. The scale of the images and cell size varies slightly, but the chloroplasts are a uniform 5 μm in these protoplasts.

Fig. 2.

In vitro import into organelles of targeting sequence-GFP fusions. In vitro transcriptions/translations products (lane 1) were incubated in the presence of mitochondria or of chloroplasts and were partially processed into smaller polypeptides (lane 2), which corresponded to the fusion proteins upon cleavage of the predicted targeting sequences. The addition of proteinase K to the import medium reduced the signals corresponding to the preproteins but did not affect the signals corresponding to the processed proteins that were protected by mitochondrial or chloroplastic membranes (lane 3). The addition of valinomycin, which is known to inhibit mitochondrial protein import, or darkness, which is known to inhibit chloroplastic protein import, prevented the formation of the processed proteins (lanes 4 and 5). In these conditions, all radioactive signals were digested by proteinase K (lanes 5), showing that the signal observed in lane 3 represented genuine imported proteins.

Fig. 3.

Western blot analysis. Total (Total), cytosolic (Cyto), chloroplastic (Chloro), or mitochondrial (Mito) proteins extracts were prepared from A. thaliana suspension cells. Immunodetection was performed with bean chloroplastic leucyl-tRNA synthetase (@LeuRS) (19), A. thaliana alanyl-tRNA synthetase (@AlaRS-1) (7), maize mitochondrial pyruvate dehydrogenase (@PDH E1-α) (GT Monoclonal Antibodies), Chlamydomonas chloroplastic light harvesting complex II (@LHC II) and α-tubuline (@α-tubuline) (Amersham Pharmacia Biotech).

Fig. 4.

Putative origins of Arabidopsis aaRSs and their cognate tRNAs. Extensive sharing between the different cellular compartments are observed in A. thaliana at the level of tRNAs (A) and aaRSs (B). In most cases, the tRNA/aaRS couples are in accordance with the generally admitted coevolution between aaRSs and tRNAs, but numerous mismatches are also found (C). Some aaRSs have cognate tRNAs of different origins, and all of the cases are represented in C. For example, mitochondrial tRNAs^Ser are native or cp-like, so the SerRS is represented twice, once associated with native tRNAs and once with cp-like tRNAs. ^*, one is similar to mitochondrial aaRSs. ^**, two are similar to mitochondrial aaRSs.