Characterization of two bifunctional Arabdopsis thaliana genes coding for mitochondrial and cytosolic forms of valyl‐tRNA synthetase and threonyl‐tRNA synthetase by alternative use of two in‐frame AUGs

Abstract

We characterized two Arabidopsis thaliana cDNAs coding for class I valyl-tRNA synthetase and class II threonyl-tRNA synthetase. The proteins display characteristics of cytosolic enzymes, yet possess an N-terminal extension relative to their prokaryotic homologs. The proximal part of the N-terminal extension is a mitochondrial-targeting signal. Through transient expression of GFP fusions in tobacco cells, we demonstrated that both genes encode the cytosolic and mitochondrial forms of the enzymes by alternative use of two in-frame initiation codons. A long, mitochondrial form of the enzyme is translated from a first initiation codon at reduced levels because of a poor sequence context and a shorter, cytosolic form is translated from a second in-phase AUG, which is in a better context for translation initiation. Primer extension experiments revealed several transcript ends mapping upstream of the first AUG and between the two AUGs. Distal to the mitochondrial transit peptide both valyl-tRNA synthetase and threonyl tRNA synthetase possess an NH 2 -appended domain compared with their prokaryotic counterparts. This domain's amphiphilic helix is conserved between yeast and A. thaliana valyl-tRNA synthetase, suggesting an important role in translation. Based on the high structural similarities between yeast and A. thaliana valyl-tRNA synthetase, we propose that the acquisition of bifunctionality of valyl-tRNA synthetase predates the divergence of these two organisms.

Translation Initiation from a Naturally Occurring Non-AUG Codon in Saccharomyces cerevisiae

“…These two isoforms cannot substitute for each other because of different localization (15). Similar observations have been made for the Arabdopsis thaliana genes that encode alanyl-tRNA synthetase, threonyl-tRNA synthetase, and valyl-tRNA synthetase (16). It should be addressed that except for some algae, all aminoacyl-tRNA synthetases are encoded by nuclear genes and imported post-translationally into their respective compartments.…”

Translational Efficiency of a Non-AUG Initiation Codon Is Significantly Affected by Its Sequence Context in Yeast

“…Because the isozymes are targeted to different compartments, the two isoforms of valyl-tRNA synthetase, for example, cannot functionally substitute for each other in vivo (7,8). A similar scenario has been observed for the genes encoding the mitochondrial and cytoplasmic forms of Arabidopsis thaliana alanyl-tRNA synthetase (AlaRS), 2 threonyl-tRNA synthetase, and valyl-tRNA synthetase (9). Although two isoforms of AlaRS are generated in a similar manner in Candida albicans, the longer form per se appears dual-targeted and thus can be functional in both compartments (10).…”

Widespread dual targeting of proteins in land plants: When, where, how and why

“…1 Since then the number of proteins shown to be dual targeted in plants is more than 250 ( Table 1). Dual targeting can refer to a protein targeted to any two locations, such as mitochondria and chloroplasts, mitochondria and the nucleus, 2 mitochondria and peroxisomes, 3 mitochondria and cytoplasm, 4 mitochondria and endoplasmic reticulum, 5 mitochondria and golgi, 6 mitochondria and plasma…”