|dc.description.abstract||Previous studies showed that cytoplasmic methionyl-tRNA synthetase (MetRS) and glutamyl-tRNA synthetase (GluRSc) form a ternary complex with an aaRS cofactor, Arc1p, thereby enhancing their aminoacylation activities. In addition, Arc1p also regulates the subcellular distribution of these two associated enzymes. Upon dissociation from the ternary complex, GluRSc and MetRS are targeted into the mitochondria and nucleus for functioning. The structure of Arc1p can be divided into three domains, N, M, and C domains. The N domain interacts with GluRSc and MetRS, while the M plus C domains form a non-specific tRNA-binding domain. A recent report demonstrated that a SSKD motif in the N domain of Arc1p can be biotinylated through post-translational modification in vivo. However, the biological significance of this modification remained unclear. We show herein that Arc1p was biotinylated (15%) under normal growth conditions. However, biotinylation had little effect on its ability to interact with tRNA or GluRSc/MetRS. In contrast, Arc1p was almost biotin free at a high temperature. Non-biotinylated Arc1p was more heat-tolerant and more efficiently promoted the aminoacylation activity of GluRSc. Perhaps the structure and function of Arc1p can be modulated via biotinylation in response to temperature changes.
WHEP domains exist in certain eukaryotic aminoacyl-tRNA synthetases (aaRSs) and play roles in tRNA or protein binding. We show herein that cytoplasmic and mitochondrial forms of Caenorhabditis elegans glycyl-tRNA synthetase (CeGlyRS) are encoded by the same gene (CeGRS1) through alternative initiation of translation. As a result, the cytoplasmic form possessed an N-terminal WHEP domain, while its mitochondrial counterpart possessed an extra N-terminal sequence (aa 1~64) consisting of a mitochondrial targeting signal (MTS； aa 1~20) and an appended domain (aa 21~64). Cross-species rescue assays showed that this dual-functional gene effectively rescued the cytoplasmic and mitochondrial defects of a yeast GRS1 (which encodes GlyRS) knockout strain. While both forms of CeGlyRS efficiently charged the cytoplasmic tRNAsGly of C. elegans, the mitochondrial form was much more efficient than its cytoplasmic counterpart in charging the mitochondrial tRNAGly isoacceptor, which carries a defective TψC hairpin. Despite the WHEP domain per se lacking tRNA-binding activity, deletion of this domain reduced the enzyme’s catalytic efficiency. Most interestingly, the deletion mutant possessed a higher thermal stability and a somewhat lower structural flexibility. Our study suggests the WHEP domain may act in cis to regulate the enzyme’s dynamic structure and activity.||en_US|