Thermus thermophilus possesses an aspartyl-tRNA synthetase (AspRS2) able to aspartylate efficiently tRNA ^Asp and tRNA ^Asn . Aspartate mischarged on tRNA ^Asn then is converted into asparagine by an ω amidase that differs structurally from all known asparagine synthetases. However, aspartate is not misincorporated into proteins because the binding capacity of aminoacylated tRNA ^Asn to elongation factor Tu is only conferred by conversion of aspartate into asparagine. T. thermophilus additionally contains a second aspartyl-tRNA synthetase (AspRS1) able to aspartylate tRNA ^Asp and an asparaginyl-tRNA synthetase able to charge tRNA ^Asn with free asparagine, although the organism does not contain a tRNA-independent asparagine synthetase. In contrast to the duplicated pathway of tRNA asparaginylation, tRNA glutaminylation occurs in the thermophile via the usual pathway by using glutaminyl-tRNA synthetase and free glutamine synthesized by glutamine synthetase that is unique. T. thermophilus is able to ensure tRNA aminoacylation by alternative routes involving either the direct pathway or by conversion of amino acid mischarged on tRNA. These findings shed light on the interrelation between the tRNA-dependent and tRNA-independent pathways of amino acid amidation and on the processes involved in fidelity of the aminoacylation systems.

It is impossible to predict which pathway, direct glutaminylation of tRNA^Gln or tRNA-dependent transamidation of glutamyl-tRNA^Gln, generates mitochondrial glutaminyl-tRNA^Gln for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNA^Gln has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNA^Gln is in fact generated by a transamidation pathway involving a novel type of trimeric tRNA-dependent amidotransferase (AdT). More surprising is the fact that cytosolic glutamyl-tRNA synthetase (_cERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the mitochondrial mischarged glutamyl-tRNA^Gln substrate for the AdT. We show that dual localization of _cERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for _cERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of _cERS to satisfy a higher demand of mitochondrial glutaminyl-tRNA^Gln for mitochondrial protein synthesis. This novel strategy that enables a single protein to be localized in both the cytosol and mitochondria provides a new paradigm for regulation of the dynamic subcellular distribution of proteins between membrane-separated compartments.

Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNA ^Gln is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNA ^Gln is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNA ^Gln , which is the only tRNA ^Gln present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNA ^Gln by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNA ^Gln formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts.