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 ((c)ERS) 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 (c)ERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for (c)ERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of (c)ERS 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.