Saccharomyces cerevisiae imports the cytosolic pathway for Gln-tRNA synthesis into the mitochondrion

Abstract

Aminoacyl-tRNA (aa-tRNA) formation, an essential process in protein biosynthesis, is generally achieved by direct attachment of an amino acid to tRNA by the aa-tRNA synthetases. An exception is Gln-tRNA synthesis, which in eukaryotes is catalyzed by glutaminyl-tRNA synthetase (GlnRS), while most bacteria, archaea, and chloroplasts employ the transamidation pathway, in which a tRNA-dependent glutamate modification generates Gln-tRNA. Mitochondrial protein synthesis is carried out normally by mitochondrial enzymes and organelle-encoded tRNAs that are different from their cytoplasmic counterparts. Early work suggested that mitochondria use the transamidation pathway for Gln-tRNA formation. We found no biochemical support for this in Saccharomyces cerevisiae mitochondria, but demonstrated the presence of the cytoplasmic GlnRS in the organelle and its involvement in mitochondrial Gln-tRNA synthesis. In addition, we showed in vivo localization of cytoplasmic tRNA^Gln in mitochondria and demonstrated its role in mitochondrial translation. We furthermore reconstituted in vitro cytoplasmic tRNA^Gln import into mitochondria by a novel mechanism. This tRNA import mechanism expands our knowledge of RNA trafficking in the eukaryotic cell. These findings change our view of the evolution of organellar protein synthesis.