In vitro mutagenesis of the mitochondrial leucyl-tRNA synthetase of S. cerevisiae reveals residues critical for its in vivo activities

The mitochondrial leucyl-tRNA synthetase (mLRS) of Saccharomyces cerevisiae is involved in both mitochondrial protein synthesis and pre-mRNA splicing. We have created mutations in the regions HIGH, GWD and KMSKS, which are involved in ATP-, amino acid-and tRNA-binding respectively, and which have been conserved in the evolution of group I tRNA synthetases. The mutants GRD and NMSKS have no discernible phenotype. The mutants AWD and ARD act as null alleles and lead to the production of 100% cytoplasmic petites. The mutants HIGN, NIGH and KMSNS are unable to grown on glycerol even in the presence of an intronless mitochondrial genome and accumulate petites to a greater extent than the wild-type but less than 40%. Experiments with an imported bI4 maturase indicate that the lesion in these mutations primarily affects the synthetase and not the splicing functions.