Identification and Characterization of Human Mitochondrial Tryptophanyl-tRNA Synthetase*

A full-length cDNA clone encoding the human mitochondrial tryptophanyl-tRNA synthetase (h_mtTrpRS) has been identified. The deduced amino acid sequence shows high homology to both the mitochondrial tryptophanyl-tRNA synthetase (_mtTrpRS) from Saccharomyces cerevisiae and to different eubacterial forms of tryptophanyl-tRNA synthetase (TrpRS). Using the baculovirus expression system, we have expressed and purified the protein with a carboxyl-terminal histidine tag. The purified His-tagged h_mtTrpRS catalyzes Trp-dependent exchange of PP_i in the PP_i-ATP exchange assay. Expression of h_mtTrpRS in both human and insect cells leads to high levels of h_mtTrpRS localizing to the mitochondria, and in insect cells the first 18 amino acids constitute the mitochondrial localization signal sequence. Until now the human cytoplasmic tryptophanyl-tRNA synthetase (hTrpRS) was thought to function as the h_mtTrpRS, possibly in the form of a splice variant. However, no mitochondrial localization signal sequence was ever detected and the present identification of a different_mtTrpRS almost certainly rules out that possibility. The h_mtTrpRS shows kinetic properties similar to human mitochondrial phenylalanyl-tRNA synthetase (h_mtPheRS), and h_mtTrpRS is not induced by interferon-γ as is hTrpRS.