Identification and characterization of human mitochondrial tryptophanyl-tRNA synthetase.
A full-length cDNA clone encoding the human mitochondrial tryptophanyl-tRNA synthetase (h(mt)TrpRS) has been identified. The deduced amino acid sequence shows high homology to both the mitochondrial tryptophanyl-tRNA synthetase ((mt)TrpRS) 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(mt)TrpRS catalyzes Trp-dependent exchange of PP(i) in the PP(i)-ATP exchange assay. Expression of h(mt)TrpRS in both human and insect cells leads to high levels of h(mt)TrpRS 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(mt)TrpRS, possibly in the form of a splice variant. However, no mitochondrial localization signal sequence was ever detected and the present identification of a different (mt)TrpRS almost certainly rules out that possibility. The h(mt)TrpRS shows kinetic properties similar to human mitochondrial phenylalanyl-tRNA synthetase (h(mt)PheRS), and h(mt)TrpRS is not induced by interferon-gamma as is hTrpRS.
Jorgensen
R
R
Department of Molecular and Structural Biology, University of Aarhus, 8000 Aarhus C, Denmark.
Søgaard
T M
TM
Rossing
A B
AB
Martensen
P M
PM
Justesen
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014369:Tryptophan-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D014369:Tryptophan-tRNA Ligase