The C-terminal appended domain of human cytosolic leucyl-tRNA synthetase is indispensable in its interaction with arginyl-tRNA synthetase in the multi-tRNA synthetase complex.
Human cytosolic leucyl-tRNA synthetase is one component of a macromolecular aminoacyl-tRNA synthetase complex. This is unlike prokaryotic and lower eukaryotic LeuRSs that exist as free soluble enzymes. There is little known about it, since the purified enzyme has been unavailable. Herein, human cytosolic leucyl-tRNA synthetase was heterologously expressed in a baculovirus system and purified to homogeneity. The molecular mass (135 kDa) of the enzyme is close to the theoretical value derived from its cDNA. The kinetic constants of the enzyme for ATP, leucine, and tRNA(Leu) in the ATP-PP(i) exchange and tRNA leucylation reactions were determined, and the results showed that it is quite active as a free enzyme. Human cytosolic leucyl-tRNA synthetase expressed in human 293 T cells localizes predominantly to the cytosol. Additionally, it is found to have a long C-terminal extension that is absent from bacterial and yeast LeuRSs. A C-terminal 89-amino acid truncated human cytosolic leucyl-tRNA synthetase was constructed and purified, and the catalytic activities, thermal stability, and subcellular location were found to be almost identical to native enzyme. In vivo and in vitro experiments, however, show that the C-terminal extension of human cytosolic leucyl-tRNA synthetase is indispensable for its interaction with the N-terminal of human cytosolic arginyl-tRNA synthetase in the macromolecular complex. Our results also indicate that the two molecules interact with each other only through their appended domains.
Ling
Chen
C
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031.
Yao
Yong-Neng
YN
Zheng
Yong-Gang
YG
Wei
Hui
H
Wang
Lie
L
Wu
Xiang-Fu
XF
Wang
En-Duo
ED
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001126:Arginine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007313:Insecta; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D007935:Leucine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013696:Temperature
D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D010710:Phosphates; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D001126:Arginine-tRNA Ligase; D007935:Leucine-tRNA Ligase; D007930:Leucine