The processing of human mitochondrial leucyl‐tRNA synthetase in the insect cells

Abstract

A His-tagged full-length cDNA of human mitochondrial leucyl-tRNA synthetase was expressed in a baculovirus system. The N-terminal sequence of the enzyme isolated from the mitochondria of insect cells was found to be IYSATGKWT-KEYTL, indicating that the mitochondrial targeting signal peptide was cleaved between Ser39 and Ile40 after the enzyme precursor was translocated into mitochondria. The enzyme puri¢ed from mitochondria catalyzed the leucylation of Escherichia coli tRNA Leu 1 (CAG) and Aquifex aeolicus tRNA Leu (GAG) with higher catalytic activity in the leucylation of E. coli tRNA Leu than that previously expressed in E. coli without the N-terminal 21 residues. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Degenerate Connective Polypeptide 1 (CP1) Domain from Human Mitochondrial Leucyl-tRNA Synthetase

“…E. coli tRNA Leu (CAG) (EctRNA Leu ) was isolated from an overexpression strain constructed in our laboratory (34). hmtLeuRS can leucylate EctRNA Leu efficiently (19,25); therefore, EctRNA Leu was used to assay the aminoacylation activity of hmtLeuRS.…”

“…(19). Although hmtLeuRS preserves the key conserved Asp residue in the CP1 domain, the GTG region and Thr-rich region have variations to different extents ( Fig.…”

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

“…The Bac-to-Bac baculovirus expression system, pCDNA3, pEGFP-N2, and recombinant Protein G-agarose were purchased from Invitrogen. The plasmid pFastBac1 of the Bac-to-Bac baculovirus system was modified into pFastBac1H in order to add an Ala 3 -His 6 tag at the C terminus of the recombinant protein (23). Calf liver tRNA and pET-22b(ϩ) were purchased from Novagen (EMD Biosciences, Inc., Germany).…”

“…After 3 days, Tn5 insect cells were harvested and washed twice with phosphate-buffered saline. Fractionation of sub- cellular cytosol/mitochondria was carried out as described (23)(24)(25). Cytosol from the two cultures was further centrifuged at 150,000 ϫ g for 30 min.…”

“…Enzymatic Kinetic Assays-The activity and kinetic constants in the ATP-PP i exchange or leucylation of the tRNA Leu reaction were determined at 37°C as described in a previous report (23). Prior to the determination of substrate kinetic constants, the pH value and magnesium ion concentration were optimized for hcLeuRS activity to 7.6 and 2 mM.…”