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

Chen Ling,Yong-Neng Yao,Yong-Gang Zheng,Hui Wei,Lie Wang,Xiang-Fu Wu,En-Duo Wang

doi:10.1074/jbc.m413511200

Abstract

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.

Highlights

Plex has been discovered consisting of 11 polypeptides and containing activities of nine aaRSs: bifunctional glutamylprolyl, isoleucyl, leucyl, methionyl, glutaminyl, lysyl, arginyl, and aspartyl-tRNA synthetases and three nonsynthetase components, p18, p38, and p43 [3, 4]
The high molecular weight form is a part of the multi-tRNA synthetase complex, whereas the low molecular weight form, which is short of the N-terminal extension, is a free enzyme [16]
We determined the kinetic constants of hcArgRS and ⌬NhcArgRS, the subcellular location of hcLeuRS, ⌬ChcLeuRS, hcArgRS, and ⌬NhcArgRS and proved that the C-terminal extension of hcLeuRS is able to interact with the N terminus of human cytosolic ArgRS

Summary

Interaction of C Terminus of hcLeuRS with hcArgRS

Not shown). hcLeuRS has been suggested to interact with LysRS, ArgRS, and IleRS [13, 15] by chemical cross-linking and yeast two-hybrid methods. Like many other mammalian and human aaRSs, hcLeuRS has a long C-terminal extension, unlike bacterial and yeast enzymes (Fig. 1). His-tagged hcLeuRS and ⌬ChcLeuRS from the cytosol were purified with Ni2ϩ-NTA column chromatography, and their catalytic kinetic constants and thermal stability were determined. We determined the kinetic constants of hcArgRS and ⌬NhcArgRS, the subcellular location of hcLeuRS, ⌬ChcLeuRS, hcArgRS, and ⌬NhcArgRS and proved that the C-terminal extension of hcLeuRS is able to interact with the N terminus of human cytosolic ArgRS (hcArgRS). The role of this specific interaction in the complex assembly is discussed

EXPERIMENTAL PROCEDURES

Forward Reverse Reverse Forward Reverse Forward Forward Reverse

Percentage of units of total

TABLE FOUR

RESULTS

DISCUSSION