No 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. Aminoacyl-tRNA synthetases (aaRSs)3 catalyze specific esterification of an amino acid with the 3Ј-terminal hydroxyl group of its cognate tRNA (1, 2). The reaction can be separated into two steps: 1) the ATP-PP i exchange for amino acid activation and 2) aminoacylation of tRNA (1, 2). Based on structural features, aaRSs have been divided into two classes, I and II; there are three subclasses in each class (2).In higher eukaryotes, from flies to humans, a macromolecular complex 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 structure of the aaRS complex has been analyzed through various approaches. Electron microscopy has revealed that the complex has a "cup" or elongated U-shaped structure (5). Chaotropic salts, detergents (6, 7), and hydrophobic chromatography (8 -10) partially dissociate the components of the complex, suggesting that hydrophobic interactions between the enzymes are responsible for the assembly of the complex (11). Interactions between adjacent aaRSs have been determined with chemical cross-linking (12, 13). Additionally, genetic approaches such as the yeast two-hybrid approach have been applied to detect the interactions between the components of the complex (14). The function of the terminal extensions of the aaRSs in the complex assembly has also been tested (15)...