Reconstitution in vitro of the valyl-tRNA synthetase-elongation factor (EF) 1 beta gamma delta complex. Essential roles of the NH2-terminal extension of valyl-tRNA synthetase and of the EF-1 delta subunit in complex formation

• Published in: The Journal of biological chemistry Vol. 269; no. 3; pp. 2086 - 2092
• Main Authors: Bec, G, Kerjan, P, Waller, J P
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 21.01.1994
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Hominidae - metabolism; Humans; Liver - enzymology; Macromolecular Substances; Molecular Sequence Data; Molecular Weight; Pancreatic Elastase; Peptide Elongation Factor 1; Peptide Elongation Factors - isolation & purification; Peptide Elongation Factors - metabolism; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Rabbits; Saccharomyces cerevisiae - enzymology; Sequence Homology, Amino Acid; Valine-tRNA Ligase - chemistry; Valine-tRNA Ligase - isolation & purification; Valine-tRNA Ligase - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)42139-9

Valyl-tRNA synthetase from mammalian cells is isolated exclusively as a complex with elongation factor (EF) 1H (the "heavy" form of eukaryotic EF-1, composed of subunits alpha, beta, gamma, and delta). In a previous study, the 140-kDa valyl-tRNA synthetase subunit dissociated from the purified rabbit liver complex was shown to display hydrophobic properties, unlike the corresponding yeast cytoplasmic enzyme of 125 kDa (Bec, G., and Waller, J.-P. (1989) J. Biol. Chem. 264, 21138-21143). Compared to the sequence of yeast cytoplasmic valyl-tRNA synthetase, that of the human enzyme displays an NH2-terminal extension of approximately 200 amino acid residues that bears strong sequence similarity to the NH2-terminal moiety of EF-1 gamma (Hsieh, S. L., and Campbell, R. D. (1991) Biochem. J. 278, 809-816). We now show that this NH2-terminal extension can be selectively excised by elastase treatment of the isolated rabbit valyl-tRNA synthetase, without impairing catalytic activity. To examine the role of the NH2-terminal extension of mammalian valyl-tRNA synthetase in complex formation and to identify the subunit(s) of EF-1H responsible for binding the enzyme, reconstitution experiments were undertaken. Native or truncated valyl-tRNA synthetases were incubated with the isolated EF-1 subunits beta gamma and delta, either separately or in combination, and the ensuing products were analyzed by chromatography on DEAE-Sepharose FF and Superose 6. The results demonstrate that the NH2-terminal extension of valyl-tRNA synthetase is required for complex formation and that the enzyme-binding site(s) resides on the EF-1 delta subunit. Moreover, although the EF-1 beta gamma binary complex does not bind valyl-tRNA synthetase, it is nevertheless required for assembly of a complex of defined quaternary structure by preventing the formation of high molecular weight aggregates generated in the presence of EF-1 delta alone.