Abstract
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.
Highlights
ESSENTIAL ROLES OF THE NH2-TERMINAL EXTENSIONOF VALm-tRNA SYNTHETASE AND O F THE elongation factor (EF)-16 SUBUNIT IN COMPLEX FORMATION*
Compared to the synthetase underlying its tight association with EF-lH, we sequence of yeast cytoplasmic valyl-tRNA synthetase, previously compared the propertiesof the 140-kDa monomeric that of the human enzyme displaysan NH,terminal ex- valyl-tRNA synthetase dissociated from the purified rabbit tension of -200 amino acid residues that bears strong liver complex to thoseof the corresponding 125-kDamonomeric sequencesimilarity to the NHz-terminal moietoyf EF-1y
The results demonstrate that the NHz-terminal extension of valyl-tRNAsynthetase is required for complex formation and that the enzyme-bindingsite(s) reyeast cytoplasmic enzymethat does notoccur as a complex with EF-1H [5].Both were shown to display strong affinity for the polyanionic carrier heparin-Ultrogela, property not manifested by the corresponding 110-kDa prokaryotic enzyme
Summary
10 to 300 mM KC1 in bufferA was initiated. Fractions were analyzed for polypeptide composition by SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis of subunits isolated from rabbit liver valyl-tRNA synthetase-EF-1H complex. The reaction mixture contained, per 0.1-ml were removed, adjusted to 1 m~ diisopropyl fluorophosphate, and as- volume, 50 mM Tris-HC1, pH 7.5, 75 mM KC],7.5 mM magnesium acsayed for valyl-tRNA synthetase activity and for polypeptidecomposi- etate, 0.4 mM dithioerythritol, 1mM GTP or GMP-PNP, 25 pgof poly(U), tion by SDS-polyacrylamide gel electrophoresis. After a 70-min incubation a t 25 "C, the reaction was stoppebdy addition addition of 5 pmol of EF-la or of the valyl-tRNA synthetase-EF-1H or of 1 m~ diisopropyl fluorophosphate, and the reactionmixturewas EF-1H complexes. Automated Edman degradiluted to a final volume of 0.12 ml with 25 m~ potassium phosphate dation of the purifiedelastase-modified valyl-tRNA synthetase (300 buffer, pH 7.5, 1mM dithioerythritol, 0.1% Triton X-100, and 50% glyc- pmol)wasperformed by J. DEAE-Sepharose FF chromatography was performed at 4 "C on a column (1.1 x 3.2 cm)
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