title: Aspartyl-tRNA synthetase from rat: in vitro functional analysis of its assembly into the multisynthetase complex.
abstract: In mammalian cells, nine aminoacyl-tRNA synthetases, including aspartyl-tRNA synthetase, are associated within a multienzyme complex. Rat aspartyl-tRNA synthetase has a N-terminal polypeptide extension of about 40 amino acid residues which can be removed without impairing its catalytic activity. Earlier, in vivo studies showed that enzymes deprive of this N-terminal segment behave in vivo as free entities. We designed an experimental in vitro approach, based on the exchange of the complexed endogenous enzyme by free recombinant species, to assess the contribution of that domain in the association of aspartyl-tRNA synthetase to the complex. A phosphorylation site was introduced at the N-terminus of rat aspartyl-tRNA synthetase. The enzyme served as a reporter protein to evaluate the dissociation constants of native and N-terminal-truncated species towards the complex. Our data show that a moderate but significant drop in affinity is inferred by the removal of the N-terminal domain. The results suggest that this domain binds to another component of the complex, but might primarily serve a targeting function absolutely required in vivo for the assembly within the multienzyme structure.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
authors.forename: F
authors.initials: F
authors.lastname: Agou
authors.forename: M
authors.initials: M
authors.lastname: Mirande
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001226:Aspartate-tRNA Ligase / Q000737:chemistry*; D001483:Base Sequence; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
chemical_list: D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D014364:Tryptophan; D001226:Aspartate-tRNA Ligase