Macromolecular assemblage of aminoacyl-tRNA synthetases: Identification ofprotein-protein interactions and characterization of a core protein

In eukaryotes, from fly to human, nine aminoacyl-tRNA synthetases contribut e a multienzyme complex of defined and conserved structural organization. T his ubiquitous multiprotein assemblage comprises a unique bifunctional amin oacyl-tRNA synthetase, glutamyl-prolyl-tRNA synthetase, as well as the mono specific isoleucyl, leucyl, glutaminyl, methionyl, lysyl, arginyl, and aspa rtyl-tRNA synthetases. Three auxiliary proteins of apparent molecular masse s of 18, 38 and 43 kDa are invariably associated with the nine tRNA synthet ase components of the complex. As part of an inquiry into the molecular and functional organization of this macromolecular assembly, we isolated the c DNA encoding the p38 non-synthetase component and determined its function. The 320 amino acid residue encoded protein has been shown to have no homolo g in yeast, bacteria and archaea, according to the examination of the compl ete genomic sequences available. The p38 protein is a moderately hydrophobi c protein, displays a putative leucine-zipper motif, and shares a sequence pattern with protein domains that are involved in protein-protein interacti ons. We used the yeast two-hybrid system to register protein connections be tween components of the complex. We performed an exhaustive search of inter active proteins, involving 10 of the 11 components of the complex. Twenty-o ne protein pairs have been unambiguously identified, leading to a global vi ew of the topological arrangement of the subunits of the multisynthetase co mplex. In particular, p38 was found to associate with itself to form a dime r, but also with p43, with the class I tRNA synthetases ArgRS and GLnRS, wi th the class II synthetases AspRS and LysRS, and with the bifunctional GluP roRS. We generated a series of deletion mutants to localize the regions of p38 mediating the identified interactions. Mapping the interactive domains in p38 showed the specific association of p38 with its different protein pa rtners. These findings suggest that p38, for which no homologous protein ha s been identified to date in organisms devoid of multisynthetase complexes, plays a pivotal role for the assembly of the subunits of the eukaryotic tR NA synthetase complex. (C) 1999 Academic Press.