Macromolecular Assemblage of Aminoacyl-tRNA Synthetases: Identification of Protein-Protein Interactions and Characterization of a Core Protein Sophie Quevillon, Jean-Charles Robinson, Eric Berthonneau, Miroslawa Siatecka and Marc Mirande* Laboratoire d'Enzymologie et Biochimie Structurales UPR 9063 du Centre National de la Recherche Scienti®que 91190 Gif-sur-Yvette, France In eukaryotes, from ¯y to human, nine aminoacyl-tRNA synthetases contribute a multienzyme complex of de®ned and conserved struc- tural organization. This ubiquitous multiprotein assemblage comprises a unique bifunctional aminoacyl-tRNA synthetase, glutamyl-prolyl- tRNA synthetase, as well as the monospeci®c isoleucyl, leucyl, gluta- minyl, methionyl, lysyl, arginyl, and aspartyl-tRNA synthetases. Three auxiliary proteins of apparent molecular masses of 18, 38 and 43 kDa are invariably associated with the nine tRNA synthetase components of the complex. As part of an inquiry into the molecular and func- tional organization of this macromolecular assembly, we isolated the cDNA encoding the p38 non-synthetase component and determined its function. The 320 amino acid residue encoded protein has been shown to have no homolog in yeast, bacteria and archaea, according to the examination of the complete genomic sequences available. The p38 protein is a moderately hydrophobic protein, displays a putative leucine-zipper motif, and shares a sequence pattern with protein domains that are involved in protein-protein interactions. We used the yeast two-hybrid system to register protein connections between components of the complex. We performed an exhaustive search of interactive proteins, involving 10 of the 11 components of the com- plex. Twenty-one protein pairs have been unambiguously identi®ed, leading to a global view of the topological arrangement of the sub- units of the multisynthetase complex. In particular, p38 was found to associate with itself to form a dimer, but also with p43, with the class I tRNA synthetases ArgRS and GlnRS, with the class II synthe- tases AspRS and LysRS, and with the bifunctional GluProRS. We generated a series of deletion mutants to localize the regions of p38 mediating the identi®ed interactions. Mapping the interactive domains in p38 showed the speci®c association of p38 with its different pro- tein partners. These ®ndings suggest that p38, for which no homolo- gous protein has been identi®ed to date in organisms devoid of multisynthetase complexes, plays a pivotal role for the assembly of the subunits of the eukaryotic tRNA synthetase complex. # 1999 Academic Press Keywords: aminoacyl-tRNA synthetase; macromolecular assemblage; topology; core protein; two-hybrid system *Corresponding author E-mail address of the corresponding author: marc.mirande@lebs.cnrs-gif.fr Abbreviations used: AspRS, aspartyl-tRNA synthetase; ArgRS, arginyl-tRNA synthetase; GlnRS, glutaminyl-tRNA synthetase; GluProRS, glutamyl-prolyl-tRNA synthetase; IleRS, isoleucyl-tRNA synthetase; LysRS, lysyl-tRNA synthetase; MetRS, methionyl-tRNA synthetase; ValRS, valyl-tRNA synthetase; RS, amino acyl-tRNA synthetase; ORF, open reading frame. Article No. jmbi.1998.2316 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 285, 183±195 0022-2836/99/010183±13 $30.00/0 # 1999 Academic Press