It has become evident that the process of protein synthesis is performed by many cellular polypeptides acting in concert within the structural confines of protein complexes. In multicellular eukaryotes, one of these assemblies is a multienzyme complex composed of eight proteins that have aminoacyl-tRNA synthetase activities as well as three non-synthetase proteins (p43, p38, and p18) with diverse functions. This study uses electron microscopy and three-dimensional reconstruction to explore the arrangement of proteins and tRNA substrates within this "core" multisynthetase complex. Binding of unfractionated tRNA establishes that these molecules are widely distributed on the exterior of the structure. Binding of gold-labeled tRNALeu places leucyl-tRNA synthetase and the bifunctional glutamyl-/prolyl-tRNA synthetase at the base of this asymmetric "V"-shaped particle. A stable cell line has been produced that incorporates hexahistidine-labeled p43 into the multisynthetase complex. Using a gold-labeled nickel-nitrilotriacetic acid probe, the polypeptides of the p43 dimer have been located along one face of the particle. The results of this and previous studies are combined into an initial three-dimensional working model of the multisynthetase complex. This is the first conceptualization of how the protein constituents and tRNA substrates are arrayed within the structural confines of this multiprotein assembly.It has been known for many years that a "core" complex of aminoacyltRNA synthetases can be isolated from all multicellular eukaryotes (1). Its characteristic composition is three non-synthetase proteins and eight polypeptides having nine aminoacyl-tRNA synthetase activities. With the enzymes abbreviated as the three-letter amino acid name plus RS for the enzyme, the components are ArgRS, AspRS, GluProRS, GlnRS, IleRS, LeuRS, LysRS, MetRS, p43, p38, and p18. Several of these are known dimers, so the total polypeptide count in the multisynthetase complex is at least fifteen. This is a particularly intriguing protein assembly as all of the enzymes catalyze the same reaction, albeit with individual and highly specific substrates. The reaction catalyzed is the covalent attachment of an amino acid to either the 2Ј-or 3Ј-hydroxyl of the 3Ј-terminal adenosine of tRNA. Recent increased interest in these enzymes has been generated by studies indicating that they have roles in a variety of other cellular processes (2-4). Such activities range from cytokine-mediated chemoattraction to priming of reverse transcription of the human immunodeficiency virus, type 1 genome. These enzymes are also viewed as targets in the development of new antimicrobial agents (5). The multisynthetase complex also contains three non-synthetase proteins. The first of these, p18, has been shown to interact with EF-1␥, which may facilitate transfer of aminoacyl-tRNAs to the ribosome (6). Data from yeast two-hybrid screens (7), in vitro binding assays (8), and deletion analysis (9) indicate that p38 functions as a scaffolding protein and appears to be...