minoacyl-tRNA Synthetase Complex Toxoplasma MARS complex displays significant heterogeneity in solution The large upper size limit of the Toxoplasma MARS complex was confirmed by extensive size-exclusion chromatographic separa- tions of FLAG-tagged material which also revealed a distribution of molecular weight sub-species. The size distribution of proteins co-eluting with the scaffold protein, Tg-p43-Myc-FLAG, displayed an almost bimodal distribution of species with a high molecular weight peak centred on 1.0 MDa and a low molecular Toxoplasma Multi-Aminoacyl-tRNA Synthetase Complex around the circumference leading a much weaker ring of averaged density. This is further exemplified by the rotational alignment and averaging of images which resulted in the appearance of weak and diffuse density arranged in spatially separated regions around the periphery of the particle. The core region, on the other hand, produces more homogenous class averages, which display subdivisions consistent with an oligomeric arrangement of proteins, when only this central region is aligned and averaged. Similar alignments and classifications of all the particles in the micrographs confirmed this structural flexibility by showing a strong dominance of the central region in the highly heterogeneous averages. weight peak occurring around 0.3 MDa, with lower concentrations of subunits found in the intervening fractions. The lower molecular weight peak, the position of which was similar to the measurements from recombinant Tg-p43, contained the largest fraction of Tg-p43. Most interestingly, however, all of the detectable YRS protein was found in the high molecular weight fractions and similar results were obtained from separations of Tg-p43DC-HA-FLAG and MRS-HA-FLAG. This preferential association with the high PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19631915 molecular weight complexes was subsequently confirmed by isolations of YRS-HA-FLAG-tagged material which were highly enriched in this species with a conspicuous absence of intermediate and low molecular weight species. The Tg-p43DC-HA-FLAG complex displayed a similar enrichment but with a broader distribution of QRS and ERS proteins occurring towards lower MWs. Discussion Presence and features of Tg-p43 Our preliminary isolations of tagged endogenous Tg-p43 confirmed the bioinformatics analyses of the Toxoplasma genome which predicted the presence of a protein with both an Nterminal, all-alpha helical, glutathione-S-transferase C-terminallike domain and a C-terminal EMAPIIlike tRNA binding domains. Our results however showed that the latter domain is not cleaved to become an extracellular cytokine, as in the case of the human p43 protein. Furthermore, the human p43 protein does not possess a GST C-terminal like domain. Instead, these motifs are contributed by the p38 and p18 accessory proteins. In this sense, the domain structure of Tg-p43 more closely resembles the yeast Arc1p protein which is also a hybrid scaffold protein containing sequence features OPC 8212 site resembling different regions of the human AIMPs. This similarity is reflected at the sequence PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19632179 level where these proteins share 20 and 
25% identity between their matching GST-C and tRNA-binding domains, respectively. In comparison, the divergence is much larger between the Tg-43 GST domain and the human p43 and p38 proteins which only share 17% sequence identity. The remainder of the Tg-p43 sequence shares very little homology with known proteins and was predicted to be largely disordered based on the percenta
