Dissection of the Structural Organization of the Aminoacyl-tRNA Synthetase Complex

Abstract

The spatio-temporal organization of proteins within the cytoplasm of eukaryotic cells rests in part on the assembly of stable and transient multiprotein complexes. Here we examined the assembly of the multiaminoacyl-tRNA synthetase complex (MARS) in human cells. This complex contains nine aminoacyltRNA synthetases and three auxiliary proteins and is a hallmark of metazoan species. Isolation of the complexes has been performed by tandem affinity purification from human cells in culture. To understand the rules of assembly of this particle, expression of the three nonsynthetase components of MARS, p18, p38, and p43, was blocked by stable small interfering RNA silencing. The lack of these components was not lethal for the cells, but cell growth was slightly reduced. The residual complexes that could form in vivo in the absence of the auxiliary proteins were isolated by tandem affinity purification. From the repertoire of the subcomplexes that could be isolated, a comprehensive map of protein-protein interactions mediating complex assembly is deduced. The data are consistent with a structural role of the three nonsynthetase components of MARS, with p38 connecting two subcomplexes that may form in the absence of p38.Multiprotein complexes are molecular machines that are essential for organization of the proteome and for integration of cellular functions. Translation of genetic information involves several supramolecular assemblies, including the ribosome and multiprotein complexes involved in the initiation and elongation steps of the protein biosynthesis process (1). A complex containing the four subunits of elongation factor 1 (EF1A and the guanine nucleotide exchange factors EF1B␣, EF1B␤, and EF1B␥) and ValRS (valyl-tRNA synthetase) was described (2, 3). Several structural models of the ValRS-EF1A-guanine nucleotide exchange factor assembly have been proposed (2,4,5). This complex is believed to play a role in channeling of tRNA Val during translation (6). A multiaminoacyl-tRNA synthetase complex (MARS) 4 of about 1.5 MDa was described more than 20 years ago, but its physical and functional organization remain elusive (7). This complex is ubiquitous from Drosophila to mammals and contains the nine aminoacyl-tRNA synthetases ArgRS, AspRS, GlnRS, GluRS, IleRS, LeuRS, LysRS, MetRS, ProRS, and the three nonsynthetase components p18, p38, and p43 (8). Initial protein-protein interaction maps have been determined by using the yeast two-hybrid system (9, 10) or by in vitro cross-linking (11). A structural working model has been proposed by electron microscopy and three-dimensional reconstruction (12). The p38 component forms the platform for complex assembly (9, 13). A p38 deficiency is lethal in mice (14). Recent studies have indicated that some of the components of this complex lead a double life. They are essential components of translation when associated within MARS but may also play noncanonical functions after dissociation from the complex or following transport in other cellular compartments. LysRS associates wit...

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A Multiple Aminoacyl-tRNA Synthetase Complex That Enhances tRNA-Aminoacylation in African Trypanosomes

“…In eukaryotes, these enzymes are organized in a multiprotein complex called the multiple aminoacyl-tRNA synthetase (MARS) complex (3)(4)(5)(6). A MARS complex that is composed of nine cytoplasmic aaRSs and three accessory proteins, p38, p43, and p18 (also called aminoacyl-tRNA synthetase-interacting multifunctional proteins 1, 2, and 3, respectively), has been characterized in mammalian cells (6,7).…”

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An Archaeal tRNA-Synthetase Complex that Enhances Aminoacylation under Extreme Conditions

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