Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3

Abstract

The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome.hepatitis C virus internal ribosome entry site ͉ subunit organization model3 ͉ top-down analysis of macromolecular complexes ͉ translation regulation ͉ in-solution disruption S ince its identification in the 1970s (1-3), the translation initiation factor eIF3 has been subjected to intense scrutiny. Despite considerable interest, knowledge of many aspects of its structure and function remain elusive because of its overall structural complexity and the lack of facile genetic approaches. It is established, however, that eIF3 is involved in both ribosome biogenesis and protein synthesis in eukaryotes (4). Concerted binding of initiation factors is required to initiate protein synthesis and recruit transfer and messenger RNAs to the 40S subunit before assembly of active ribosomes (5). eIF3 binding may take place initially during this process, together with eIF1 and eIF1A to the 40S, followed by binding of the Met-tRNA i -eIF2-GTP complex. Then mRNA binding, scanning, and AUG recognition occur, enabling the 60S subunit to join to form elongation-competent 80S ribosomes (6). An alternative pathway of initiating protein synthesis, often used by viruses, involves a structured sequence in the 5Ј untranslated region of mRNA known as the internal ribosome entry site (IRES). These sequences promote translation initiation without requiring the full complement of eukaryotic initiation factors (5-7). The hepatitis C virus (HCV) IRES is recognized specifically by the small ribosomal subunit and eIF3 before viral translation initiation, forming stable complexes ...

Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: The effect of ligand binding on conformational stability

“…In many cases, binding affinities determined using ESI-MS show good agreement with values obtained using other means, potentially validating MS methods for use in early-stage screening in the drug discovery process [13,14]. ESI-MS is not only suitable for the analysis of protein-ligand interactions, but has widespread utility in studying large protein-protein complexes [15,16], and has been applied to the preservation and detection of very large biomolecular assemblies, including the ribosome [17,18] and the tobacco mosaic virus (Ͼ40 MDa) [19].A key underlying issue in ESI-MS studies of ligand binding is to what extent these findings can be related to solution behavior. Given the great importance of water to the protein fold [20], it seems clear that desolvation should result in catastrophic loss of native structure, with accompanying consequences for ligand binding.…”

Stepwise evolution of protein native structure with electrospray into the gas phase, 10 −12 to 10 2 s

“…1 C and D) on the protein surface can transiently stabilize the native fold of large, compact protein complexes. Critical conformer stabilization during ESI can also be provided by noncovalent binding of intermolecular additives (56)(57)(58). For a native structure with substantial stabilization by hydrophobic bonding in solution, ESI denaturing can form an unstable transient structure (Fig.…”

“…However, ESI solution additives such as imidazole can preserve the complex, possibly because of enhanced cooling from imidazole evaporation that delays the dissociation. In a recent innovative approach to a similar problem, Robinson and colleagues (57,58) show that detergent micelles can protect membrane protein complexes from dissociating during ESI. The micelles could prevent or retard all of the steps in Fig.…”

In the land of the rising sun with the COP9 signalosome and related Zomes

“…Nedd8 activates CRLs by triggering a conformational change of the carboxy-terminal part of the cullin that frees Rbx1, allowing it to adopt a variable conformation that brings the activated E2 enzyme in close proximity to the substrate (Fig 2; Duda et Sharon et al (2006), Zhou et al (2008) and Sharon et al (2009). CRL, cullin-RING ligase; CSN, COP9 signalosome; eIF3, eukaryotic initiation factor 3; MPN, Mpr1-Pad1-amino terminal; N8, Nedd8; PCI, proteasome, COP9, eukaryotic initiation factor 3; Rpn, proteasome regulatory particle, non-ATPase-like; Ub, ubiquitin.…”