title: Novel insights into the architecture and protein interaction network of yeast eIF3.
abstract: Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs). The largest of these factors, eIF3, forms the scaffold for other initiation factors, promoting their binding to the 40S ribosomal subunit. Biochemical and structural studies on eIF3 need highly pure eIF3. However, natively purified eIF3 comprise complexes containing other proteins such as eIF5. Therefore we have established in vitro reconstitution protocols for Saccharomyces cerevisiae eIF3 using its five recombinantly expressed and purified subunits. This reconstituted eIF3 complex (eIF3(rec)) exhibits the same size and activity as the natively purified eIF3 (eIF3(nat)). The homogeneity and stoichiometry of eIF3(rec) and eIF3(nat) were confirmed by analytical size exclusion chromatography, mass spectrometry, and multi-angle light scattering, demonstrating the presence of one copy of each subunit in the eIF3 complex. The reconstituted and native eIF3 complexes were compared by single-particle electron microscopy showing a high degree of structural conservation. The interaction network between eIF3 proteins was studied by means of limited proteolysis, analytical size exclusion chromatography, in vitro binding assays, and isothermal titration calorimetry, unveiling distinct protein domains and subcomplexes that are critical for the integrity of the protein network in yeast eIF3. Taken together, the data presented here provide a novel procedure to obtain highly pure yeast eIF3, suitable for biochemical and structural analysis, in addition to a detailed picture of the network of protein interactions within this complex.
authors.affiliation: Department of Molecular Structural Biology, Institute of Microbiology and Genetics, Georg-August University Göttingen, 37077 Göttingen, Germany.
authors.forename: Sohail
authors.initials: S
authors.lastname: Khoshnevis
authors.forename: Florian
authors.initials: F
authors.lastname: Hauer
authors.forename: Pohl
authors.initials: P
authors.lastname: Milón
authors.forename: Holger
authors.initials: H
authors.lastname: Stark
authors.forename: Ralf
authors.initials: R
authors.lastname: Ficner
mesh_terms: D001483:Base Sequence; D039621:Eukaryotic Initiation Factor-3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D046912:Multiprotein Complexes; D010442:Peptide Chain Initiation, Translational; D054730:Protein Interaction Domains and Motifs; D060066:Protein Interaction Maps; D021122:Protein Subunits; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054682:Ribosome Subunits, Small, Eukaryotic / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D039621:Eukaryotic Initiation Factor-3; D046912:Multiprotein Complexes; C077915:NIP1 protein, S cerevisiae; D021122:Protein Subunits; C050998:Prt1 protein, S cerevisiae; D012331:RNA, Fungal; D012333:RNA, Messenger; C111889:RPG1 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C102947:TIF34 protein, S cerevisiae