title: Identification of intersubunit domain interactions within eukaryotic initiation factor (eIF) 2B, the nucleotide exchange factor for translation initiation.
abstract: In eukaryotic translation initiation, eIF2B is the guanine nucleotide exchange factor (GEF) required for reactivation of the G protein eIF2 between rounds of protein synthesis initiation. eIF2B is unusually complex with five subunits (α-ε) necessary for GEF activity and its control by phosphorylation of eIF2α. In addition, inherited mutations in eIF2B cause a fatal leukoencephalopathy. Here we describe experiments examining domains of eIF2Bγ and ε that both share sequence and predicted tertiary structure similarity with a family of phospho-hexose sugar nucleotide pyrophosphorylases. Firstly, using a genetic approach, we find no evidence to support a significant role for a potential nucleotide-binding region within the pyrophosphorylase-like domain (PLD) of eIF2Bε for nucleotide exchange. These findings are at odds with one mechanism for nucleotide exchange proposed previously. By using a series of constructs and a co-expression and precipitation strategy, we find that the eIF2Bε and -γ PLDs and a shared second domain predicted to form a left-handed β helix are all critical for interprotein interactions between eIF2B subunits necessary for eIF2B complex formation. We have identified extensive interactions between the PLDs and left-handed β helix domains that form the eIF2Bγε subcomplex and propose a model for domain interactions between eIF2B subunits.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Reid
authors.forename: Sarah S
authors.initials: SS
authors.lastname: Mohammad-Qureshi
authors.forename: Graham D
authors.initials: GD
authors.lastname: Pavitt
mesh_terms: D020134:Catalytic Domain / Q000502:physiology*; D020717:Eukaryotic Initiation Factor-2B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational / Q000502:physiology*; D010449:Peptide Mapping / Q000379:methods; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020717:Eukaryotic Initiation Factor-2B; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins