title: Critical contacts between the eukaryotic initiation factor 2B (eIF2B) catalytic domain and both eIF2beta and -2gamma mediate guanine nucleotide exchange.
abstract: Diverse guanine nucleotide exchange factors (GEFs) regulate the activity of GTP binding proteins. One of the most complicated pairs is eukaryotic initiation factor 2B (eIF2B) and eIF2, which function during protein synthesis initiation in eukaryotes. We have mutated conserved surface residues within the eIF2B GEF domain, located at the eIF2Bepsilon C terminus. Extensive genetic and biochemical characterization established how these residues contribute to GEF activity. We find that the universally conserved residue E569 is critical for activity and that even a conservative E569D substitution is lethal in vivo. Several mutations within residues close to E569 have no discernible effect on growth or GCN4 expression, but an alanine substitution at the adjacent L568 is cold sensitive and deregulates GCN4 activity at 15 degrees C. The mutation of W699, found on a separate surface approximately 40 A from E569, is also lethal. Binding studies show that W699 is critical for interaction with eIF2beta, while L568 and E569 are not. In contrast, all three residues are critical for interaction with eIF2gamma. These data show that multiple contacts between eIF2gamma and eIF2Bepsilon mediate nucleotide exchange.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester M13 9PT, United Kingdom.
authors.forename: Sarah S
authors.initials: SS
authors.lastname: Mohammad-Qureshi
authors.forename: Raphaël
authors.initials: R
authors.lastname: Haddad
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Hemingway
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Richardson
authors.forename: Graham D
authors.initials: GD
authors.lastname: Pavitt
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D050976:Basic-Leucine Zipper Transcription Factors; D002384:Catalysis / Q000187:drug effects; D020134:Catalytic Domain; D003080:Cold Temperature; D004268:DNA-Binding Proteins / Q000378:metabolism; D020717:Eukaryotic Initiation Factor-2B / Q000737:chemistry* / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D020717:Eukaryotic Initiation Factor-2B; C501391:GCN4 protein, S cerevisiae; D006150:Guanine Nucleotides; D050505:Mutant Proteins; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts; D014157:Transcription Factors