title: eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control.
abstract: In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2β that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2β mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2β mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2β acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation.
authors.affiliation: Faculty of Biology Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Jennings
authors.affiliation: Faculty of Biology Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Kershaw
authors.affiliation: Faculty of Biology Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK.
authors.forename: Christopher
authors.initials: C
authors.lastname: White
authors.affiliation: Faculty of Biology Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK.
authors.forename: Danielle
authors.initials: D
authors.lastname: Hoyle
authors.affiliation: Faculty of Biology Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Richardson
authors.affiliation: Faculty of Biology Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Costello
authors.affiliation: Faculty of Biology Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK.
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Donaldson
authors.affiliation: Faculty of Biology Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Faculty of Biology Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK graham.pavitt@manchester.ac.uk.
authors.forename: Graham D
authors.initials: GD
authors.lastname: Pavitt
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D020717:Eukaryotic Initiation Factor-2B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D015003:Yeasts / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D020717:Eukaryotic Initiation Factor-2B; D020730:Guanine Nucleotide Dissociation Inhibitors; D012097:Repressor Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D012343:RNA, Transfer