Identification of a regulatory subcomplex in the guanine nucleotide exchange factor eIF2B that mediates inhibition by phosphorylated eIF2.
Eukaryotic translation initiation factor 2B (eIF2B) is a five-subunit complex that catalyzes guanine nucleotide exchange on eIF2. Phosphorylation of the alpha subunit of eIF2 [creating eIF2(alphaP]) converts eIF2 x GDP from a substrate to an inhibitor of eIF2B. We showed previously that the inhibitory effect of eIF2(alphaP) can be decreased by deletion of the eIF2B alpha subunit (encoded by GCN3) and by point mutations in the beta and delta subunits of eIF2B (encoded by GCD7 and GCD2, respectively). These findings, plus sequence similarities among GCD2, GCD7, and GCN3, led us to propose that these proteins comprise a regulatory domain that interacts with eIF2(alphaP) and mediates the inhibition of eIF2B activity. Supporting this hypothesis, we report here that overexpression of GCD2, GCD7, and GCN3 specifically reduced the inhibitory effect of eIF2(alphaP) on translation initiation in vivo. The excess GCD2, GCD7, and GCN3 were coimmunoprecipitated from cell extracts, providing physical evidence that these three proteins can form a stable subcomplex. Formation of this subcomplex did not compensate for a loss of eIF2B function by mutation and in fact lowered eIF2B activity in strains lacking eIF2(alphaP). These findings indicate that the trimeric subcomplex does not possess guanine nucleotide exchange activity; we propose, instead, that it interacts with eIF2(alphaP) and prevents the latter from inhibiting native eIF2B. Overexpressing only GCD2 and GCD7 also reduced eIF2(alphaP) toxicity, presumably by titrating GCN3 from eIF2B and producing the four-subunit form of eIF2B that is less sensitive to eIF2(alphaP). This interpretation is supported by the fact that overexpressing GCD2 and GCD7 did not reduce eIF2(alphaP) toxicity in a strain lacking GCN3; however, it did suppress the impairment of eIF2B caused by the gcn3c-R104K mutation. An N-terminally truncated GCD2 protein interacted with other eIF2B subunits only when GCD7 and GCN3 were overexpressed, in accordance with the idea that the portion of GCD2 homologous to GCD7 and GCN3 is sufficient for complex formation by these three proteins. Together, our results provide strong evidence that GCN3, GCD7, and the C-terminal half of GCD2 comprise the regulatory domain in eIF2B.
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892-2785, USA.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D020717:Eukaryotic Initiation Factor-2B; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D046911:Macromolecular Substances; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D017931:DNA Primers; D004268:DNA-Binding Proteins; D000939:Epitopes; D015852:Eukaryotic Initiation Factor-2; D020717:Eukaryotic Initiation Factor-2B; D005656:Fungal Proteins; C068999:GCD2 protein, S cerevisiae; C079879:GCD7 protein, S cerevisiae; C501390:GCN3 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D006153:Guanosine Diphosphate; D011494:Protein Kinases