Mutations in the GCD7 subunit of yeast guanine nucleotide exchange factor eIF-2B overcome the inhibitory effects of phosphorylated eIF-2 on translation initiation.
Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) impairs translation initiation by inhibiting the guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In Saccharomyces cerevisiae, phosphorylation of eIF-2 alpha by the protein kinase GCN2 specifically stimulates translation of GCN4 mRNA in addition to reducing general protein synthesis. We isolated mutations in several unlinked genes that suppress the growth-inhibitory effect of eIF-2 alpha phosphorylation catalyzed by mutationally activated forms of GCN2. These suppressor mutations, affecting eIF-2 alpha and the essential subunits of eIF-2B encoded by GCD7 and GCD2, do not reduce the level of eIF-2 alpha phosphorylation in cells expressing the activated GCN2c kinase. Four GCD7 suppressors were shown to reduce the derepression of GCN4 translation in cells containing wild-type GCN2 under starvation conditions or in GCN2c strains. A fifth GCD7 allele, constructed in vitro by combining two of the GCD7 suppressors mutations, completely impaired the derepression of GCN4 translation, a phenotype characteristic of deletions in GCN1, GCN2, or GCN3. This double GCD7 mutation also completely suppressed the lethal effect of expressing the mammalian eIF-2 alpha kinase dsRNA-PK in yeast cells, showing that the translational machinery had been rendered completely insensitive to phosphorylated eIF-2. None of the GCD7 mutations had any detrimental effect on cell growth under nonstarvation conditions, suggesting that recycling of eIF-2 occurs efficiently in the suppressor strains. We propose that GCD7 and GCD2 play important roles in the regulatory interaction between eIF-2 and eIF-2B and that the suppressor mutations we isolated in these genes decrease the susceptibility of eIF-2B to the inhibitory effects of phosphorylated eIF-2 without impairing the essential catalytic function of eIF-2B in translation initiation.
Vazquez de Aldana
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CR
Section on Molecular Genetics of Lower Eukaryotes, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Hinnebusch
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AG
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D020717:Eukaryotic Initiation Factor-2B; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D016153:Genes, Suppressor; D005838:Genotype; D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; D008322:Mammals; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010442:Peptide Chain Initiation, Translational; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic
D017931:DNA Primers; D015852:Eukaryotic Initiation Factor-2; D020717:Eukaryotic Initiation Factor-2B; D005656:Fungal Proteins; C079879:GCD7 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; D011506:Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins