Mutations in the GCD7 subunit of yeast guanine nucleotide exchange factor eIF-2B overcome the inhibitory effects of phosphorylated eIF-2 on translation initiation

Mol Cell Biol. 1994 May;14(5):3208-22. doi: 10.1128/mcb.14.5.3208-3222.1994.

Abstract

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.

Publication types

  • Comparative Study

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • DNA Primers
  • Eukaryotic Initiation Factor-2 / metabolism*
  • Eukaryotic Initiation Factor-2B*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Gene Deletion
  • Gene Expression Regulation, Fungal*
  • Genes, Fungal*
  • Genes, Suppressor*
  • Genotype
  • Guanine Nucleotide Exchange Factors
  • Macromolecular Substances
  • Mammals
  • Molecular Sequence Data
  • Mutagenesis
  • Peptide Chain Initiation, Translational*
  • Phosphorylation
  • Plasmids
  • Polymerase Chain Reaction
  • Protein Biosynthesis
  • Proteins / genetics
  • Proteins / metabolism*
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins*
  • Sequence Homology, Amino Acid
  • Suppression, Genetic

Substances

  • DNA Primers
  • Eukaryotic Initiation Factor-2
  • Eukaryotic Initiation Factor-2B
  • Fungal Proteins
  • GCD7 protein, S cerevisiae
  • Guanine Nucleotide Exchange Factors
  • Macromolecular Substances
  • Proteins
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins