Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid-starved cells

EMBO J. 2000 Dec 1;19(23):6622-33. doi: 10.1093/emboj/19.23.6622.

Abstract

GCN2 stimulates GCN4 translation in amino acid-starved cells by phosphorylating the alpha-subunit of translation initiation factor 2. GCN2 function in vivo requires the GCN1/GCN20 complex, which binds to the N-terminal domain of GCN2. A C-terminal segment of GCN1 (residues 2052-2428) was found to be necessary and sufficient for binding GCN2 in vivo and in vitro. Overexpression of this fragment in wild-type cells impaired association of GCN2 with native GCN1 and had a dominant Gcn(-) phenotype, dependent on Arg2259 in the GCN1 fragment. Substitution of Arg2259 with Ala in full-length GCN1 abolished complex formation with native GCN2 and destroyed GCN1 regulatory function. Consistently, the Gcn(-) phenotype of gcn1-R2259A, but not that of gcn1Delta, was suppressed by overexpressing GCN2. These findings prove that GCN2 binding to the C-terminal domain of GCN1, dependent on Arg2259, is required for high level GCN2 function in vivo. GCN1 expression conferred sensitivity to paromomycin in a manner dependent on its ribosome binding domain, supporting the idea that GCN1 binds near the ribosomal acceptor site to promote GCN2 activation by uncharged tRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / chemistry
  • Alleles
  • Anti-Bacterial Agents / pharmacology
  • Arginine / chemistry
  • Binding Sites
  • DNA-Binding Proteins*
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism
  • Fungal Proteins / chemistry*
  • Fungal Proteins / metabolism*
  • Genes, Dominant
  • Glutathione Transferase / metabolism
  • Models, Biological
  • Paromomycin / pharmacology
  • Peptide Elongation Factors
  • Peptide Initiation Factors / metabolism
  • Phenotype
  • Phosphorylation
  • Polyribosomes / metabolism
  • Prokaryotic Initiation Factor-2
  • Protein Binding
  • Protein Biosynthesis
  • Protein Kinases / chemistry*
  • Protein Kinases / metabolism*
  • Protein Serine-Threonine Kinases
  • Protein Structure, Tertiary
  • RNA, Transfer / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Ribosomes / metabolism*
  • Saccharomyces cerevisiae Proteins*
  • Yeasts / metabolism

Substances

  • Anti-Bacterial Agents
  • DNA-Binding Proteins
  • Fungal Proteins
  • GCN1 protein, S cerevisiae
  • Peptide Elongation Factors
  • Peptide Initiation Factors
  • Prokaryotic Initiation Factor-2
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Paromomycin
  • RNA, Transfer
  • Arginine
  • Glutathione Transferase
  • Protein Kinases
  • GCN2 protein, S cerevisiae
  • Protein Serine-Threonine Kinases
  • Alanine