title: Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid-starved cells.
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.
authors: Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, 6 Center Drive, Building 6A, Room B1A-13, Bethesda, MD 20892-2759, USA
authors: E
authors: E
authors: Sattlegger
authors: A G
authors: AG
authors: Hinnebusch
mesh_terms: D000409:Alanine / Q000737:chemistry; D000483:Alleles; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D005799:Genes, Dominant; D005982:Glutathione Transferase / Q000378:metabolism; D008954:Models, Biological; D010303:Paromomycin / Q000494:pharmacology; D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D011132:Polyribosomes / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2; D011485:Protein Binding; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015003:Yeasts / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C501389:GCN1 protein, S cerevisiae; D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D010303:Paromomycin; D012343:RNA, Transfer; D001120:Arginine; D005982:Glutathione Transferase; D011494:Protein Kinases; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D000409:Alanine