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

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.
Mesh Terms:
Alanine, Alleles, Anti-Bacterial Agents, Arginine, Binding Sites, DNA-Binding Proteins, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Fungal Proteins, Genes, Dominant, Glutathione Transferase, Models, Biological, Paromomycin, Peptide Elongation Factors, Peptide Initiation Factors, Phenotype, Phosphorylation, Polyribosomes, Prokaryotic Initiation Factor-2, Protein Binding, Protein Biosynthesis, Protein Kinases, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, RNA, Transfer, Recombinant Fusion Proteins, Ribosomes, Saccharomyces cerevisiae Proteins, Yeasts
EMBO J.
Date: Dec. 01, 2000
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