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
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Research Support, Non-U.S. Gov't
MeSH terms
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Alanine / chemistry
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Alleles
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Anti-Bacterial Agents / pharmacology
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Arginine / chemistry
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Binding Sites
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DNA-Binding Proteins*
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli / metabolism
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Fungal Proteins / chemistry*
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Fungal Proteins / metabolism*
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Genes, Dominant
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Glutathione Transferase / metabolism
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Models, Biological
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Paromomycin / pharmacology
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Peptide Elongation Factors
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Peptide Initiation Factors / metabolism
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Phenotype
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Phosphorylation
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Polyribosomes / metabolism
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Prokaryotic Initiation Factor-2
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Protein Binding
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Protein Biosynthesis
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Protein Kinases / chemistry*
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Protein Kinases / metabolism*
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Protein Serine-Threonine Kinases
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Protein Structure, Tertiary
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RNA, Transfer / metabolism
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Recombinant Fusion Proteins / metabolism
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Ribosomes / metabolism*
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Saccharomyces cerevisiae Proteins*
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Yeasts / metabolism
Substances
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Anti-Bacterial Agents
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DNA-Binding Proteins
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Fungal Proteins
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GCN1 protein, S cerevisiae
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Peptide Elongation Factors
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Peptide Initiation Factors
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Prokaryotic Initiation Factor-2
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Recombinant Fusion Proteins
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Saccharomyces cerevisiae Proteins
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Paromomycin
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RNA, Transfer
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Arginine
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Glutathione Transferase
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Protein Kinases
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GCN2 protein, S cerevisiae
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Protein Serine-Threonine Kinases
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Alanine