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PMID:11101534

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Citation

Sattlegger, E and Hinnebusch, AG (2000) Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid-starved cells. EMBO J. 19:6622-33

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.

Links

PubMed PMC305848 Online version:10.1093/emboj/19.23.6622

Keywords

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 Structure, Tertiary; Protein-Serine-Threonine Kinases; RNA, Transfer/metabolism; Recombinant Fusion Proteins/metabolism; Ribosomes/metabolism; Saccharomyces cerevisiae Proteins; Yeasts/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

YEAST:GCN2

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P33892

F

Seeded From UniProt

complete

YEAST:GCN1

enables

GO:0043008: ATP-dependent protein binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:GCN1

enables

GO:0043022: ribosome binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

YEAST:GCN1

part_of

GO:0005844: polysome

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:GCN1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P43535

F

Seeded From UniProt

complete

YEAST:GCN1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P15442

F

Seeded From UniProt

complete

YEAST:GCN1

enables

GO:0019887: protein kinase regulator activity

ECO:0000315: mutant phenotype evidence used in manual assertion

F

  • has_input:(UniProtKB:P15442)

Seeded From UniProt

complete

YEAST:GCN20

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P33892

F

Seeded From UniProt

complete


See also

References

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