PMID:22888004

Citation	Visweswaraiah, J, Lee, SJ, Hinnebusch, AG and Sattlegger, E (2012) Overexpression of eukaryotic translation elongation factor 3 impairs Gcn2 protein activation. J. Biol. Chem. 287:37757-68
Abstract	In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn(-) phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn(-) phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn(-) phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes.
Links	PubMed PMC3488051 Online version:10.1074/jbc.M112.368266
Keywords	Binding Sites/genetics; Enzyme Activation; Eukaryotic Initiation Factor-2/metabolism; Gene Expression Regulation, Fungal; Immunoblotting; Mutation; Peptide Elongation Factors/genetics; Peptide Elongation Factors/metabolism; Phenotype; Phosphorylation; Protein Biosynthesis; Protein-Serine-Threonine Kinases/genetics; Protein-Serine-Threonine Kinases/metabolism; Ribosomes/genetics; Ribosomes/metabolism; Saccharomyces cerevisiae/genetics; Saccharomyces cerevisiae/growth & development; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae Proteins/genetics; Saccharomyces cerevisiae Proteins/metabolism; Substrate Specificity
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
YEAST:EF3A	involved_in	GO:0006469: negative regulation of protein kinase activity	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
YEAST:EF3A	part_of	GO:0042788: polysomal ribosome	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete
YEAST:EF3A	involved_in	GO:0001933: negative regulation of protein phosphorylation	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
YEAST:GCN2	part_of	GO:0042788: polysomal ribosome	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete

References

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