The protein kinase GCN2 mediates translational control of gene expression in amino acid-starved cells by phosphorylating eukaryotic translation initiation factor 2alpha (eIF2alpha). In Saccharomyces cerevisiae, activation of GCN2 by uncharged tRNAs in starved cells requires its direct interaction with both the GCN1/GCN20 regulatory complex and ribosomes. GCN1 also interacts with ribosomes in cell extracts, but it was unknown whether this activity is crucial for its ability to stimulate GCN2 function in starved cells. We describe point mutations in two conserved, noncontiguous segments of GCN1 that lead to reduced polyribosome association by GCN1/GCN20 in living cells without reducing GCN1 expression or its interaction with GCN20. Mutating both segments simultaneously produced a greater reduction in polyribosome-binding by GCN1/GCN20, and a stronger decrease in eIF2alpha phosphorylation, than did mutations in one segment alone. These findings provide strong evidence that ribosome-binding by GCN1 is required for its role as a positive regulator of GCN2. A particular mutation in the GCN1 domain related in sequence to translation elongation factor 3 (eEF3) decreased GCN2 activation much more than it reduced ribosome binding by GCN1. Hence, the eEF3-like domain appears to have an effector function in GCN2 activation. This conclusion supports the model that an eEF3-related activity of GCN1 influences occupancy of the ribosomal decoding site by uncharged tRNA in starved cells.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|