Polyribosome binding by GCN1 is required for full activation of eukaryotic translation initiation factor 2{alpha} kinase GCN2 during amino acid starvation.
The protein kinase GCN2 mediates translational control of gene expression in amino acid-starved cells by phosphorylating eukaryotic translation initiation factor 2alpha. 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 eukaryotic translation initiation factor 2alpha phosphorylation than did mutating 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.
Sattlegger
Evelyn
E
Laboratory of Gene Regulation and Development, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hinnebusch
Alan G
AG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors; D011132:Polyribosomes / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D004268:DNA-Binding Proteins; C501389:GCN1 protein, S cerevisiae; D010445:Peptide Elongation Factors; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases