Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid-starved cells

Abstract

GCN2 stimulates GCN4 translation in amino acid‐starved cells by phosphorylating the α‐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 gcn1Δ, 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.

YIH1 Is an Actin-binding Protein That Inhibits Protein Kinase GCN2 and Impairs General Amino Acid Control When Overexpressed

“…Protein Techniques-In vitro and in vivo binding assays using GST and His 6 fusion proteins and co-immunoprecipitation and GST pulldown assays were performed as described previously (15). Proteins were separated by SDS-PAGE using gradient gels (4 -12 or 4 -20%; Invitrogen).…”

“…Genetic evidence further suggests that GCN1 binds near the decoding (A) site of the ribosome. These findings have led to a model in which GCN1 and GCN2 are tethered to the ribosome in a GCN1-GCN20-GCN2 complex, wherein GCN1 facilitates transfer of uncharged tRNA from the A-site to the tRNA-binding domain in GCN2 for kinase activation (15,18). This proposed mechanism has similarities with that demonstrated in Escherichia coli for activation of RelA protein by uncharged tRNA in the A site, which mediates the stringent response to amino acid starvation (19,20).…”

“…The GCN2 NTD is required for kinase function in vivo, and its overexpression impairs complex formation between native GCN1 and GCN2 and produces a dominant Gcn Ϫ (general control noninducible) phenotype (13,14). A C-terminal segment in GCN1 is necessary and sufficient for complex formation with the GCN2 NTD and, when overexpressed in vivo, competes with full-length GCN1 for GCN2 binding and likewise confers a dominant Gcn Ϫ phenotype (14,15). Arginine 2259 in the GCN2-binding domain of GCN1 was shown to be essential for GCN1/GCN2 association in vivo, and the Gcn Ϫ phenotype produced by mutating this residue was suppressed by overexpressing GCN2 (15).…”

“…A C-terminal segment in GCN1 is necessary and sufficient for complex formation with the GCN2 NTD and, when overexpressed in vivo, competes with full-length GCN1 for GCN2 binding and likewise confers a dominant Gcn Ϫ phenotype (14,15). Arginine 2259 in the GCN2-binding domain of GCN1 was shown to be essential for GCN1/GCN2 association in vivo, and the Gcn Ϫ phenotype produced by mutating this residue was suppressed by overexpressing GCN2 (15). Other point mutations in the GCN2-NTD and GCN1 C-terminal region that impair interaction of these segments in the yeast two-hybrid assay also conferred a Gcn Ϫ phenotype and diminished eIF2␣ phosphorylation in starved cells (16).…”

“…GCN1 and GCN2 can interact independently with ribosomes, and these interactions are thought to be crucial for activation of GCN2 in starved cells (8,15,17). Genetic evidence further suggests that GCN1 binds near the decoding (A) site of the ribosome.…”