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

Dec 2, 2000The EMBO journal

Separate parts of GCN1 needed to bind GCN2 and ribosomes for activating GCN2 when cells lack amino acids

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Abstract

GCN2 binding to the C-terminal domain of GCN1, dependent on Arg2259, is required for high level GCN2 function in vivo.

GCN2 stimulates translation of GCN4 in cells lacking amino acids by modifying a translation initiation factor.
The GCN1/GCN20 complex is necessary for GCN2 function in living organisms.
A specific segment of GCN1 is both necessary and sufficient for binding to GCN2.
Overexpression of this GCN1 segment disrupts GCN2's association with native GCN1, leading to a dominant negative effect.
Changes to a specific amino acid in GCN1 eliminate its ability to form a complex with GCN2 and regulate its function.
GCN1's role in enhancing sensitivity to paromomycin is linked to its interaction with ribosomes.

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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.

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

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