Polyribosome Binding by GCN1 Is Required for Full Activation of Eukaryotic Translation Initiation Factor 2α Kinase GCN2 during Amino Acid Starvation

Evelyn Sattlegger,Alan G Hinnebusch

doi:10.1074/jbc.m414566200

Abstract

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.

Highlights

Polyribosome Binding by GCN1 Is Required for Full Activation of Eukaryotic Translation Initiation Factor 2␣ Kinase GCN2 during Amino Acid Starvation*
Mutating both segments simultaneously produced a greater reduction in polyribosome binding by GCN11⁄7GCN20 and a stronger decrease in eukaryotic translation initiation factor 2␣ phosphorylation than did mutating in one segment alone
Basic Residues in Area B of GCN1 Are Required for Optimal Polyribosome Binding in Vivo—We showed previously that ribosome binding by GCN1 was impaired by deletions of areas A, B, or C, which together comprise 2052 amino acids or roughly two-thirds of the protein (Fig. 1A) [5]

Summary

Introduction

Polyribosome Binding by GCN1 Is Required for Full Activation of Eukaryotic Translation Initiation Factor 2␣ Kinase GCN2 during Amino Acid Starvation*. We describe point mutations in two conserved, noncontiguous segments of GCN1 that lead to reduced polyribosome association by GCN11⁄7GCN20 in living cells without reducing GCN1 expression or its interaction with GCN20 Mutating both segments simultaneously produced a greater reduction in polyribosome binding by GCN11⁄7GCN20 and a stronger decrease in eukaryotic translation initiation factor 2␣ phosphorylation than did mutating in one segment alone. Expression of transcription factor ATF4 is stimulated at the translational level in response to eIF2␣ phosphorylation by a mechanism involving upstream open reading frames very similar to that elucidated for GCN4 mRNA in yeast [2, 3]

Methods

Results

Conclusion