Purification and Analysis of eIF2α Phosphorylation by Stress-Activated Protein Kinase Gcn2 from S. cerevisiae

Gcn2 is the sole eIF2α kinase in budding yeast, responsible for inhibiting general translation while inducing translation of transcriptional activator Gcn4, a master regulator of amino acid biosynthesis, in nutrient-starved cells. Gcn2 is activated by interactions between multiple regulatory domains that overcome the inherent latency of its protein kinase domain, including a pseudokinase domain, one related to histidyl-tRNA synthetase, a ribosome-binding and dimerization domain, and a region that binds the trans-acting activators Gcn1/Gcn20, which respond to deacylated tRNAs engendered by amino acid starvation or other impediments to translation elongation that lead to ribosome stalling and collisions. Here, we describe methods for purifying Gcn2 from yeast cells and assaying its protein kinase activity against a recombinant segment of eIF2α.