Eukaryotic Ribosome GCN2 interaction Hydrogen/Deuterium Exchange Mass Spectrometry

General control nonderepressible 2 (GCN2) phosphorylates eIF2α, regulating translation in response to nutritional stress. Here, we show that mammalian ribosomes are potent GCN2 activators. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) showed GCN2 interacting with domain II of the uL10 P-stalk protein. The P-stalk is a uL10/P12/P22 pentameric complex that is part of the ribosomal GTPase-associated center. Recombinant human P-stalk greatly stimulates GCN2. Both domain II of uL10 and the C-terminal tails of P1 and P2 are necessary for maximal GCN2 activation. On actively translating ribosomes, the C-terminal tails of P1 and P2 are sequestered by elongation factors, suggesting P-stalk availability could link translational stress to GCN2 activation.