Abstract
Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) mapped GCN2-ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.
Highlights
Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments
Mutation of two residues in the HisRS-like domain that are analogous to residues important for tRNA binding in tRNA synthetases generated the General control nonderepressible 2 (GCN2) mutant known as m2 [13]
We show that human GCN2 interacts directly with ribosomes and by using a combination of hydrogen/deuterium exchange–mass spectrometry (HDX-MS) and truncation analysis, we have identified domain II of the ribosomal P-stalk protein uL10 [previously known as P0 [36]] as the principal GCN2 binding site
Summary
Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. The kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). General control nonderepressible 2 (GCN2) is one of four related kinases that respond to these cellular stresses by phosphorylating the translation initiation factor eIF2α [2]. Cognate deacylated tRNA binds in the ribosome acceptor site (A site), recruits RelA to the ribosome, and stimulates RelA-mediated (p)ppGpp synthesis This ribosome-mediated nutrient-sensing mechanism led to early efforts to examine whether GCN2 could be analogously regulated. Mutation of two residues in the HisRS-like domain that are analogous to residues important for tRNA binding in tRNA synthetases generated the GCN2 mutant known as m2 [13] This mutant greatly decreases binding to deacylated tRNA, decreases activity in vitro, and completely abolishes GCN2 activation in cells. The C-terminal tails of P1 and P2 are sequestered by elongation factors, suggesting P-stalk availability could link translational stress to GCN2 activation
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