Structural insights into the eukaryotic General Amino Acid Control pathway

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All living cells must adapt to a variety of different environmental stresses in a rapid and efficient way to survive. Critical to this adaptation is the integrated stress response (ISR), a central signalling network that enables cells to maintain cellular homeostasis or enter into apoptosis. In metazoans, the ISR comprises four different kinases that each phosphorylate serine 51 (Ser51) of the alpha subunit of eukaryotic initiation factor 2 (eIF2). In yeast and mammalian cells, the ancestral Gcn2 (general control nonderepressible-2) kinase modulates the response to nutrient deprivation. In mammals, Gcn2 is important for long-term memory formation, feeding behaviour and immune system regulation, and has also been implicated in various diseases, including neurological disorders (such as Alzheimers), cancer as well as viral infection. The prevailing model for Gcn2 activation during nutrient deprivation is that Gcn2 recognizes and binds ribosomes that have become stalled during translation due to the accumulation of uncharged (deacylated) tRNAs binding to the ribosomal A-site. The activation of Gcn2 strictly requires its co-activator Gcn1, a large protein (2,672 amino acids or 297 kDa in yeast) conserved from yeast to humans. Despite the high conservation and importance of the Gcn pathway for the ISR, as well as decades of research into the Gcn proteins, a structural basis for their mechanism of action on the ribosome has been lacking. In this proposal, we plan to determine cryo-EM structures of native Gcn1-Gcn2-ribosome complexes using strains bearing endogenously tagged proteins, coupled with affinity chromatography. These structures will provide much needed insight into how Gcn1 monitors and sense stalled ribosomes, as well as recruit and activate Gcn2 to elicit the downstream ISR.