Snf1 Promotes Phosphorylation of the α Subunit of Eukaryotic Translation Initiation Factor 2 by Activating Gcn2 and Inhibiting Phosphatases Glc7 and Sit4

Abstract

Snf1 is the ortholog of mammalian AMP-activated kinase and is responsible for activation of glucose-repressed genes at low glucose levels in budding yeast. We show that Snf1 promotes the formation of phosphorylated α subunit of eukaryotic translation initiation factor 2 (eIF2α-P), a regulator of general and gene-specific translation, by stimulating the function of eIF2α kinase Gcn2 during histidine starvation of glucose-grown cells. Thus, eliminating Snf1 or mutating its activation loop lowers Gcn2 kinase activity, reducing the autophosphorylation of Thr-882 in the Gcn2 activation loop, and decreases eIF2α-P levels in starved cells. Consistently, eliminating Reg1, a negative regulator of Snf1, provokes Snf1-dependent hyperphosphorylation of both Thr-882 and eIF2α. Interestingly, Snf1 also promotes eIF2α phosphorylation in the nonpreferred carbon source galactose, but this occurs by inhibition of protein phosphatase 1α (PP1α; Glc7) and the PP2A-like enzyme Sit4, rather than activation of Gcn2. Both Glc7 and Sit4 physically interact with eIF2α in cell extracts, supporting their direct roles as eIF2α phosphatases. Our results show that Snf1 modulates the level of eIF2α phosphorylation by different mechanisms, depending on the kind of nutrient deprivation existing in cells.

We thank Marian Carlson, Martin Schmidt, Sergei Kuchin, Kim Arndt, George Sprague, and John Cannon for gifts of strains and plasmids and Richard Maraia for kindly providing laboratory space for several experiments. We thank Karen Arndt for discussing results prior to publication.

This work was supported in part by the Intramural Program of the National Institutes of Health.