Mutations in the αI helix of the YKD constitutively activates Gcn2 <i>in vivo</i>.

<p>(<b>A</b>) Transformants of <i>gcn2</i>Δ strain H1149 containing p722 derivatives with WT <i>GCN2</i>, <i>gcn2-m2</i>, <i>GCN2^c-M788V</i>, or mutations affecting residues in helix αI of the YKD were replica-plated to SC-Ura, SC-Ura plus 30 mM 3-AT, or SD plus 0.5 mM 5-FT and 0.125 mM TRA (5FT/TRA) and incubated for 3 d at 30°C. (<b>B</b>) Cultures of strains from panel A were analyzed for levels of eIF2α-P as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004326#pgen-1004326-g003" target="_blank">Fig. 3B</a>. Western signals on the upper panel (P-eIF2α) were quantified by scanning densitometry of exposed films using ImageJ software, normalized for the corresponding signals in the middle panel (total eIF2α), and the ratios of the two signals (eIF2α-P; eIF2α) are indicated below the lanes. Standard errors are less than 6.5% of the mean values shown. (<b>C</b>) Gcd⁻ phenotypes of the indicated mutants were quantified by measuring <i>HIS4-lacZ</i> expression. Strains from (A) were cultured in nonstarvation conditions as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004326#s4" target="_blank">Materials and Methods</a> and WCEs were prepared and assayed for β-galactosidase activities. Results are the means and S.E.M.s calculated from three transformants, with activity expressed as nanomoles of <i>o</i>-nitrophenyl-β-D-galactopyronoside hydrolyzed per minute per milligram of protein. (<b>D</b>) Locations on the predicted structure of the YKD domain of Gcd⁻ substitutions (from panel A; green) and a subset of Gcn⁻ substitutions in αE and αI (red) determined as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004326#pgen-1004326-g003" target="_blank">Fig. 3C</a>.