Accumulation of ASA lowers Gcn4 abundance and stability.

<p>(<b>A</b>) WT (BY4741) and <i>hom6Δ</i> (YR001) strains transformed with vector (pRS313) and <i>hom6Δ hom3Δ</i> strain YR003 transformed with vector or lc <i>HOM3^fbr</i> plasmid pYPR030 (indicated as <i>hom6Δ hom3Δ</i> and <i>hom6Δ HOM3^fbr</i> respectively) were cultured in SC-His/Ile/Val for at least two doublings to A₆₀₀ = 0.4–0.6 and subjected to SM treatment (0.5 µg/ml) for the indicated times. WCEs were extracted under denaturing conditions and subjected to Western analysis with α-Gcn4 and α-Gcd6 antibodies. (<b>B</b>) Western signals from (A) were quantified, Gcn4 signals were normalized to Gcd6 signals, and the Gcn4/Gcd6 ratios were expressed relative to that measured for the WT strain without SM treatment. Means and S.E.Ms were calculated from three independent transformants of each strain. (<b>C</b>) Yeast strains described in Fig. 3D transformed with p180 were analyzed for <i>GCN4-lacZ</i> expression as in Fig. 1C. Means and S.E.Ms were calculated from three independent transformants of each strain. (<b>D</b>) p180 transformants of strains from (C) and <i>gcn2Δ</i> strain H2931 were analyzed for <i>GCN4-lacZ</i> expression after SM treatment for the indicated times. Means and S.E.Ms were calculated from three independent transformants of each strain. (<b>E</b>) The WT and <i>hom6Δ HOM3^fbr</i> strains from (A) were cultured in SC-His/Ile/Val, collected and resuspended in SC-His/Ile/Val/Met containing SM (1 µg/ml) for 15 min, and labeled with [³⁵S] methionine/cysteine for 15 min. Cells were collected and resuspended in SC-His/Ile/Val containing Met and Cys (both at 10 mM) and aliquots were removed after the indicated times of chase. Aliquots of WCEs containing equal cpm were immunoprecipitated with α-Gcn4 antibodies, immunocomplexes were collected with protein A-agarose beads and resolved by SDS-PAGE, and the [³⁵S]-Gcn4 signals were quantified by phosphorimaging. (<b>F</b>) Gcn4 signals from (E) are plotted relative to the value at 0 min of chase (100%). Means and S.D.s were calculated from two independent experiments.