FIG. 7.

Macbecin I reduces Gcn2 levels in vivo. Strain HH1a-pHCA/Hsp82 was transformed with plasmids pYes/Gcn2 (A) and pYes/Gcn2ΔN (B). Gcn2 variants were induced for 24 h by 2% galactose in the presence or absence of macbecin I (50 μM) and immunoprecipitated with an anti-Gcn2 antiserum. (A) Gcn2 was revealed by immunoblotting with an antiserum against Gcn2. Lanes 1 and 2, induction in the presence of galactose (10-fold less material was loaded in lane 2 than in lane 1; lane 3, cells grown with 2% glucose; lane 4, induction with galactose in the presence of 50 μM macbecin I. (B) Gcn2ΔN was either visualized by immunoblot analysis with an antibody against Gcn2 or used for an in vitro kinase assay. Lanes 1 and 2, parent strain; lanes 3 and 4, strain transformed with the plasmid pYes/Gcn2ΔN. (C) The WT strain was transformed with the plasmid pLG/LUC and grown with glucose or galactose in the presence or absence of macbecin I for 24 h. The plotted luciferase activity represents the mean values from two independent experiments.