FIG. 8.

Inactivation of Hsp90 abolishes Gcn2 accumulation and activity in vivo. Strain HH1a-G170D was transformed with plasmids pYes/Gcn2ΔN (A) and pLG/LUC (B). Cells grown to mid-logarithmic phase at 30°C in glucose were shifted to galactose and 37°C simultaneously for 6 h to induce the expression of Gcn2ΔN (A) or luciferase (B). (A) Cell extracts from the different treatments were immunoprecipitated with an anti-Gcn2 antiserum, and an immunoblotting experiment with the same antibody (top) and a kinase assay (bottom) were performed. Lanes 1 and 2, shift to galactose. Cells were grown at 30°C (lanes 1 and 3) or at 37°C (lane 2). (B) Cells were grown with galactose (lanes 1, 2, and 3) or glucose (lane 4) at 30°C (lanes 1, 3, and 4) or at 37°C (lane 2) for 6 h. Lane 3, cells treated also with macbecin I (50 μM) for 24 h. Following immunoprecipitation with an anti-luciferase antiserum, luciferase was revealed with the same antibody. IgH, immunoglobulin heavy chain.