Mediator Acts Upstream of the Transcriptional Activator Gal4
Figure 5
Mediator controls galactose-induced Gal80 degradation.
(A) JD52 cells expressing Srb7 fused to Cub-RUra3 and Nub-HA fused to the indicated derivatives of Skp1 were titrated onto the depicted plates and incubated for 3 d (Skp1dM = Skp1V90A,E129A). Protein-protein interaction between Srb7 and Skp1 is indicated by lack of growth on the uracil-depleted plate and by growth on the FOA-containing plate. (B) JD52 cells whose chromosomal SRB7 gene had been replaced by HIS3 and that expressed wild-type Srb7 under the control of its own promoter from the single-copy vector YCplac22 (22-SRB7; lines 1 and 8) or GST-Srb7 lacking the first 40 amino acids of Srb7 from the multi-copy vector YG1u under the control of the ADH1 promoter (GST-Srb7Δ40; lines 2 to 4 and lines 9 to 11) and BY4741ΔW cells of the indicated genotype (lines 5 to 7) were 10-fold serially diluted, titrated onto the depicted plates, and incubated for 3 d on the glucose plate and for 6 d on the galactose plate containing 0.1 mg/l AA. Cells in lines 3 and 6 over-expressed Gal3 from RS315 under the control of the ACT1 promoter, cells in line 10 over-expressed α2 from RS315 under the control of the ACT1 promoter, cells in lines 4 and 7 lacked GAL80, and cells in line 11 lacked MIG2. (C) JD52 cells whose chromosomal SRB7 gene had been replaced by HIS3 and that expressed wild-type Srb7 under the control of its own promoter from the single-copy vector YCplac22 (22-SRB7) or GST-Srb7 lacking the first 40 amino acids of Srb7 from the multi-copy vector YG1u under the control of the ADH1 promoter (GST-Srb7Δ40) were transformed with the single-copy vector RS316 expressing HA-tagged Gal80 under the control of the ACT1 promoter. Cells were grown in glucose liquid media to OD600 nm = 1 and induced with galactose liquid media for 1 h. Cycloheximide was added at time = 0 and the amount of Gal80 protein remaining in the cells after the indicated number of minutes was determined by Western blot with the help of an anti-HA antibody (upper panels). The membranes were stripped and stained with Coomassie Blue as loading controls (lower panels). (D) The ratio of the amount of HA-Gal80 protein to total protein (Coomassie) in 22-SRB7 cells and GST-Srb7Δ40 cells for each time point in part A was determined with Image J. The ratio of the band intensities before the addition of cycloheximide (time = 0) was set as 1 and the error bars indicate the deviations between duplicates. (E) Cells of part B, lines 1 to 4, were grown in glucose liquid media to OD600 nm = 1 and induced with galactose liquid media for 8 h. Total RNA was isolated and GAL1 mRNA was determined relative to ACT1 mRNA by quantitative real-time PCR. The value determined for 22-SRB7 cells grown with glucose liquid media was set as 1 and the error bars indicate the standard deviations between three replicates. (F) Whole-cell extracts of JD52 cells expressing the indicated Srb7 derivatives in place of endogenous Srb7 and Nub-HA-Skp1 were incubated with glutathione beads, and precipitates were washed five times. Inputs and precipitates were analyzed by Western blot with an anti-HA antibody (upper panel) and with an anti-GST antibody (lower panels).