Mediator Acts Upstream of the Transcriptional Activator Gal4
Figure 2
Galactose induction requires SCF-mediated Gal80 degradation.
(A) JD52 cells whose chromosomal SKP1 gene had been replaced by HIS3 and that expressed Nub-Skp1 (SKP1wt) or Nub-Skp1V90A,E129A (skp1dM) under the control of its own promoter from the single-copy vector PSCNX were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). (B) The ratio of the amount of HA-Gal80 protein to total protein (Coomassie) in SKP1wt cells (white bars) and skp1dM cells (black bars) 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. (C) JD52 cells expressing Nub-Skp1wt (line 1) and Nub-Skp1dM (lines 2 to 7) in place of endogenous Skp1 were 10-fold serially diluted, titrated onto the indicated plates, and incubated for 3 d on glucose plates and for 6 d on galactose plates containing 0.1 mg/l of the respiration inhibitor Antimycin A (AA). Cells over-expressed the indicated proteins from the multi-copy vector YEplac112 under the control of their own respective promoters. Cells in line 7 expressed Skp1 from the single-copy vector YCplac22 under the control of its own promoter. (D) JD52 cells of the indicated genotype were grown in glucose liquid media to OD600 nm = 1 and induced with galactose liquid media for 8 h. Cells over-expressed Gal3 from RS314 under the control of the ACT1 promoter or lacked GAL80 as indicated. Total RNA was isolated and GAL1 mRNA was determined relative to ACT1 mRNA by quantitative real-time PCR. The value determined for SKP1wt cells grown with glucose liquid media was set as 1 and the error bars indicate the standard deviations between three replicates.