The protein kinase Snf1 is required for galactose-induced protein degradation of Gal80.

<p>(A) <i>BY4742ΔW</i> (lanes 1 to 12), <i>BY4742ΔWΔSNF1</i> (lanes 13 to 24), and <i>BY4741ΔWΔSNF4</i> (lanes 25 to 36) cells expressing HA-tagged Gal80 under the control of the <i>ACT1</i> promoter were grown in glucose liquid media to OD_{600 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 reprobed with an anti-CPY antibody (middle panels), followed by a second stripping and staining with Coomassie Blue as loading controls (lower panels). (B) The ratio of the amount of HA-Gal80 protein to CPY protein in <i>BY4742ΔW</i> cells (white bars), <i>BY4742ΔWΔSNF1</i> cells (black bars), and <i>BY4741ΔWΔSNF4</i> cells (grey 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) <i>BY4742ΔW</i> cells (lines 1 to 3) and <i>BY4741ΔW</i> cells (lines 4 to 6) of the indicated genotype were 10-fold serially diluted, dropped onto the depicted plates, and incubated for 6 d at 28°C. The galactose plate contained 1 mg/l Antimycin A. (D) Cells of part C, lines 1 to 3, were grown in glucose liquid media to OD_{600 nm} = 1 and induced with galactose liquid media for the indicated number of hours. Total RNA was isolated and <i>GAL1</i> mRNA was determined relative to <i>ACT1</i> mRNA by quantitative real-time PCR. The value determined for <i>BY4742ΔW</i> cells grown with glucose liquid media was set as 1 and the error bars indicate the standard deviations between three replicates.