6 research outputs found
A galactose-stable Gal80 deletion derivative inhibits galactose induction of the <i>GAL1</i> gene.
<p>(A) <i>BY4742ΔW</i> cells over-expressing the indicated HA-Gal80 deletion derivatives from <i>RS316</i> under the control of the <i>ACT1</i> promoter were 10-fold serially diluted, titrated onto the indicated plates, and incubated for 3 d (Glucose) and 6 d (Galactose+AA = 1 mg/l Antimycin A), respectively. (B) Cells of part A, lines 1, 2 and 6, were grown in glucose liquid media to OD<sub>600 nm</sub> = 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 containing <i>RS316</i> grown with glucose liquid media was set as 1 and the error bars indicate the standard deviations between three replicates. (C) <i>BY4742ΔW</i> cells expressing HA-tagged wild-type Gal80 or Gal80ΔN12 from <i>RS317</i> under the control of the <i>ACT1</i> promoter were grown in glucose liquid media to OD<sub>600 nm</sub> = 1 and induced with galactose liquid media for 1 h. Cycloheximide was added at time = 0 and the amount of HA-Gal80 and HA-Gal80ΔN12 proteins 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). (D) The ratio of the amount of HA-Gal80 protein to CPY protein (white bars) and of HA-Gal80ΔN12 protein to CPY protein (black bars) in <i>BY4742ΔW</i> cells for each time point in part C 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.</p
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<sub>600 nm</sub> = 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<sub>600 nm</sub> = 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.</p
Galactose induction requires SCF-mediated Gal80 degradation.
<p>(A) <i>JD52</i> cells whose chromosomal <i>SKP1</i> gene had been replaced by <i>HIS3</i> and that expressed Nub-Skp1 (SKP1wt) or Nub-Skp1V90A,E129A (skp1dM) under the control of its own promoter from the single-copy vector <i>PSCNX</i> were transformed with the single-copy vector <i>RS316</i> expressing HA-tagged Gal80 under the control of the <i>ACT1</i> promoter. Cells were grown in glucose liquid media to OD<sub>600 nm</sub> = 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 <i>SKP1wt</i> cells (white bars) and <i>skp1dM</i> 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) <i>JD52</i> 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 <i>YEplac112</i> under the control of their own respective promoters. Cells in line 7 expressed Skp1 from the single-copy vector <i>YCplac22</i> under the control of its own promoter. (D) <i>JD52</i> cells of the indicated genotype were grown in glucose liquid media to OD<sub>600 nm</sub> = 1 and induced with galactose liquid media for 8 h. Cells over-expressed Gal3 from <i>RS314</i> under the control of the <i>ACT1</i> promoter or lacked <i>GAL80</i> as indicated. 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>SKP1wt</i> cells grown with glucose liquid media was set as 1 and the error bars indicate the standard deviations between three replicates.</p
Gal80 is the main target of Mdm30.
<p>(A) HA-tagged Gal80 was expressed in <i>BY4741ΔW</i> cells of the indicated genotype. Cells were grown in glucose liquid media to OD<sub>600 nm</sub> = 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 hours 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 <i>BY4741ΔW</i> cells (white bars), <i>BY4741ΔWΔMDM30</i> cells (black bars), and <i>BY4741ΔWΔGAL11</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>BY4741ΔW</i> cells of the indicated genotype were 10-fold serially diluted, titrated onto the indicated plates, and incubated for 3 d on the glucose plate and for 6 d on the galactose plate. (D) <i>BY4741ΔW</i> cells of the indicated genotype were grown in glucose liquid media to OD<sub>600 nm</sub> = 1 (Glu) and induced with galactose liquid media for 8 h (Gal). 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>BY4741ΔW</i> wild-type cells grown with glucose liquid media was set as 1 and the error bars indicate the standard deviations between three replicates. (E) HA3-tagged Mdm30 and GST or GST-Gal80 were co-expressed in <i>BY4741ΔW</i> cells under the control of the <i>ACT1</i> promoter from the multi-copy vectors <i>RS423</i> and <i>RS424</i>, respectively. Cells were grown with glucose liquid media to OD<sub>600 nm</sub> = 1 (odd lanes) and induced in galactose liquid media for 1 h (even lanes). GST and GST-Gal80 were pulled down from cell extracts with the help of glutathione beads, and Inputs and GST Pulldowns were analyzed by Western blots with the help of an anti-HA antibody (upper panel) and an anti-GST antibody (middle panel). The membrane was stripped and stained with Coomassie in order to compare the amount of protein loaded for Input and GST Pulldown (bottom panel). (F) Histidine-tagged ubiquitinated proteins were precipitated with Ni-beads from extracts of glucose-grown (Glu) and galactose-induced (Gal) <i>SUB288ΔWL</i> (lanes 1, 2, 5, 6, 7) and <i>SUB288ΔWLΔMDM30</i> (lanes 3, 4, 8, 9) cells expressing HA-Gal80 and ubiquitin (lane 5) or histidine-tagged ubiquitin (lanes 1 to 4 and 6 to 9) in place of endogenous ubiquitin. Inputs (lanes 1 to 4) and precipitates (lanes 5 to 9) were analyzed by Western blot with the help of an anti-HA antibody. The ubiquitinated bands appear as doublets, indicating that more than one lysine in Gal80 is subject to ubiquitination.</p
How Mediator orchestrates its own recruitment to the <i>GAL1</i> promoter.
<p>(A) The protein kinase SNF1 senses the absence of glucose and phosphorylates Mig2. Phosphorylated Mig2 is ubiquitinated by the E3 ubiquitin ligases SCF<sup>Das1/Ufo1 </sup><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001290#pbio.1001290-Lim1" target="_blank">[37]</a>. SNF1 also signals Mediator the absence of glucose via the Snf1-Srb11 interaction. Mediator transduces the signal to the E3 ubiquitin ligases SCF<sup>Mdm30/Ufo1/Das1</sup> via the Srb7-Skp1 interaction. SCF<sup>Mdm30/Ufo1/Das1</sup> ubiquitinate Gal80 via the interaction of Gal80 with the F-box proteins Mdm30, Ufo1, and Das1. (B) Poly-ubiquitinated Gal80 and Mig2 are degraded by the 26S proteasome. (C) Gal4 recruits chromatin-remodeling and chromatin-modifying complexes as well as the Holoenzyme of Transcription (consisting of RNA Polymerase II, the General Transcription Factors, and Mediator) to the <i>GAL1</i> promoter genes via the Gal4-Gal11 interaction <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001290#pbio.1001290-Lim2" target="_blank">[43]</a>.</p
Gal80 is stable in galactose-induced <i>H<sub>10</sub>UbD58A</i> cells.
<p>(A) <i>SUB288GAL3ΔWL</i> cells expressing the indicated ubiquitin derivative in place of endogenous ubiquitin were 10-fold serially diluted, titrated onto glucose and onto galactose plates, and incubated at 28°C for 6 d. Cells in lines 3 and 6 over-expressed Gal3 from the <i>ACT1</i> promoter, while cells in lines 4 and 7 lacked <i>GAL80</i>. (B) <i>SUB288GAL3ΔL</i> cells expressing H<sub>10</sub>Ub and H<sub>10</sub>UbD58A in place of endogenous ubiquitin as indicated were grown in glucose liquid media to OD<sub>600 nm</sub> = 1 (Glu) and induced with galactose liquid media for 6 h (Gal). Cells over-expressed Gal3 from the <i>ACT1</i> promoter or lacked <i>GAL80</i> as indicated. Total RNA was isolated and the amount of <i>GAL1</i> mRNA relative to <i>ACT1</i> mRNA was determined with reverse transcription-coupled real-time PCR. The value determined for cells expressing H<sub>10</sub>Ub grown with glucose was set as 1, and the error bars indicate the standard deviations between three replicates. (C) <i>SUB288GAL3ΔL</i> cells expressing H<sub>10</sub>Ub (lanes 1 to 8) or H<sub>10</sub>UbD58A (lanes 9 to 16) in place of endogenous ubiquitin were transformed with the single-copy vector <i>RS316</i> expressing HA-Gal80 under the control of the <i>ACT1</i> promoter. Cells were grown in glucose liquid media to OD<sub>600 nm</sub> = 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 hours was determined by Western blot with the help of an anti-hemagglutinin (HA) antibody (upper panels). The membranes were stripped and reprobed with an anti-carboxypeptidase Y (CPY) antibody (middle panels), followed by a second stripping and staining with Coomassie Blue as loading controls (lower panels). (D) The ratio of the amount of HA-Gal80 protein to CPY protein in <i>H<sub>10</sub>-Ub</i> cells (white bars) and <i>H<sub>10</sub>-UbD58A</i> cells (black bars) for each time point in part C 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.</p