Multiple signals regulate GAL transcription in yeast - PubMed (original) (raw)

Multiple signals regulate GAL transcription in yeast

J R Rohde et al. Mol Cell Biol. 2000 Jun.

Abstract

Gal4p activates transcription of the Saccharomyces GAL genes in response to galactose and is phosphorylated during interaction with the RNA polymerase II (Pol II) holoenzyme. One phosphorylation at S699 is necessary for full GAL induction and is mediated by Srb10p/CDK8 of the RNA Pol II holoenzyme mediator subcomplex. Gal4p S699 phosphorylation is necessary for sensitive response to inducer, and its requirement for GAL induction can be abrogated by high concentrations of galactose in strains expressing wild-type GAL2 and GAL3. Gal4p S699 phosphorylation occurs independently of Gal3p and is responsible for the long-term adaptation response observed in gal3 yeast. SRB10 and GAL3 are shown to represent parallel mechanisms for GAL gene induction. These results demonstrate that Gal4p activity is controlled by two independent signals: one that acts through Gal3p-galactose and a second that is mediated by the holoenzyme-associated cyclin-dependent kinase Srb10p. Since Srb10p is regulated independently of galactose, our results suggest a function for CDK8 in coordinating responses to specific inducers with the environment through the phosphorylation of gene-specific activators.

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Figures

FIG. 1

FIG. 1

Phosphorylation of Gal4p S699 is required for efficient GAL gene induction. (A) Schematic representation of Gal4p and the location of identified and predicted phosphorylations. Abbreviations: DNA, DNA-binding domain; AR1, activating region 1; AR2, activating region 2; GRD, glucose response domain. (B) Yeast strain YT6G80 bearing a control plasmid (⧫) or expressing WT GAL4 (■), GAL4 S699A (▴), GAL4 S699E (●), or GAL4 bearing alanine substitutions of serines 691, 696, and 837 (□) from plasmid YCpG4 were induced with 2% galactose for the indicated times. GAL expression was measured by assaying β-galactosidase activity produced by the GAL1-lacZ reporter gene. (C) Yeast strain YJR10::131 bearing a vector control (⧫), expressing WT GAL4 (■ and □), or GAL4 S699A (▴ and ▵) were induced with either 2% galactose (closed symbols) or 0.02% galactose (open symbols), and GAL1-lacZ activity was measured at the indicated times.

FIG. 2

FIG. 2

Weak S288C GAL2 and GAL3 alleles contribute to dependence on the Gal4p S699 phosphorylation. (A) Yeast strain ISY45 (gal2 W303-1A) bearing a vector control (⧫) and expressing WT GAL4 (■) or GAL4 S699A (▴) was induced with 2% galactose, and GAL1-lacZ activity was measured at the indicated times. (B) Diploid strains produced by crosses between YT6G80 and a WT W303-1A derivative (YJR53 YT6/WT), gal2 W303-1A (ISY46 YT6/gal2), or gal3 W303-1A (YJR54 YT6/gal3) were transformed with a vector control (−) or plasmids expressing WT GAL4 or GAL4 S699A (S669A). Cultures were induced with 2% galactose for 2.5 h before GAL1-lacZ expression was measured.

FIG. 3

FIG. 3

Gal4p S699 phosphorylation is required for sensitive response to inducer. The W303-1A-derived strains YJT1 (WT GAL4) and YJT2 (GAL4 S699A) bearing an integrated GAL1-HIS3 reporter gene (A) were grown in YEPD, were washed with sterile water, and were plated in equivalent numbers in top agar on His− plates containing glycerol as the sole source of carbon. Sterile discs were placed in the centers of the plates onto which 5 μl of 2% galactose was spotted. The plates were photographed after 3 days growth at 30°C (B).

FIG. 4

FIG. 4

Gal4p S699 phosphorylation occurs independently of the Gal3p-galactose signaling mechanism. (A) Yeast strain YJR14::131 (gal3) bearing a vector control (▵), YCpG4 expressing WT GAL4 (■ and □), or GAL4 S699A (▴) was grown in minimal medium containing glycerol and were induced with 2% galactose or left uninduced (□). GAL1-lacZ reporter gene expression was measured at the indicated times postinduction. (B) W303-1A (WT) and YJR58 (gal1 gal3) yeast expressing GAL4Δ683 from a plasmid were labeled with [32P]orthophosphate in the presence of galactose. Tryptic phosphopeptides from labeled Gal4p were resolved in 2 dimensions and were visualized by autoradiography. Phosphopeptides 1 and 5 represent S699 and S837 phosphorylation, respectively. The major phosphopeptide 2 is derived from the DNA-binding domain (Fig. 1A). We do not know the origin of phosphopeptide 8 nor if the apparent increase in the gal1 gal3 strain is significant.

FIG. 5

FIG. 5

Gal3p and Srb10p represent independent mechanisms for GAL induction. Yeast strains W303-1A (WT), H617 (srb10), YJR7 (gal3), and YJR47 (gal3 srb10) were grown to saturation in minimal medium containing glycerol and lactate as the sole sources of carbon, and 5 μl was used to inoculate YEPD-glucose (A) or YEPD-galactose (B) plates containing ethidium bromide. Plates were photographed after incubation at 30°C for 5 days.

FIG. 6

FIG. 6

Gal4p activity is regulated by two independent signals. Under noninducing conditions (A), Gal4p activity is inhibited by the negative regulator Gal80p. Upon galactose addition (B), Gal3p-galactose interacts with Gal80p to cause a transient conformational alteration that allows Gal4p to activate transcription. During interaction with the RNA Pol II holoenzyme (C), Gal4p is phosphorylated at S699 by Srb10p; this phosphorylation stabilizes the active Gal4p-Gal80p conformation induced by Gal3p-galactose. The ability of Srb10p to phosphorylate Gal4p is regulated by independent environmental signals, thus modulating GAL induction to levels appropriate for the cellular environment.

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