GAL3 gene product is required for maintenance of the induced state of the GAL cluster genes in Saccharomyces cerevisiae

Gal4p regulates expression of genes necessary for galactose catabolism in Saccharomyces cerevisiae. We have previously shown that phosphorylation of Gal4p requires both its DNA binding and transcriptional-activation functions and have suggested that phosphorylation occurs as a consequence of interaction with general transcription factors. In this study, we show that phosphorylation occurs rapidly on a limited fraction of overexpressed Gal4p present in a sodium dodecyl sulfate-extractable subcellular fraction while a significant fraction remains stably unphosphorylated. Taken together with our previous observations, we conclude that Gal4p is phosphorylated only if it becomes localized to the nucleus and is capable of both DNA binding and transcriptional activation. We demonstrate that Gal4p is multiply phosphorylated at both the C and N termini, and we identify the precise locations of three sites of phosphorylation at serines 691, 696, and 699. Of these sites, only serine 699 must be phosphorylated for galactose-inducible transcription to occur. Mutation of S-699 to alanine significantly impairs GAL induction by galactose in GAL80 ؉ cells but does not affect transcriptional activation by Gal4p in gal80 ؊ cells. In gal80 ؊ cells, Gal4p phosphorylation, including that of serine 699, is stimulated by the presence of both galactose and glucose, indicating that phosphorylation at this site is not specifically activated by galactose. Serine 699 phosphorylation requires Gal4p's DNA binding function and is influenced by the function of the RNA polymerase II holoenzyme component Gal11p. These results suggest that a phosphorylation on Gal4p, likely resulting from interaction with the holoenzyme, modulates the induction process by regulating interaction between Gal4p and Gal80p.

supporting

confidence: 78%

Phosphorylation of Gal4p at a Single C-Terminal Residue Is Necessary for Galactose-Inducible Transcription

Sadowski

Costa

Dhanawansa

1996

“…When galactose is absent, the transcription activity of the Gal4 protein is inhibited by the Gal80 protein by virtue of the direct interaction of Gal80p with a short sequence within the activation domain (amino acids 768 to 881) of the Gal4 protein (28,33,34,40,54,62). Relief of this inhibition state in response to galactose involves the function of the GAL3 gene product (6,7,10,15,38,55). In vitro studies have shown that Gal3p directly interacts with Gal80p in a galactose-dependent manner (8,52,61,63).…”

mentioning

confidence: 99%

Evidence for Gal3p's Cytoplasmic Location and Gal80p's Dual Cytoplasmic-Nuclear Location Implicates New Mechanisms for Controlling Gal4p Activity in Saccharomyces cerevisiae

Peng¹,

Hopper

2000

Genetics and in vitro studies have shown that the direct interaction between Gal3p and Gal80p plays a central role in galactose-dependent Gal4p-mediated GAL gene expression in the yeast Saccharomyces cerevisiae. Precisely how Gal3p-Gal80p interaction effects induction is not clear. It has been assumed that Gal3p interacts with Gal80p in the nucleus upon galactose addition to release Gal80p inhibition of Gal4p. Although Gal80p has been shown to possess nuclear localization signal (NLS) peptides, the subcellular distribution of neither Gal80p nor Gal3p was previously determined. Here we report that Gal3p is located in the cytoplasm and apparently excluded from the nucleus. We show that Gal80p is located in both the cytoplasm and the nucleus. Converting Gal80p into a nucleus-localized protein (NLS-Gal80p) by exogenous NLS addition impairs GAL gene induction. The impaired induction can be partially suppressed by targeting Gal3p to the nucleus (NLS-Gal3p). We document a very rapid association between NLS-Gal3p and Gal80p in vivo in response to galactose, illustrating that the nuclear import of Gal80p is very rapid and efficient. We also demonstrate that nucleus-localized NLS-Gal80p can move out of the nucleus and shuttle between nuclei in yeast heterokaryons. These results are the first indication that the subcellular distribution dynamics of the Gal3 and Gal80 proteins play a role in regulating Gal4p-mediated GAL gene expression in vivo.

“…In noninducing conditions, Gal4p is bound to the upstream activating sequences for galactose (UAS G ) but is prevented from activating transcription by the inhibitor Gal80p (32,43). Rapid induction by galactose requires the product of GAL3 (40,52,57), which is a regulatory protein with similarity to the galactokinase encoded by GAL1 (3,9,50), although Gal3p does not have galactokinase activity (9). Recent experiments demonstrate that Gal3p, when bound to galactose, directly interacts with Gal80p in the presence of ATP (42,50,59,60).…”

mentioning

confidence: 99%

Multiple Signals Regulate GALTranscription in Yeast

Rohde

Trinh

Sadowski

2000

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 cyclindependent 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.Eukaryotic cells react to their environment by regulating transcription factors bound to promoters of responsive genes (28). Cells grown in culture are typically provided with sufficient essential nutrients and factors to ensure unchecked propagation. However, in their natural environment, cell growth is ordinarily limited by the scarcity of one or more factors or nutrients. In such circumstances, there must be mechanisms to ensure that transcriptional responses to one signal do not surpass what the cell can accommodate with its limited growth potential. This issue has not yet been addressed in eukaryotes. In this report, we demonstrate that the prototypical transactivator Gal4p is regulated by two separate signals represented by the specific inducer galactose and the RNA polymerase II (Pol II) holoenzyme-associated cyclin-dependent kinase Srb10p/ CDK8. These observations suggest a mechanism whereby responses to a specific inducer can be coordinated with the physiological environment.Gal4p regulates expression of the yeast GAL genes in response to galactose. In noninducing conditions, Gal4p is bound to the upstream activating sequences for galactose (UAS G ) but is prevented from activating transcription by the inhibitor Gal80p (32,43). Rapid induction by galactose requires the product of GAL3 (40, 52, 57), which is a regulatory protein with similarity to the galactokinase encoded by GAL1 (3, 9, 50), although Gal3p does not have galactokinase activity (9). Recent experiments demonstrate that Gal3p, when bound to galactose, directly interacts with Gal80p in the presence of ATP (42,50,59,60). Gal3p-galactose is thought to cause induction of the GAL genes by producing a conformational change in the Gal4p-Gal80p complex that allows interaction of the Gal4p activating domains with the general transcription factors (42, 59). The induced conformation may ...