Repression of transcription by Rgt1 in the absence of glucose requires Std1 and Mth1 (original) (raw)
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Journal of Cellular Biochemistry, 2011
The yeast Rgt1 repressor is a bifunctional protein that acts as a transcriptional repressor and activator. Under glucose-limited conditions, Rgt1 induces transcriptional repression by forming a repressive complex with its corepressors Mth1 and Std1. Here, we show that Rgt1 is converted from a transcriptional repressor into an activator under high glucose conditions and this occurs through two independent but consecutive events mediated by two glucose signaling pathways: (1) disruption of the repressive complex by the Rgt2/Snf3 pathway; (2) phosphorylation of Rgt1 by the cAMP-PKA (cAMP-dependent protein kinase) pathway. Rgt1 is phosphorylated by PKA at four serine residues within its amino-terminal region, but this does not occur until the repressive complex is disrupted. While phosphorylation of any one of these sites is sufficient to enable Rgt1 to induce transcriptional activation, phosphorylation of all the sites results in the release of Rgt1 from DNA. We discuss how the bifunctional properties of Rgt1 are regulated through differential phosphorylation.
Understanding the mechanism of glucose-induced relief of Rgt1-mediated repression in yeast
FEBS open bio, 2014
The yeast Rgt1 repressor inhibits transcription of the glucose transporter (HXT) genes in the absence of glucose. It does so by recruiting the general corepressor complex Ssn6-Tup1 and the HXT corepressor Mth1. In the presence of glucose, Rgt1 is phosphorylated by the cAMP-activated protein kinase A (PKA) and dissociates from the HXT promoters, resulting in expression of HXT genes. In this study, using Rgt1 chimeras that bind DNA constitutively, we investigate how glucose regulates Rgt1 function. Our results show that the DNA-bound Rgt1 constructs repress expression of the HXT1 gene in conjunction with Ssn6-Tup1 and Mth1, and that this repression is lifted when they dissociate from Ssn6-Tup1 in high glucose conditions. Mth1 mediates the interaction between the Rgt1 constructs and Ssn6-Tup1, and glucose-induced downregulation of Mth1 enables PKA to phosphorylate the Rgt1 constructs. This phosphorylation induces dissociation of Ssn6-Tup1 from the DNA-bound Rgt1 constructs, resulting i...
Biochemical evidence for glucose-independent induction of HXT expression in Saccharomyces cerevisiae
FEBS Letters, 2007
The yeast glucose sensors Rgt2 and Snf3 generate a signal in response to glucose that leads to degradation of Mth1 and Std1, thereby relieving repression of Rgt1-repressed genes such as the glucose transporter genes (HXT). Mth1 and Std1 are degraded via the Yck1/2 kinase-SCF Grr1 -26S proteasome pathway triggered by the glucose sensors. Here, we show that RGT2-1 promotes ubiquitination and subsequent degradation of Mth1 and Std1 regardless of the presence of glucose. Site-specific mutagenesis reveals that the conserved lysine residues of Mth1 and Std1 might serve as attachment sites for ubiquitin, and that the potential casein kinase (Yck1/2) sites of serine phosphorylation might control their ubiquitination. Finally, we show that active Snf1 protein kinase in high glucose prevents degradation of Mth1 and Std1.
Eukaryotic Cell, 2006
Expression of the HXT genes encoding glucose transporters in the budding yeast Saccharomyces cerevisiae is regulated by two interconnected glucose-signaling pathways: the Snf3/Rgt2-Rgt1 glucose induction pathway and the Snf1-Mig1 glucose repression pathway. The Snf3 and Rgt2 glucose sensors in the membrane generate a signal in the presence of glucose that inhibits the functions of Std1 and Mth1, paralogous proteins that regulate the function of the Rgt1 transcription factor, which binds to the HXT promoters. It is well established that glucose induces degradation of Mth1, but the fate of its paralogue Std1 has been less clear. We present evidence that glucose-induced degradation of Std1 via the SCF Grr1 ubiquitin-protein ligase and the 26S proteasome is obscured by feedback regulation of STD1 expression. Disappearance of Std1 in response to glucose is accelerated when glucose induction of STD1 expression due to feedback regulation by Rgt1 is prevented. The consequence of relieving feedback regulation of STD1 expression is that reestablishment of repression of HXT1 expression upon removal of glucose is delayed. In contrast, degradation of Mth1 is reinforced by glucose repression of MTH1 expression: disappearance of Mth1 is slowed when glucose repression of MTH1 expression is prevented, and this results in a delay in induction of HXT3 expression in response to glucose. Thus, the cellular levels of Std1 and Mth1, and, as a consequence, the kinetics of induction and repression of HXT gene expression, are closely regulated by interwoven transcriptional and posttranslational controls mediated by two different glucose-sensing pathways.
Journal of Biological Chemistry, 2006
The yeast Saccharomyces cerevisiae deploys two different types of glucose sensors on its cell surface that operate in distinct glucose signaling pathways: the glucose transporter-like Snf3 and Rgt2 proteins and the Gpr1 receptor that is coupled to Gpa2, a G-protein ␣ subunit. The ultimate target of the Snf3/ Rgt2 pathway is Rgt1, a transcription factor that regulates expression of HXT genes encoding glucose transporters. We have found that the cAMP-dependent protein kinase A (PKA), which is activated by the Gpr1/Gpa2 glucose-sensing pathway and by a glucose-sensing pathway that works through Ras1 and Ras2, catalyzes phosphorylation of Rgt1 and regulates its function. Rgt1 is phosphorylated in vitro by all three isoforms of PKA, and this requires several serine residues located in PKA consensus sequences within Rgt1. PKA and the consensus serine residues of Rgt1 are required for glucose-induced removal of Rgt1 from the HXT promoters and for induction of HXT expression. Conversely, overexpression of the TPK genes led to constitutive expression of the HXT genes. The PKA consensus phosphorylation sites of Rgt1 are required for an intramolecular interaction that is thought to regulate its DNA binding activity. Thus, two different glucose signal transduction pathways converge on Rgt1 to regulate expression of glucose transporters.
Journal of Biological Chemistry, 2004
Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser 85 ) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker's yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport.
Biochemical Journal, 2005
Expression of HXK2, a gene encoding a Saccharomyces cerevisiae bifunctional protein with catalytic and regulatory functions, is controlled by glucose availability, being activated in the presence of glucose and inhibited when the levels of the sugar are low. In the present study, we identified Rgt1 as a transcription factor that, together with the Med8 protein, is essential for repression of the HXK2 gene in the absence of glucose. Rgt1 represses HXK2 expression by binding specifically to the motif (CGGAAAA) located at −395 bp relative to the ATG translation start codon in the HXK2 promoter. Disruption of the RGT1 gene causes an 18-fold increase in the level of HXK2 transcript in the absence of glucose. Rgt1 binds to the RGT1 element of HXK2 promoter in a glucose-dependent manner, and the repression of target gene depends on binding of Rgt1 to DNA. The physiological significance of the connection between two glucose-signalling pathways, the Snf3/Rgt2 that causes glucose induction and the Mig1/Hxk2 that causes glucose repression, was also analysed.