Std1 and Mth1 Proteins Interact with the Glucose Sensors To Control Glucose-Regulated Gene Expression in Saccharomyces cerevisiae (original) (raw)
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Repression of transcription by Rgt1 in the absence of glucose requires Std1 and Mth1
Current Genetics, 2003
In the yeast Saccharomyces cerevisiae, glucose induces expression of the hexose transporter (HXT) genes by inhibiting the repressor function of the transcription factor Rgt1. We have previously shown that Rgt1 binds to the HXT gene promoters only in the absence of glucose. In the presence of glucose, Rgt1 becomes phosphorylated and is unable to bind to the HXT promoters and repress their transcription. We report that Rgt1 interacts with Std1 and Mth1 in a yeast two-hybrid assay and co-immunoprecipitates with both proteins in vivo only when glucose is absent. In addition, we demonstrate that repression of HXT gene expression by Rgt1 is abolished in the std1 mth1 double mutant. While Rgt1 is normally phosphorylated only in the presence of high concentrations of glucose, it is constitutively modified in the std1 mth1 double mutant. Based on these data, we conclude that, in the absence of glucose, Rgt1 associates with Std1 and Mth1 to repress HXT gene expression.
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.
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.
Glucose Sensing and Signal Transduction in Saccharomyces cerevisiae
Molecular Mechanisms in Yeast Carbon Metabolism, 2014
Cells of the yeast Saccharomyces cerevisiae have an exquisite preference for high concentrations of glucose compared to other sugars or carbon sources. The likely explanation is that glucose is the best fermentable sugar, i.e., the sugar that allows the yeast to accumulate most rapidly high levels of ethanol, which are strongly inhibitory to competing microorganisms. To accomplish rapid fermentation of glucose, S. cerevisiae has evolved multiple glucose sensing and signaling pathways, which stimulate both fermentation and rapid cell proliferation. The latter is important for rapid fermentation in order to recycle the ATP generated in glycolysis to ADP. Downregulation of respiration to maximize ethanol production is accomplished by the main glucose repression pathway, in which the Snf1 protein kinase is a central regulator. It is inactivated by dephosphorylation upon glucose addition, and its reactivation upon glucose exhaustion is essential for induction of genes sustaining respiration, gluconeogenesis, and the catabolism of alternative carbon sources. Stimulation of fermentation and growth is mainly exerted by the protein kinase A pathway, which senses glucose with an extracellular and intracellular sensing mechanism that activates protein kinase A in a concerted manner through stimulation of cAMP synthesis. Sensing of other nutrients by plasma membrane transceptors integrates with this glucose-sensing mechanism to maintain high protein kinase A activity throughout fermentative growth. Induction of appropriate glucose transporters during fermentative growth is controlled by plasma membrane transporter-like proteins, which function as glucose sensors. Although detailed knowledge has been gained on the molecular mechanisms involved in glucose signaling, multiple important questions still remain.
Journal of Biological Chemistry, 2004
In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes. Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression. Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression. Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in ⌬bmh1⌬bmh2 cells uses the same activation pathway that operates in ⌬reg1 cells. Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression. A twohybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi. Deleting this region partially releases SUC2 from glucose repression. This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process. An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA 3 -TAP-tagged Reg1. The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA 3 -TAP-tagged Reg1. Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression. . The abbreviations used are: PP1c, type 1 protein phosphatase catalytic subunit; HA 3 -TAP, a C-terminal epitope tag having three repeats of the influenza hemagglutinin epitope followed by one TAP tag consisting of a calmodulin binding peptide and Protein A; GAD, Gal4 activation domain; cAPK, cAMP-dependent protein kinase; ADHI, alcohol dehydrogenase isozyme I; ADHII, glucose-repressible alcohol dehydrogenase isozyme II; PP1c, type 1 protein phosphatase catalytic subunit; HPLC, high pressure liquid chromatography; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
Role of casein kinase 1 in the glucose sensor-mediated signaling pathway in yeast
2010
Background: In yeast, glucose-dependent degradation of the Mth1 protein, a corepressor of the glucose transporter gene (HXT) repressor Rgt1, is a crucial event enabling expression of several HXT. This event occurs through a signaling pathway that involves the Rgt2 and Snf3 glucose sensors and yeast casein kinase 1 and 2 (Yck1/2). In this study, we examined whether the glucose sensors directly couple with Yck1/2 to convert glucose binding into an intracellular signal that leads to the degradation of Mth1. Results: High levels of glucose induce degradation of Mth1 through the Rgt2/Snf3 glucose signaling pathway. Fluorescence microscopy analysis indicates that, under glucose-limited conditions, GFP-Mth1 is localized in the nucleus and does not shuttle between the nucleus and cytoplasm. If glucose-induced degradation is prevented due to disruption of the Rgt2/Snf3 pathway, GFP-Mth1 accumulates in the nucleus. When engineered to be localized to the cytoplasm, GFP-Mth1 is degraded regardless of the presence of glucose or the glucose sensors. In addition, removal of Grr1 from the nucleus prevents degradation of GFP-Mth1. These results suggest that glucoseinduced, glucose sensor-dependent Mth1 degradation occurs in the nucleus. We also show that, like Yck2, Yck1 is localized to the plasma membrane via C-terminal palmitoylation mediated by the palmitoyl transferase Akr1. However, glucose-dependent degradation of Mth1 is not impaired in the absence of Akr1, suggesting that a direct interaction between the glucose sensors and Yck1/2 is not required for Mth1 degradation. Conclusion: Glucose-induced, glucose sensor-regulated degradation of Mth1 occurs in the nucleus and does not require direct interaction of the glucose sensors with Yck1/2.
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 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.
Metabolic Signals Trigger Glucose-Induced Inactivation of Maltose Permease in Saccharomyces
Journal of Bacteriology, 2000
Organisms such as Saccharomyces capable of utilizing several different sugars selectively ferment glucose when less desirable carbon sources are also available. This is achieved by several mechanisms. Glucose downregulates the transcription of genes involved in utilization of these alternate carbon sources. Additionally, it causes posttranslational modifications of enzymes and transporters, leading to their inactivation and/or degradation. Two glucose sensing and signaling pathways stimulate glucose-induced inactivation of maltose permease. Pathway 1 uses Rgt2p as a sensor of extracellular glucose and causes degradation of maltose permease protein. Pathway 2 is dependent on glucose transport and stimulates degradation of permease protein and very rapid inactivation of maltose transport activity, more rapid than can be explained by loss of protein alone. In this report, we characterize signal generation through pathway 2 using the rapid inactivation of maltose transport activity as an assay of signaling activity. We find that pathway 2 is dependent on HXK2 and to a lesser extent HXK1. The correlation between pathway 2 signaling and glucose repression suggests that these pathways share common upstream components. We demonstrate that glucose transport via galactose permease is able to stimulate pathway 2. Moreover, rapid transport and fermentation of a number of fermentable sugars (including galactose and maltose, not just glucose) are sufficient to generate a pathway 2 signal. These results indicate that pathway 2 responds to a high rate of sugar fermentation and monitors an intracellular metabolic signal. Production of this signal is not specific to glucose, glucose catabolism, glucose transport by the Hxt transporters, or glucose phosphorylation by hexokinase 1 or 2. Similarities between this yeast glucose sensing pathway and glucose sensing mechanisms in mammalian cells are discussed.
The yeast Saccharomyces cerevisiae senses glucose through two transmembrane glucose sensors, Snf3 and Rgt2. Extracellular glucose causes these sensors to generate an intracellular signal that induces expression of HXT genes encoding glucose transporters by inhibiting the function of Rgt1, a transcriptional repressor of HXT genes. We present the following evidence that suggests that the glucose sensors are coupled to the membrane-associated protein kinase casein kinase I (Yck1). (i) Overexpression of Yck1 leads to constitutive HXT1 expression; (ii) Yck1 (or its paralogue Yck2) is required for glucose induction of HXT1 expression; (iii) Yck1 interacts with the Rgt2 glucose sensor; and (iv) attaching the C-terminal cytoplasmic tail of Rgt2 to Yck1 results in a constitutive glucose signal. The likely targets of Yck1 in this signal transduction pathway are Mth1 and Std1, which bind to and regulate function of the Rgt1 transcription factor and bind to the C-terminal cytoplasmic domain of glucose sensors. Potential casein kinase I phosphoryla-tion sites in Mth1 and Std1 are required for normal glucose regulation of HXT1 expression, and Yck1 catalyzes phosphoryla-tion of Mth1 and Std1 in vitro. These results support a model of glucose signaling in which glucose binding to the glucose sensors causes them to activate Yck1 in the cell membrane, which then phosphorylates Mth1 and Std1 bound to the cytoplasmic face of the glucose sensors, triggering their degradation and leading to the derepression of HXT gene expression. Our results add nutrient sensing to the growing list of processes in which casein kinase I is involved.