Glucose-promoted inactivation of Snf1 is necessary for degradation of Mth1 and Std1 Removal of the gene prevents Mth1 degradation in high glucose [20], which may give an explanation of why expression of the gene is constitutively repressed in gene is disrupted in expression via the Rgt2/Snf3-Rgt1 pathway [8, 19]. of glucose, the Rgt1 DNA-binding repressor represses expression in conjunction with Mth1 and Std1, paralogous proteins that actually interact with Rgt1 [3-5]. Glucose disrupts this conversation by promoting degradation of Mth1 and Std1 [6-8], thereby relieving repression of expression [6, 9, 10]. Mth1 and Std1 are subject to phosphorylation-driven ubiquitination and subsequent degradation when glucose levels are high. According to a current working model, glucose binding to the glucose sensors activates the Yck1/2 kinases, which phosphorylate Mth1 and Std1 [7]. Phosphorylated Mth1 and Std1 are ubiquitinated by the SCFGrr1 ubiquitin protein ligase, targeting them for degradation by the 26S proteasome [6, 8]. Dominant mutations in the glucose sensor genes, (Arg-231 to Lys) and (Arg-229 to Lys), confer the glucose-independent induction of expression [11, 12]. This obtaining suggests that glucose transport is not required for generation of signal; rather glucose directly binds and activates the glucose sensors, which initiate receptor (sensor)-mediated signaling [13]. However, it has not been exhibited whether and cause induction of expression by promoting degradation of Mth1 and Std1. Here, we show that promotes degradation of Mth1 and Std1 independent of the presence of glucose. This MethADP sodium salt supports the view that locks the protein in the glucose-bound conformation, and thus causing constitutive activation of the glucose sensor signaling pathway [11]. The Snf1 kinase plays a crucial role in signaling glucose limitations. Glucose regulates activity and subcellular localization of Snf1 kinase [14]. Snf1 is usually active and present in the nucleus upon phosphorylation on threonine 210 when glucose is usually depleted in the medium [15]. However, addition of glucose promotes dephosphorylation of Snf1 by the Reg1/Glc7 phosphatase, leading to conversion of the kinase from an active to an inactive conformation [16]. Deletion of causes inhibition of expression [3]. In this study, we show that glucose-promoted inactivation of Snf1 is necessary for degradation of Mth1 and Std1 2. Materials and Methods 2.1. Yeast strains and gene deletions strains used in this study are listed in Table 1. Except where indicated, yeast strains were produced in YP (2% bacto-peptone, 1% yeast extract) or SYNB (synthetic yeast nitrogen base media; 0.17% yeast nitrogen base with 0.5% ammonium sulfate) supplemented with the appropriate amino acids. Genes were disrupted by homologous recombination using NatMX cassettes [17]. Table 1 Yeast strains used in this study or to amplify the 5 and 3 portions of the genes in individual reactions, using pBM4748 (strain (YM6292) was cotransformed with the PCR products and the plasmid pUG34 or pUG36 [8] cut with and strains by Western blotting and confocal microscopy. MethADP sodium salt Mth1-myc and Std1-myc are not or barely detected by Western blotting in the and strains produced in the medium lacking glucose (Fig. 1A, Gal). Fluorescence intensities of GFP-Mth1 and GFP-Std1 are strong in the wild-type cells but are MethADP sodium salt profoundly MethADP sodium salt diminished in the and FA3 strains, in the absence MethADP sodium salt of glucose (Fig. 1B, Gal). These results suggest that and promote degradation of Mth1 and Std1 in a glucose-independent manner. Mth1 degradation is usually reinforced by glucose repression of expression by Mig1, whereas Std1 degradation is usually obscured by glucose induction of expression through the Rgt2/Snf3-Rgt1 pathway (Fig. 1A, WT) [19]. Indeed, and induce expression of gene 3- and 10-fold, respectively, in the absence of glucose [19]. However, Std1 degradation is usually accelerated and Mth1 degradation is usually slowed when glucose regulation of and expression is usually interrupted by replacing their promoters with the promoter, which is not regulated by glucose [8]. Open in a separate window Fig. 1 and promote glucose-independent degradation of Mth1 and Std1. (A) Yeast cells expressing Mth1-myc or Std1-myc under the control of their own promoters [7] were produced to mid-log phase in a selective medium made up of 2% galactose. Aliquots were then transferred to 2% galactose medium (Gal) or 4% glucose medium (Glu) and incubated for 60 min. Levels of Mth1-myc and Std1-myc were determined by Western blotting using anti-myc antibody. (B) Yeast cells expressing GFP-Mth1 or GFP-Std1 under the control of the promoter [8] were grown as described above. Cells were observed under the Zeiss LSM 510 META confocal laser scanning microscope. DIC and.