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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
FEBS Lett. Author manuscript; available in PMC Oct 22, 2007.
Published in final edited form as:
PMCID: PMC2040036
NIHMSID: NIHMS26796

Biochemical evidence for glucose-independent induction of HXT expression in Saccharomyces cerevisiae

Abstract

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-SCFGrr1-26S 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.

1. Introduction

The budding yeast S. cerevisiae growing on high levels of glucose induces HXT expression, which facilitates the rate-limiting step of glucose utilization—glucose uptake. This is achieved by derepressing the Rgt1-repressed HXT expression via the Rgt2/Snf3-Rgt1 signaling pathway [1, 2]. In the absence of glucose, the Rgt1 DNA-binding repressor represses HXT expression in conjunction with Mth1 and Std1, paralogous proteins that physically interact with Rgt1 [3-5]. Glucose disrupts this interaction by promoting degradation of Mth1 and Std1 [6-8], thereby relieving repression of HXT 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, RGT2-1 (Arg-231 to Lys) and SNF3-1 (Arg-229 to Lys), confer the glucose-independent induction of HXT expression [11, 12]. This finding 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 demonstrated whether RGT2-1 and SNF3-1 cause induction of HXT expression by promoting degradation of Mth1 and Std1. Here, we show that RGT2-1 promotes degradation of Mth1 and Std1 independent of the presence of glucose. This supports the view that RGT2-1 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 active and present in the nucleus upon phosphorylation on threonine 210 when glucose is 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 REG1 causes inhibition of HXT1 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

S. cerevisiae strains used in this study are listed in Table 1. Except where indicated, yeast strains were grown 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

2.2. Plasmids

Plasmids expressing the mutant Mth1 and Std1 proteins were generated using gap-repair [18] and subcloning protocols. Briefly two oligonucleotides carrying complementary nucleotide changes that result in a single nucleotide substitution were used as primers along with the oligonucleotides flanking MTH1 or STD1 to amplify the 5′ and 3′ portions of the genes in separate reactions, using pBM4748 (MTH1) or pBM4747 (STD1) [8] as a template. The mth1Δstd1Δ strain (YM6292) was cotransformed with the PCR products and the plasmid pUG34 or pUG36 [8] cut with BamHI. All mutations were confirmed by sequencing (SeqWright, TX).

2.3. Western blotting and immunoprecipitation (IP)

For Western blotting, yeast lysates were resolved by SDS-PAGE, transferred to Polyvinylidene fluoride membrane (Millipore), and detected with appropriate antibodies [10]. For IP, yeast lysates were incubated with appropriate antibodies at 4°C for 3 h and further incubated with protein A/G-conjugated agarose beads (Santa Cruz) for 1 h [10].

2.4. Fluorescence microscopy

Cells expressing fluorescent proteins were visualized using a Zeiss LSM 510 META confocal laser scanning microscope with a 63x Plan-Apochromat 1.4 NA Oil DIC objective lens. Images were acquired with the Zeiss LSM 510 software version 3.2.

3. Results and Discussion

3.1. RGT2-1 and SNF3-1 cause degradation of Mth1 and Std1 independent of the presence of glucose

To address glucose-independent degradation of Mth1 and Std1, we determined cellular levels of Mth1 and Std1 in the RGT2-1 and SNF3-1 strains by Western blotting and confocal microscopy. Mth1-myc and Std1-myc are not or barely detected by Western blotting in the RGT2-1 and SNF3-1 strains grown 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 profoundly diminished in the RGT2-1 and SNF3-1 strains, in the absence of glucose (Fig. 1B, Gal). These results suggest that RGT2-1 and SNF3-1 promote degradation of Mth1 and Std1 in a glucose-independent manner. Mth1 degradation is reinforced by glucose repression of MTH1 expression by Mig1, whereas Std1 degradation is obscured by glucose induction of STD1 expression through the Rgt2/Snf3-Rgt1 pathway (Fig. 1A, WT) [19]. Indeed, RGT2-1 and SNF3-1 induce expression of STD1 gene 3- and 10-fold, respectively, in the absence of glucose [19]. However, Std1 degradation is accelerated and Mth1 degradation is slowed when glucose regulation of MTH1 and STD1 expression is interrupted by replacing their promoters with the MET25 promoter, which is not regulated by glucose [8].

Fig. 1
RGT2-1 and SNF3-1 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 grown to mid-log phase in a selective medium containing 2% galactose. Aliquots ...

3.2. RGT2-1 promotes ubiquitination of Mth1 and Std1 by SCFGrr1

To determine whether RGT2-1 promotes ubiquitination of Mth1 and Std1, the extracts of the RGT2-1 strain expressing Mth1-myc and Std1-myc, grown in the absence of glucose, were analyzed IP-Western blotting (Fig. 2). In Western blotting of the wild-type cell extracts, anti-myc antibody detects a single band that corresponds to Mth1-myc (Fig. 2A, lane 2), whereas anti-Ub antibody is cross-reactive to a high-molecular mass ladder, typical of a polyubiquitin chain (Fig. 2A, lane 7). Next, Mth1-myc in cell extracts were precipitated using anti-myc antibody-conjugated beads and then subjected to Western blotting using either anti-myc antibody (Fig. 2A, lanes 3-5) or anti-Ub antibody (Fig. 2A, lanes 8-10). Compared to its wild-type allele (Fig. 2A, lanes 8), RGT2-1 greatly enhances Mth1 ubiquitination (Fig. 2A, lane 9). However, ubiquitination is largely impaired when GRR1 is disrupted in the RGT2-1 strain (RGT2-1grr1Δ; Fig. 2, lane 10). Similar observations are also made with Std1 (Fig. 2B). Therefore, we concluded that RGT2-1 promotes ubiquitination of Mth1 and Std1 by SCFGrr1 in the absence of glucose.

Fig. 2
RGT2-1 promotes ubiquitination of Mth1 and Std1 in vivo. The wild-type and RGT2-1 strains expressing Mth1-myc (A) or Std1-myc (B) were grown in 2% galactose medium. Yeast cell extracts were resolved on an SDS-gel and analyzed by Western blotting using ...

3.3. The evolutionarily conserved lysine residues of Mth1 and Std1 are required for degradation

The ClustalW protein alignment (Saccharomyces Geneome Database) shows that Mth1 and Std1 contain ~19 lysines which are well conserved in their orthologs from other yeast species (Fig. 3A). We have previously shown that conversion of 9 out of the 19 conserved lysines in Std1 to arginine (Std1-9KR) reduces induction of HXT1 expression by impairing degradation of Std1 (Fig. 3C, [8]). Individual mutations of the 19 conserved lysines to alanine do not prevent degradation of Mth1 and Std1 (data not shown). However, simultaneous mutation of 5 lysines in the carboxy terminal region of Mth1 (5KA; positions K326, K333, K334, K336, and K343) severely impairs degradation of Mth1 (Fig. 3B). Both Mth1-5KA and Std1-9KR [8] are not degraded in the RGT2-1 strain (Fig. 3B and Fig. 3C). These results suggest that the evolutionarily conserved lysine residues might serve as attachment sites for ubiquitin, which is required for both the glucose-promoted and -independent degradation of Mth1 and Std1.

Fig. 3
The evolutionarily conserved lysine residues of Mth1 and Std1 are required for glucose-independent degradation. (A) Mth1 and Std1 contain ~19 evolutionarily conserved lysine residues. Individual mutations of the conserved lysine residues to alanine ...

3.4. Glucose-independent degradation of Mth1 and Std1 requires the putative Yck1/2 phosphorylation sites

Yck1/2 appear to phosphorylate Mth1 and Std1 at a conserved cluster of serine residues, known as the Yck1/2 phosphorylation sites (SXXS [7]). Deletion of the sites in Mth1 (Δ118-138) and Std1 (Δ129-148) prevents both the glucose-promoted and -independent degradation of Mth1 and Std1 (Fig. 4). It has been proposed that a conformational change in the glucose sensors upon glucose binding causes activation of Yck1/2 that is tethered to the cell membrane through a C-terminal palmitate moiety in the sequence [7]. Our results suggest that RGT2-1 converts the protein into the glucose-bound form, as proposed previously [11], which activates Yck1/2 even in the absence of glucose. Thus Yck1/2 interaction with the glucose sensors appears to be crucial for activation of the kinases. However, Yck1/2 seem to interact with the glucose sensors in both the presence and absence of glucose [7]. The molecular mechanism underlying activation of Yck1/2 in response to glucose remains elusive.

Fig. 4
The putative Yck1/2 phosphorylation sites of Mth1 and Std1 are required for glucose-independent degradation of Mth1 and Std1. GFP-Mth1(Δ118-138; KP90) and GFP-Std1 (Δ129-148; KP91) lacking the Yck1/2 phosphorylation sites were expressed ...

3.5. Glucose-promoted inactivation of Snf1 is necessary for degradation of Mth1 and Std1

Removal of the REG1 gene prevents Mth1 degradation in high glucose [20], which may give an explanation of why expression of the HXT1 gene is constitutively repressed in reg1Δ [3]. Snf1 is constitutively active in reg1Δ, probably due to a failure in converting the kinase from an active into an inactive conformation [16]. Therefore, we determined if Snf1 is involved in the stability of Mth1 and Std1 in reg1Δ by Western blotting. As seen in Fig. 5, considerable amounts of Mth1 are detected in reg1Δ grown in high glucose as reported previously [20]; in contrast, Mth1 is not detected when the SNF1 gene is disrupted in reg1Δ (snf1Δreg1Δ) (Fig. 5). As aforementioned, glucose not only promotes degradation of Std1, but also induces STD1 expression via the Rgt2/Snf3-Rgt1 pathway [8, 19]. This obscures disappearance of Std1 (Std1-myc in Fig. 1A and Fig. 5, WT, glu). However, Std1 levels are increased by REG1 deletion (reg1Δ) but decreased again by SNF1 deletion in reg1Δ (snf1Δreg1Δ) (Fig. 5), suggesting that Std1 degradation is also prevented when Snf1 is not inactivated by glucose. The Sak1 kinase is known to promote activation and nuclear localization of Snf1 upon glucose depletion [21, 22]. Indeed, overexpression of SAK1 prevents degradation of Mth1 and Std1 (Fig. 5, pSAK1). These results suggest that artificially activated Snf1 plays an important role in blocking the glucose-promoted degradation of Mth1 and Std1. In addition, a hyperactive Snf1, Snf1-G53R [15], prevents degradation of Mth1 and Std1 in high glucose (Fig. 6).

Fig. 5
Artificial activation of the Snf1 kinase prevents degradation of Mth1 and Std1. Yeast cells of the indicated genotype expressing Mth1-myc or Std1-myc were grown in 2% galactose medium (Gal) or 4% glucose medium (Glu) as described in Fig.1. The Sak1 kinase ...
Fig. 6
The hyperactive Snf1 kinase prevents degradation of Mth1 and Std1. The hyperactive Snf1 (Snf1-G53R [15]) was coexpressed with GFP-Mth1 or GFP-Std1 in snf1Δ, and levels of GFP-Mth1 and GFP-Std1 were determined by confocal microscopy (A) and Western ...

It is not known how Snf1 prevents degradation of Mth1 and Std1, when it is not inactivated by high levels of glucose. The proposed model for degradation of Mth1 and Std1 includes nuclear export of the proteins, because they must undergo phosphorylation by the membrane-tethered Yck1/2 prior to being ubiquitinated [7]. It is possible that Snf1 regulates nuclear export of Mth1 and Std1, because Mth1 and Std1 are found in the nucleus of the cells harboring active Snf1. Snf1 plays a crucial, decisive role in Snf1-Mig1 signaling that leads to establishment of glucose repression of gene expression. Glucose repression of SUC2 expression is defective when Mth1 is not degraded [8, 23]. Therefore, these observations imply a functional link between inactivation of Snf1 and degradation of Mth1 and Std1. This cross-talk may play a key role as a molecular switch that efficiently triggers two functionally distinct glucose signaling pathways—the Rgt2/Snf3-Rgt1 glucose induction pathway and the Snf1-Mig1 glucose repression pathway—in response to glucose.

Acknowledgments

We thank Glen Shearer for critical reading of the manuscript and Marian Carlson for plasmid. We also thank Sammujjwwal Chakraborty for making some plasmids. This work was supported by NIH Grant RR016476-04 from the MS INBRE Program of the National Center for Research Resources.

Abbreviations

SCF
Skp1p-Cullin-F-box protein
GFP
Green Fluorescent Protein

Footnotes

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