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Casein kinases are required for the stability of the glucose-sensing receptor Rgt2 in yeast

In yeast, glucose induction of HXT (glucose transporter gene) expression is achieved via the Rgt2 and Snf3 glucose sensing receptor (GSR)-mediated signal transduction pathway. The membrane-associated casein kinases Yck1 and Yck2 (Ycks) are involved in this pathway, but their exact role remains unclear. Previous work suggests that the Ycks are activated by the glucose-bound GSRs and transmit the glucose signal from the plasma membrane to the nucleus. However, here we provide evidence that the YCks are constitutively active and required for the stability of the Rgt2 receptor. Cell surface levels of Rgt2 are significantly decreased in a yck1Δyck2ts mutant, but this is not due to endocytosis-mediated vacuolar degradation of the receptor. Similar observations are made in an akr1Δ mutant, where the Ycks are no longer associated with the membrane, and in a sod1Δ mutant in which the kinases are unstable. Of note, in an akr1Δ mutant, both the Ycks and Rgt2 are mislocalized to the cytoplasm, where Rgt2 is stable and functions as an effective receptor for glucose signaling. We also demonstrate that Rgt2 is phosphorylated on the putative Yck consensus phosphorylation sites in its C-terminal domain (CTD) in a Yck-dependent manner and that this glucose-induced modification is critical for its stability and function. Thus, these results indicate a role for the Ycks in stabilizing Rgt2 and suggest that Rgt2 may use glucose binding as a molecular switch not to activate the Ycks but to promote Yck-dependent interaction and phosphorylation of the CTD that increases its stability.


Results
The Ycks are required for the stability of the Rgt2 receptor. The Ycks are involved in the GSR-mediated glucose sensing and signaling, but their exact role remains unclear. To examine the role for the Ycks in the GSR pathway, we first assessed the cell surface levels of Rgt2 in cells lacking Yck activity (yck1Δyck2 ts ) by Western blot analysis. Rgt2-HA levels are significantly lower in yck1Δyck2 ts cells grown on glucose (+) or galactose (−) as compared with wild-type cells (Fig. 1A). However, no significant difference is observed in transcriptional activity of the RGT2 promoter (measured with an RGT2-lacZ reporter), suggesting that the decreased Rgt2 protein levels in yck1Δyck2 ts cells may be not due to transcriptional repression of the RGT2 gene (Fig. 1B). This is confirmed by expressing GFP-Rgt2 from the MET25 promoter, which is not regulated by glucose 15 . The membrane-bound GFP-Rgt2 levels in yck1Δyck2 ts cells grown with or without glucose are found to be significantly low compared to those in wild-type cells (Fig. 1C, left). The rgt2 snf3 double mutant grows poorly on glucose-containing media 26 . This growth defect is complemented by expression of Rgt2-HA 25 and GFP-Rgt2, suggesting that both Rgt2-HA and GFP-Rgt2 are functional (Fig. 1C, right).
To further explore the possibility of mislocalization of Rgt2 in a yck1Δyck2 ts mutant, Rgt2-HA was immunoprecipitated from cell lysates and analyzed by Western blotting. The results reveal two distinct forms of Rgt2-HA from wild-type cells: a major slower-migrating (upper) band, corresponding to the membrane-bound Rgt2-HA (Fig. 1D, lane 1) and a minor faster-migrating (lower) band (Fig. 1D, lane 3). By contrast, Rgt2-HA from yck1Δyck2 ts cells shows only the minor, lower band, implicating a role for the Ycks in the stability of Rgt2 at the cell surface.
However, the previous work by Snowdon and Johnston showed that Rgt2 levels are not significantly different between wild type and yck1Δyck2 ts strains 24 . To address this discrepancy, we monitored changes of Rgt2 abundance after shifting yck1Δyck2 ts cells from 26 °C to 37 °C for various periods of time. Rgt2-HA was immunoprecipitated from the cell extracts and analyzed by Western blotting. The results show that Rgt2-HA levels are decreased by ~ 50% within 30 min and indicate that Rgt2 stability may directly correlate with Yck activity (Fig. 1E). While we do not know the exact nature of this discrepancy, we shifted yck1Δyck2 ts cells to a restrictive temperature (37 °C) before adding glucose to completely inactivate the kinases, whereas they shifted them to 30℃. However, we find that Rgt2 is unstable in yck1Δyck2 ts cells at 30 °C (Fig. 1F).
Secondly, we examined Rgt2 levels in cells lacking Sod1, an upstream regulator of the Ycks. Previous work showed that Sod1 (Cu/Zn superoxide dismutase) interacts with and stabilizes the Ycks, and consequently, the kinases are undetectable in a sod1Δ mutant 27 . Thus, like yck1Δyck2 ts cells, sod1Δ cells are unable to properly activate HXT1 27 . Expectedly, we find very low levels of Rgt2-HA in sod1Δ cells, supporting the view that the Ycks may be required for the stability of Rgt2 (Fig. 1G,H).
Glucose starvation-induced Rgt2 endocytosis requires the EH-domain containing protein End3 25 . Our results show that Rgt2 is apparently downregulated at the protein level in a yck1Δyck2 ts mutant, but this does not occur through End3-mediated endocytosis (Fig. 1I, yck1Δyck2 ts end3Δ). Thus, the Ycks may not be involved in this process.
Cytoplasmic Rgt2 is stable in an akr1Δ strain where Ycks are cytoplasmic. Akr1 is a palmitoyl transferase that tethers proteins to the plasma membrane 28 . The Ycks are targeted to the plasma membrane through palmitoylation of the C-terminal Cys-Cys sequence by Akr1 29 . Since the yck1∆yck2∆ strain is not viable 30 , akr1 mutations are often used to model loss of the Ycks 27,31 . As reported previously 31 , GFP-Yck1 is found to be localized to the plasma membrane in wild-type cells and uniformly distributed throughout the cytoplasm in akr1Δ cells ( Fig. 2A,B). When expressed in an akr1Δ strain, cell surface levels of Rgt2-HA are dramatically decreased (Fig. 2C), without noticeable changes in transcriptional activity of the RGT2 promoter, suggesting that the membrane-bound Rgt2 is downregulated at the protein level in akr1Δ cells (Fig. 2D).
Interestingly, Rgt2-HA from akr1Δ cells are detected in immunoprecipitates of cell lysates and its levels are comparable to those of wild-type cell lysates, suggesting that Rgt2-HA is stable in the cytoplasm (Fig. 2E). This is confirmed by confocal microscopy. GFP-Rgt2 is localized in the plasma membrane of glucose-grown wild-type cells, with visible GFP signals in the vacuole, and targeted to the vacuole when the cells were shifted from glucose to galactose (Fig. 2F) 25,32 . However, GFP-Rgt2 expressed in akr1Δ cells is not properly localized to the plasma membrane but is distributed to the cytoplasm (Fig. 2F,G)  www.nature.com/scientificreports/ Cytoplasmic Rgt2 functions as an effective receptor for glucose signaling. Next, we examined whether the cytoplasmic Rgt2 is functional by monitoring glucose signaling markers, including Mth1 degradation and HXT expression. These markers are blocked by yck1Δyck2 ts mutations, confirming that the Ycks are required for glucose signaling (Fig. 3A,C). However, Mth1-myc levels and GFP-Mth1 signals are significantly decreased in response to high glucose in akr1Δ cells, indicating that the cytoplasmic Rgt2 is fully functional as a glucose receptor (Fig. 3A,B). Because extracellular glucose is unlikely to bind to the Rgt2 in the cytoplasm, we suspect that the cytoplasmic Rgt2 may directly interact with the cytoplasmic Ycks and that this interaction does not require glucose binding to Rgt2. Of note, glucose-induced Mth1 degradation does not lead to HXT1 expression in akr1Δ cells (Fig. 3C), consistent with the previous report that akr1Δ cells cannot properly activate HXT1 27  www.nature.com/scientificreports/ glucose-induced events occur for Rgt1 to be released from the HXT promoters via two different glucose sensing pathways: degradation of Mth1 via the GSR pathway and, phosphorylation and inactivation of Rgt1 via the Gpr1-PKA pathway 21 . Thus, two glucose sensing pathways converge on Rgt1 to regulate expression of HXT genes. Furthermore, PKA phosphorylation of Rgt1 does not occur until Mth1 is degraded 22 . For this reason, we suspect that in the akr1Δ mutant Mth1 is degraded, but Rgt1 is not phosphorylated.

Rgt2 is phosphorylated in the C-terminal domain in a Yck-dependent manner. The Ycks phos-
phorylate and regulate the stability of many cell surface receptors and transporters [33][34][35][36][37] . To address whether the Ycks phosphorylate Rgt2, Rgt2-HA proteins from wild-type and yck1Δyck2 ts mutant cells were treated with lambda phosphatase and analyzed by Western blotting. Rgt2 from wild-type cells migrates as two bands: a major slower-migrating (upper) band and a minor faster-migrating (lower) band (Fig. 4A, lanes 1 and 3). Phosphatase treatment causes the upper band to disappear and results in the accumulation of the lower band, indicating that the band shift is due to phosphorylation (Fig. 4A, lanes 2 and 4). However, Rgt2 from yck1Δyck2 ts mutant cells does not clearly show the major upper band (Fig. 4A, lane 7); instead, it exhibits faint, smeared bands that are collapsed into a single, lower band upon treatment with phosphatase (Fig. 4A, lane 8), which migrates similarly to phosphatase-treated Rgt2 from wild-type cells (Fig. 4A, lane 2).
Previous studies indicate that the C-terminal domains (CTDs) of the GSRs play an important role in glucose signaling 26,38,39 . The CTDs of the Rgt2 and Snf3 receptors are quite dissimilar, except for a stretch of 25 amino acids (called a signaling motif) that occurs once in the Rgt2-CTD and twice in the Snf3-CTD 26,40 . The Ycks catalyze phosphorylation of a serine or threonine residue in its consensus sequence (SXXS/T*, where the asterisk indicates the phosphorylated residue) 41 , and there are two clusters of potential Yck phosphorylation sites www.nature.com/scientificreports/ in Rgt2-CTD (Fig. 4B, Clusters I and II). To examine the effect of the deletion of these clusters on the stability, phosphorylation, and function of Rgt2, we expressed deletion constructs of individual motifs in wild-type and yck1Δyck2 ts strains. Deletion of each cluster individually has minor or no effect on the stability of Rgt2 or its Yck-mediated phosphorylation (Fig. 4C,D). However, a deletion of up to 97-amino-acids from its C-terminus (Δ667-763) containing both clusters significantly reduces the stability of Rgt2-HA, and the resulting mutant Rgt2-HA expressed in wild-type cells migrates similarly to the protein when it is expressed in yck1Δyck2 ts cells. Thus, this region may contain the Yck phosphorylation site(s). Indeed, deletion of both clusters abolishes the ability of Rgt2 to activate the HXT1 promoter (measured by P HXT1 -lacZ expression), whereas deletion of either cluster alone partially perturbs Rgt2 function, resulting in ~ 70% (cluster I) and ~ 28% (cluster II) reduced HXT1 expression, respectively (Fig. 4E). Thus, Cluster I may have a more important role than Cluster II in glucose signaling. Similarly, when expressed in the P HXT1 -hph reporter strain, Rgt2 with the 97-amino acid deletion (Δ667-763) is unable to activate the HXT1 promoter, confirming an essential role for this region in glucose signaling (Fig. 4F).  The WT strain (BY4742) strain was cotransformed the HXT1-lacZ reporter (pBM3212) with plasmid expressing either the wild type Rgt2 or the indicated mutant Rgt2 proteins. Cells were grown in glucose (2%) or galactose (2%) to mid-log phase and cell extracts were used to assay β-galactosidase activity. (F) The P HXT1 -hph reporter strain (KLS76) expressing indicated Rgt2-HA proteins was scored for growth in a SC-2% glucose plate supplemented with 200 µg/ml hygromycin. The first spot of each row represents a count of 5 × 10 7 cell/ml, which is diluted 1:10 for each spot thereafter. www.nature.com/scientificreports/ 538 Cys ) in the yeast two-hybrid assay (Fig. 5A). Positive interaction between the two proteins was confirmed by expression of the reporter genes HIS3 and lacZ. We find that Yck1 interacts with the Rgt2-CTD and that this interaction is abolished by deletion of the C-terminal half of the Rgt2-CTD (Δ625-763) or the conserved signaling motif (Δ665-Δ696) (Fig. 5B). These results are interesting because the deletion of this motif has little effect on Rgt2 stability and abolishes the ability of Rgt2 to activate the P HXT1 -lacZ reporter (Fig. 5C,D). Furthermore, expression of the Rgt2 receptor lacking this motif (Δ665-Δ696) does not activate the P HXT1 -hph reporter (Fig. 5E) and hence cannot restore the growth defect of the rgt2Δsnf3Δ strain on glucose (Fig. 5F). This motif is known to be required for signaling function, but its role remains unknown 26 . Our results lead us to suggest that Rgt2 may use this motif to interact with the Ycks.

Yck1 interacts with the C-terminal domain (CTD) of
The Ycks are constitutively active and are not regulated by the glucose sensing receptors. Previous work suggested that glucose metabolism is not necessary for glucose signaling by the GSRs: there are dominant mutations in the GSR genes (RGT2-1 and SNF3-1) that cause the receptors to constitutively generate a glucose signal 26,42 . Based on these observations, it has been postulated that the GSRs undergo a conformational change in them in response to glucose that activates the associated Ycks 19 . However, whether the catalytic activity of the Ycks has not been experimentally proven. To address this question, we assessed the kinase activity of Yck1 (7His-ProA-Yck1) from wild-type, rgt2Δsnf3Δ, RGT2-1, and SNF3-1 cells using in vitro kinase assay. If the Ycks are activated by the glucose-bound GSRs, we would expect Mth1 phosphorylation by Yck1 from wild-type cells grown on glucose, but not on galactose, and Yck1 from Rgt2-1 and Snf3-1 mutant cells grown on glucose or galactose. We also would expect Yck1 from rgt2Δsnf3Δ mutant cells not to phosphorylate Mth1. We find, however, that Mth1-myc is phosphorylated by His-ProA-Yck1 from all strains tested, grown on glucose or galactose (Fig. 6A). Cells were grown in glucose (2%) or galactose (2%) and processed from Western blotting as described in Fig. 1A. (D) The WT strain (BY4742) strain was cotransformed the HXT1-lacZ reporter (pBM3212) with plasmid expressing either wild-type Rgt2 (JKP253) or a truncated Rgt2 (Δ665-696, JKP408). β-Galactosidase activity was assayed as described in Fig. 1B. (E,F) The P HXT1 -hph reporter strain (KLS76) expressing indicated Rgt2-HA proteins was scored for growth in a SC-2% glucose plate supplemented with 200 µg/ml hygromycin (E). The rgt2 snf3 double mutant (MSY441) expressing indicated Rgt2-HA proteins were spotted on 2% glucose plate supplemented with Antimycin-A (AA, 1 µg/ml) and SC-2% galactose plate (F). www.nature.com/scientificreports/ To examine whether YCK expression is regulated by the GSRs, we assessed protein levels of Yck1 in wild type and rgt2Δsnf3Δ strains using Western blotting and confocal microscopy. Protein levels of Yck1 (7His-ProA-Yck1) are not significantly different between the wild-type and mutant strains grown with or without glucose, and treatment of the protein synthesis inhibitor cycloheximide (CHX) does not affect Yck1 expression (Fig. 6B). To further explore transcriptional regulation of the YCK1 gene, Yck1 was expressed from the MET25 promoter, which is not regulated by glucose 15 . We find no significant differences in the protein levels of GFP-Yck1 between wild-type and rgt2Δsnf3Δ strains (Fig. 6C). These results provide evidence that the Ycks are constitutively active and that their catalytic activity is not stimulated by the GSRs.

Discussion
The Ycks are widely known as nutrient sensors that regulate cell surface abundance of many nutrient receptors and transporters 33,[35][36][37] . The Ycks are required for glucose signaling through the GSR pathway, but their role is controversial 19,24,31 . Early studies suggest that glucose binding to the GSRs induces a conformational change in them that activates the protein kinase activity of the Ycks, which phosphorylates and inactivates the HXT corepressors Mth1 and Std1, direct targets of the GSR pathway 19,26,42,43 . These results indicate the role of the Yck as downstream kinases of the GSRs that transmit the glucose signal from the cell surface to the nucleus. However, here we provide evidence that the Ycks are not stimulated by the GSRs but are constitutively expressed and active. Mammalian Casein Kinases (CK1 and CK2) including CK1γ (mammalian homolog of Yck1 and Yck2) are constitutively active 44 , and their functions are regulated via targeting to specific subcellular locations 45 . Their activity could be modified by second messengers; however, this has not yet been documented in yeast. Instead, we find that Rgt2 is phosphorylated on the putative Yck consensus phosphorylation sites in its CTD in a Yckdependent manner and that this phosphorylation increases its stability. Thus, the Ycks are likely to act upstream, but not downstream, of the GSRs.
Analysis of constitutively-signaling RGT2 mutations suggests that glucose binding to the GSRs may convert their structures from an outward-facing to an inward-facing, signaling conformation 43 . We believe that the glucose-bound Rgt2, which is in the signaling state, is phosphorylated and stabilized by Ycks. Interestingly, however, we also find that both Rgt2 and Yck1 expressed in an akr1Δ mutant are mislocalized to the cytoplasm, where Rgt2 remains stable and active as a functional receptor. Since many transporters and receptors are destroyed when removed from the plasma membrane [46][47][48][49][50][51] , it is plausible that the cytoplasmic Rgt2 is protected from destruction by interacting with the cytoplasmic Ycks. While this intracellular interaction may occur without www.nature.com/scientificreports/ binding of extracellular glucose to Rgt2, we cannot exclude the possibility of binding of intracellular glucose to the cytoplasmic side of the GSRs, as their glucose binding pocket may be accessible to either the outside or the inside of the cell 43 .
The Ycks regulate stability of many membrane transporters in different ways; Yck activity is required for membrane trafficking of the multidrug transporter Pdr5 to the cell surface 36 , whereas Yck phosphorylation of the uracil permease Fur4 facilitates its ubiquitination and internalization 52 . Rgt2 is endocytosed and degraded when glucose is removed from the medium 25 , but Yck may not be involved in this process. Rgt2 is found not to be properly localized to the plasma membrane but to be colocalized with Ycks to the cytoplasm in an akr1Δ mutant, suggesting a possible role for the Ycks in membrane targeting of Rgt2. One might argue that CTD phosphorylation of Rgt2 by the Ycks may be required for membrane localization of Rgt2 and that this phosphorylation does not occur in the akr1Δ mutant. However, Rgt2 from the akr1Δ mutant migrates similarly to Rgt2 from wild-type cells, indicating that the cytoplasmic Rgt2 may be fully phosphorylated in the akr1Δ mutant (Fig. 2E).
It is not currently known how the glucose signal generated by the GSRs is transmitted from the cell surface to the nucleus. Previous work suggested that the GSR-CTDs interact with Mth1 and Std1 to bring them to the vicinity of the Ycks 19 or that Yck-dependent phosphorylation of the Rgt2-CTD stimulates Mth1 and Std1 interaction with the Rgt2 tail 24 . In this scenario, Mth1 and Std1 must shuttle between the nucleus and the plasma membrane, because they bind to Rgt1 in the nucleus and to the GSRs at the cell surface 19,22 . However, GFP-Mth1 is constitutively nuclear, and the glucose-induced Mth1 degradation occurs in the nucleus 31 . Furthermore, the Ycks are not necessary for glucose signaling in a strain overexpressing RGT2, indicating that the Ycks may not be the kinases responsible for phosphorylation and inhibition of Mth1 and Std1 24 . These observations suggest the involvement of a yet unidentified kinase that catalyzes phosphorylation of Mth1 and Std1 in the nucleus.

Methods
Yeast strains and plasmid construction. The Saccharomyces cerevisiae strains used in this study were listed in Table 1. Yeast strains were grown on YP (2% bacto-peptone, 1% yeast extract) or synthetic yeast nitrogen base medium (0.17% yeast nitrogen base and 0.5% ammonium sulfate) supplemented with appropriate amino acids and carbon sources. Genes were disrupted by homologous recombination using the Hygromycin or KanMX cassette 53,54 . The plasmids used in this study were listed in Table 2. The plasmids were constructed by using standard molecular biology techniques as described previously 25 . Plasmids expressing truncated forms of Rgt2-HA were constructed by QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to manufacturer's protocol.
Yeast membrane preparation. Membrane fractions were essentially prepared, as described previously 25,55 .
Briefly, after washing with phosphate buffer (pH 7.4), the cell pellet was resuspended in ice cold lysis buffer (100 mM Tris-Cl, pH 8, 150 mM NaCl, 5 mM EDTA) containing protease and phosphatase inhibitors and vortexed with acid-washed glass beads. After diluting the samples with the same buffer, membrane enriched fraction was collected by centrifuging the samples at 12,000 rpm for 40 min at 4 °C. The pellet was resuspended in the lysis buffer containing 5 M urea and incubated for 30 min on ice. After centrifuging at 14,000 rpm for 40 min at 4 °C, the pellet was dissolved in SDS buffer (50 mM Tris-HCl (pH, 6.8), 10% glycerol, 2% SDS, 5% β-mercaptoethanol).
Immunoprecipitation and Western blotting. Immunoprecipitation and Western blotting were carried out as described previously 13 . Briefly, yeast cells were disrupted by vortexing with acid-washed glass beads in ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.5, 140 mM NaCl, 0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate) containing protease and phosphatase inhibitors (10 mM Na-pyrophosphate, 200 µM Na-orthovanadate, www.nature.com/scientificreports/ 50 mM Na-flouride). The resulting cell lysates were incubated with appropriate antibodies at 4 °C for 3 h and further incubated with protein A/G-conjugated agarose beads at 4 °C for 1 h. The agarose beads were washed three times with RIPA buffer and boiled in SDS-PAGE buffer. The eluted proteins were subjected to Western blot analysis. For Western blotting, proteins were resolved by SDS-PAGE and transferred to polyvinylidene fluoride membrane (Millipore, Billerica, MA). The membranes were incubated with appropriate antibodies in TBST buffer (10 mM Tris-HCl, pH, 7.5, 150 mM NaCl, 1% Tween-20), and proteins were detected by the enhanced chemiluminescence (ECL) system (BioRad, Hercules, CA, USA) (Supplementary Information).
In vitro protein kinase assay. In vitro protein kinase assay was performed as described previously 21 . Mth1-9xMyc and 7X His-Protein A-tagged Yck1 were affinity-purified using agarose beads as described previously 19 and mixed in 50 µl of kinase buffer containing 0.5 µCi of [γ 32 P] ATP, 100 µM ATP, 10 mM MgCl 2 for 30 min.
After washing the beads with the kinase buffer containing 0.5 M NaCl, the proteins were eluted by boiling the beads in SDS-sample buffer for 5 min. The eluted proteins were resolved by SDS-PAGE and detected by autoradiography. Each set of in vitro kinase assays was independently repeated twice.
Yeast two-hybrid assay. To construct Gal4 DNA-binding domain hybrids (GAL4-DBD-RGT2), the C-terminal domain of RGT2 (encoding amino acids 546-763) was amplified by PCR using JKP253 as a template, and the PCR products were incorporated into the GAL4-DBD plasmid 56 . These plasmids were combined with the GAL4 activation domain hybrid (GAL4-AD-YCK1) and used to transform the yeast strain PJ69-4A 56 to Leu + Trp + . Cells were grown on selective medium (SC-leu-trp) medium lacking histidine (SC-leutrp-his + 20 mM 3-AT) to detect expression of the GAL-HIS3 reporter or medium containing X-gal (SC-leutrp + X-gal) to assay GAL-lacZ report gene expression.

β-Galactosidase assay
β-Galactosidase activity assays were performed using the yeast β-galactosidase assay kit (Pierce) according to the manufacturer's instructions 10 . Results were presented in Miller Units ((1000 × A 420 )/(T × V × A 600 ), where A420 is the optical density at 420 nm, T is the incubation time in minutes, and V is the volume of cells in milliliters). The reported lacZ activities are averages of results from triplicate of usually three different transformants.
Confocal microscopy. GFP-fusion proteins expressed in yeast cells were visualized using a Zeiss LSM 510 META confocal laser scanning microscope with a 63 × Plan-Apochromat 1.4 NA Oil DIC objective lens (Zeiss) 57 . All images documenting GFP localization were acquired with the Zeiss LSM 510 software version 3.2.