Role of casein kinase 1 in the glucose sensor-mediated signaling pathway in yeast
© Pasula et al; licensee BioMed Central Ltd. 2010
Received: 2 December 2008
Accepted: 7 March 2010
Published: 7 March 2010
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© Pasula et al; licensee BioMed Central Ltd. 2010
Received: 2 December 2008
Accepted: 7 March 2010
Published: 7 March 2010
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.
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 glucose-induced, 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.
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.
In the budding yeast Saccharomyces cerevisiae, glucose stimulates its transport across the plasma membrane by inducing expression of several HXT[1–3]. Under glucose-limited conditions, the transcriptional repressor Rgt1 binds to the HXT promoters and recruits general corepressors Ssn6 and Tup1 [4–7]. Rgt1 does this in conjunction with its corepressor Mth1, which physically interacts with Rgt1 [8–10]. Therefore, it has been proposed that Rgt1 forms a repression complex with Mth1, Ssn6, and Tup1 on the HXT promoters, inhibiting transcription . Glucose appears to prevent formation of this protein complex by causing degradation of Mth1, resulting in release of Rgt1 from HXT promoters, thereby inducing expression of HXT[6, 11–14].
The glucose signal that leads to degradation of Mth1 is generated by the plasma-membrane spanning glucose sensors Rgt2 and Snf3. Signal generation is a receptor-mediated process and does not require glucose metabolism. This idea is supported by evidence that dominant mutations exist in the glucose sensor genes that lock the sensor proteins in glucose-bound conformations, generating a constitutive signal [15, 16]. Indeed, Mth1 is constitutively degraded in cells expressing the active glucose sensor mutants . Subsequent studies have shown that the plasma membrane-tethered casein kinases Yck1/2 phosphorylate Mth1, triggering its ubiquitination and subsequent degradation . It has also been shown by yeast-two-hybrid assay that Mth1 interacts with the C-terminal tails of the glucose sensors, suggesting that Mth1 is recruited to the plasma membrane [19–21]. These observations have led to the current view of glucose-induced HXT expression. Upon glucose binding, the glucose sensors are converted from inactive to active forms through a conformational change, activating Yck1/2 in their vicinity. Mth1, recruited by the glucose sensors to the plasma membrane, is phosphorylated by Yck1/2 and, subsequently, ubiquitinated by SCFGrr1. Finally, the ubiquitinated Mth1 is targeted for degradation by the 26S proteasome [12–14, 17].
However, this hypothesis is mainly based on the following assumptions: (1) Mth1 is excluded from the nucleus upon glucose addition and recruited to the plasma membrane by any means, and (2) Yck1/2 are activated through a direct interaction with the glucose sensors. In this study, we specifically tested these assumptions and provide evidence that Yck1/2 do not directly couple to the glucose sensors during transmission of the glucose signal from the plasma membrane to the nucleus. A possible mechanism for how the glucose sensors and Yck1/2 collaborate to degrade Mth1 is discussed.
The yeast glucose sensors convert glucose binding events into an intracellular signal that leads to degradation of Mth1, which is known to require activity of the plasma membrane-tethered Yck1/2 . Because both the glucose sensors and Yck1/2 are associated with the plasma membrane, it has been hypothesized that, upon glucose addition, Yck1/2 are activated through a direct interaction with the glucose sensors and phosphorylate Mth1, triggering its proteasomal degradation.
In this study, we provide several lines of evidence that Mth1 does not shuttle between the nucleus and cytoplasm and is degraded in the nucleus when glucose is present: 1) Mth1 is not excluded from the nucleus in response to glucose (Figure 2); 2) When engineered to be localized to the cytoplasm, Mth1 is degraded in the cytoplasm regardless of the presence of glucose and the glucose sensors (Figure 3); 3) Mth1 is not degraded when Grr1 is removed from the nucleus (Figure 4).
The majority of Yck1/2 targets are plasma membrane proteins. Yck1/2 are responsible for phosphorylating the PEST-like ubiquitination-endocytosis signal of the mating pheromone receptors , and the uracil permease Fur4 . These kinases are also known to regulate the activity of the maltose permease Mal61 , the multidrug transporter Pdr5 , and plasma membrane H+-ATPase . Another important target of Yck1/2 is Ptr3, a component of the yeast amino-acid signaling pathway. Extracellular amino acids trigger activation of the Ssy1-Ptr3-Ssy5 (SPS)-amino acid signaling pathway that leads to induction of endoproteolytic processing of Stp1 and Stp2, enabling them to enter the nucleus and induce expression of the amino acid permease genes [35–38]. This processing requires Yck1/2, the novel chymotrypsin like protease Ssy5, and SCFGrr1[36, 37]. Yck1/2 have been shown to phosphorylate the peripheral plasma membrane protein Ptr3 at Thr-525, increasing Ssy5C (C-terminal activity domain)-dependent proteolytic processing of Stp1 and Stp2 . Mth1 is required to form a repression complex with Rgt1 on the HXT promoters  and appears not to contain endogenous NES-like motifs in its sequence (data not shown). These observations support the idea that Mth1 neither shuttles between the nucleus and cytoplasm nor is excluded from the nucleus. Therefore, it is unlikely that the plasma membrane-tethered Yck1/2 directly phosphorylate the nuclear-localized Mth1.
We also provide evidence that Mth1 degradation is not impaired by the mislocalization of Yck1/2 from the plasma membrane, suggesting that direct interaction between the glucose sensors and Yck1/2 is not required for Mth1 degradation (Figure 5). Although Yck1/2 have been reported to be necessary for Mth1 degradation , our results suggest that they do not exert their function through the glucose sensors. It is not known how the glucose binding to the glucose sensors is converted to an intracellular signal that leads to degradation of Mth1. In this regard, we surmise that there is a yet unidentified kinase that receives the glucose signal and converts it into an intracellular signal. In this scenario, the kinase is recruited to the glucose sensors upon glucose addition and phosphorylated by Yck1/2 at the plasma membrane. Finally, the kinase is translocated from the cytoplasm into the nucleus and catalyzes phosphorylation of Mth1, triggering its degradation by the 26S proteasome.
Glucose-induced, the glucose sensor-regulated Mth1 degradation occurs in the nucleus and requires neither nuclear export of Mth1 nor direct interaction between the glucose sensors and Yck1/2. The glucose sensors transmit their signal across the plasma membrane through a yet unidentified signaling component, not through Yck1/2.
Plasmids used in this study
Mth1-GFP fusion protein (pUG34 or pUG36)
Mth1-GFP fusion protein with NES
Mth1-GFP fusion protein with NES (L changed to A)
Grr1-GFP fusion protein
Grr1-GFP fusion protein without amino acids from 1 to 280 
Yck1-GFP fusion protein
Yck2-GFP fusion protein
Mth1 ID 1
GFP-Mth1 fusion protein without amino acids from 1 to 20
Mth1 ID 2
GFP-Mth1 fusion protein without amino acids from 81 to 90
Mth1 ID 3
GFP-Mth1 fusion protein without amino acids from 88 to 119
Mth1 ID 4
GFP-Mth1 fusion protein without amino acids from 118 to 138
Mth1 ID 5
GFP-Mth1 fusion protein without amino acids from 137 to 157
Mth1 ID 6
GFP-Mth1 fusion protein without amino acids from 156 to 180
Mth1 ID 7
GFP-Mth1 fusion protein without amino acids from 179 to 188
Mth1 ID 8
GFP-Mth1 fusion protein without amino acids from 187 to 200
Mth1 ID 9
GFP-Mth1 fusion protein without amino acids from 198 to 214
Mth1 ID 10
GFP-Mth1 fusion protein without amino acids from 213 to 236
Mth1 ID 11
GFP-Mth1 fusion protein without amino acids from 235 to 262
Mth1 ID 12
GFP-Mth1 fusion protein without amino acids from 261 to 299
Mth1 ID 13
GFP-Mth1 fusion protein without amino acids from 298 to 319
Mth1 ID 14
GFP-Mth1 fusion protein without amino acids from 318 to 327
Mth1 ID 15
GFP-Mth1 fusion protein without amino acids from 326 to 343
Mth1 ID 16
GFP-Mth1 fusion protein without amino acids from 342 to 363
Mth1 ID 17
GFP-Mth1 fusion protein without amino acids from 363 to 380
Mth1 ID 18
GFP-Mth1 fusion protein without amino acids from 390 to 433
Mth1 ID 2 tag-less
Mth1 without amino acids from 81 to 90
Mth1 ID 4 tag-less
Mth1 without amino acids from 118 to 138
Mth1 ID 6 tag-less
Mth1 without amino acids from 156 to 180
Mth1 ID 15 tag-less
Mth1 without amino acids from 326 to 343
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) . All images documenting GFP localization were acquired with the Zeiss LSM 510 software version 3.2. For FRAP of GFP-Mth1, one of the foci was bleached with a laser pulse and the subsequent recovery of fluorescence was monitored .
Western blotting was performed as described previously . Briefly, 5 ml of yeast cells (O.D600 = 1.2) were collected by centrifugation at 3,000 rpm in a table-top centrifuge for 5 min. The cell pellets were resuspended in 100 μl of SDS-buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 5 min. After the lysates were cleared by centrifugation at 12,000 rpm for 10 min., soluble proteins were resolved by SDS-PAGE and transferred to PVDF membrane (Millipore). The membranes were incubated with appropriate antibodies in TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) and proteins were detected by the enhanced chemiluminescence (ECL) system (Pierce).
To assay β-galactosidase activity with yeast cells expressing appropriate lacZ reporters, yeast cells were grown to mid-log phase and assay was performed as described previously . Results were reported in Miller Units [(1,000 × OD420)/(T × V × OD600), where OD420 was the optical density at 420 nm, T was the incubation time in minutes, and V is the volume of cells in milliliters]. The reported enzyme activities were averages of results from triplicates of three different transformants.
This work was supported by R01GM087470 (JHK) from the National Institute of General Medical Sciences (NIGMS) and RR016476-08 (MS-INBRE) from the National Center for Research Resources (NCRR).
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