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Genetics. Mar 2006; 172(3): 1427–1439.
PMCID: PMC1456300

Glc7–Reg1 Phosphatase Signals to Yck1,2 Casein Kinase 1 to Regulate Transport Activity and Glucose-Induced Inactivation of Saccharomyces Maltose Permease

Abstract

The Saccharomyces casein kinase 1 isoforms encoded by the essential gene pair YCK1 and YCK2 control cell growth and morphogenesis and are linked to the endocytosis of several membrane proteins. Here we define roles for the Yck1,2 kinases in Mal61p maltose permease activation and trafficking, using a yck1Δ yck2-2ts (yckts) strain with conditional Yck activity. Moreover, we provide evidence that Glc7–Reg1 phosphatase acts as an upstream activator of Yck1,2 kinases in a novel signaling pathway that modulates kinase activity in response to carbon source availability. The yckts strain exhibits significantly reduced maltose transport activity despite apparently normal levels and cell surface localization of maltose permease protein. Glucose-induced internalization and rapid loss of maltose transport activity of Mal61/HAp-GFP are not observed in the yckts strain and maltose permease proteolysis is blocked. We show that a reg1Δ mutant exhibits a phenotype remarkably similar to that conferred by yckts. The reg1Δ phenotype is not enhanced in the yckts reg1Δ double mutant and is suppressed by increased Yck1,2p dosage. Further, although Yck2p localization and abundance do not change in the reg1Δ mutant, Yck1,2 kinase activity, as assayed by glucose-induced HXT1 expression and Mth1 repressor stability, is substantially reduced in the reg1Δ strain.

IN Saccharomyces cerevisiae, the addition of glucose to maltose fermenting cells causes a very rapid loss of maltose transport activity and proteolysis of maltose permease (Medintz et al. 1996). This glucose-induced inactivation of maltose permease requires endocytosis, vesicle trafficking, and vacuolar proteases. Inactivation is dependent on gene functions involved in these processes, including END3, which encodes an early function in endocytosis; VPS2, an ESCRT complex component needed for delivery of membrane proteins to the endosome and vacuole; and PEP4, encoding the major vacuolar protease (Medintz et al. 1996; Babst et al. 2002). Also, Medintz et al. (1998) demonstrated glucose-stimulated ubiquitination of maltose permease and found that degradation of maltose permease requires the ubiquitin conjugation enzymes, particularly Rsp5 ubiquitin ligase, and is blocked in doa4Δ mutants, which are depleted for available intracellular ubiquitin.

Two glucose sensing and signaling pathways stimulate glucose-induced inactivation of maltose permease (Jiang et al. 1997). Pathway 1 senses high extracellular glucose concentrations by means of the integral membrane protein Rgt2p, a nontransporting homolog of the Hxt glucose transporter family of sugar transporters (Ozcan et al. 1996). Pathway 2 measures glucose uptake by monitoring the rate of glucose metabolism through the early steps of glycolysis (Hu et al. 2000; Jiang et al. 2000a). Jiang et al. (2000b) found that deletion of REG1, encoding a targeting subunit of Glc7 protein phosphatase type 1, blocks signaling via pathway 2. Additional results from this study suggested that Glc7–Reg1 phosphatase does not act directly on maltose permease, since loss of Reg1p decreases rather than increases phosphorylation of the permease. Thus, Jiang et al. (2000b) proposed that an as-yet-unidentified kinase acts downstream of Glc7–Reg1 phosphatase in pathway 2 and could be directly or indirectly responsible for maltose permease phosphorylation and possibly for glucose-induced inactivation.

Plasma-membrane-localized casein kinase 1 encoded by YCK1 and YCK2 is a likely candidate for the downstream kinase activity of pathway 2. Saccharomyces encodes four casein kinase 1 isoforms. YCK1 and YCK2 encode plasma-membrane-localized isoforms with >90% similarity between their catalytic domains (Robinson et al. 1992; Wang et al. 1992; Vancura et al. 1993). YCK1 and YCK2 have an essential redundant function in cell growth and morphology, but both YCK1 and YCK2 act as multicopy suppressors of the sucrose-nonfermenting phenotype caused by loss of SNF4 function (Robinson et al. 1992). SNF4 encodes a positive effector of the Snf1 protein kinase, which is required for derepression under glucose growth conditions.

The plasma membrane localization of the Yck1,2 kinases, and their identification as suppressors of the glucose derepression defect of the snf4 mutant, support the possibility that Yck1,2 could provide the downstream kinase activity of pathway 2. Additional studies are also consistent with this proposal. Yck1,2 kinase activity stimulates the internalization of several Saccharomyces plasma membrane proteins, including the Ste2 α-factor receptor (Hicke 1999), the Ste3 a-factor receptor (Panek et al. 1997; Feng and Davis 2000), and Fur4 uracil permease (Marchal et al. 1998). Moreover, Feng and Davis (2000) report that the Yck1,2 kinases are required for Ste3p phosphorylation. In studies of HXT gene regulation by the Rgt2 glucose sensor, evidence was presented indicating that the Yck1,2 kinases promote the phosphorylation of Rgt2p-bound Mth1p and Std1p, leading to their degradation and to the inactivation of the Rgt1 repressor (Moriya and Johnston 2004). Therefore, we explored the role of Yck1,2 casein kinase 1 activity in the glucose-induced inactivation of maltose permease and investigated the possibility that the Yck1,2 kinases act with the Glc7–Reg1 phosphatase in this glucose-signaling pathway.

MATERIALS AND METHODS

Strains and plasmids:

Strains LRB756, LRB906, and LRB1082 used here are closely related, differing at the YCK loci. Strains LRB906 (MATa YCK1 YCK2 his3 leu2 ura3) and LRB756 (MATa his3 leu2 ura3-52 yck1-1Δ::ura3 yck2-2ts) have been described (Panek et al. 1997; Babu et al. 2002). Both strains carry defective copies of MAL1 (MAL11 MAL12 mal13) and MAL3 (MAL31 MAL32 mal33) loci, encoding functional copies of maltose permease and maltase and a nonfunctional copy of the MAL-activator gene. Thus, strains LRB906 and LRB756 do not ferment maltose and require a plasmid-borne copy of the MAL-activator gene for expression of the MAL structural genes. For this we used pUN90-MAL63 and YCp50-MAL63 carrying inducible MAL63 in the CEN vectors pUN90 (Elledge and Davis 1988; Gibson et al. 1997) and YCp50 (Gibson et al. 1997), respectively.

PCR-based one-step gene replacement was used to construct CMY7000 (MATa YCK1 YCK2 his3 leu2 ura3 reg1Δ::kanR) and LRB1082 (MATa his3 leu2 ura3-52 yck1-1Δ yck2-2ts reg1Δ::kanR) from strains LRB906 and LRB756, respectively (Longtine et al. 1998).

Strain KT1112 (MATa leu2 ura3-52 his3 GLC7) and the otherwise isogenic glc7 mutant series KT1636 (glc7-133), KT1639 (glc7-132), KT1967 (glc7-127), KT1638 (glc7-109), and TW267 (glc7-256) are described in Baker et al. (1997) and Wu and Tatchell (2001) and were obtained from Kelly Tatchell, LSU Health Sciences Center, Shreveport, Louisiana.

Strain CMY1025 (MAL1 doa4Δ::LEU2) is a meiotic segregant from the cross of CMY1001 (MAL1 DOA4) and PMY270 (mal1 doa4Δ::LEU2) as described in Medintz et al. (1998).

Plasmids pYCK1 (pLJ721) and pYCK2 (pLS2.3) carry YCK1 and YCK2 on the high-copy vector YEp352 (Robinson et al. 1992). Plasmid DF041, a genomic REG1 clone (Nasmyth and Tatchell 1980) in the 2μ high-copy vector YEp13, was obtained from Kelly Tatchell.

Plasmid pRS315-MAL61/HA carries an HA-tagged MAL61 maltose permease allele under the control of its native promoter (Medintz et al. 1996). Plasmid pUN30-MAL61/HA-GFP was constructed by inserting a 0.8-kb SalI fragment encoding the GFP ORF amplified from plasmid pGFP-C-FUS (Niedenthal et al. 1996) by PCR into an XhoI site created at the 3′-end of the MAL61/HA ORF, to produce an in-frame MAL61/HA-GFP fusion. The construct was confirmed by the presence of a diagnostic NcoI site and by sequence analysis. A 4.4-kb SacI–SalI fragment containing the MAL61/HA-GFP gene was subcloned from plasmid pUN30-MAL61/HA-GFP into vector pUN70 to produce plasmid pUN70-MAL61/HA-GFP. GFP-tagged maltose permease is correctly delivered to the plasma membrane and transports maltose with the same efficiency as does Mal61/HA permease (N. Gadura and C. A. Michels, unpublished results).

Plasmid pBM3212 carries the HXT1 promoter-lacZ reporter gene in the multicopy LEU2 vector YEp367R (Ozcan et al. 1996). pBM4560 (Moriya and Johnston 2004) carries an allele of MTH1 in which a sequence encoding nine copies of the Myc-epitope (EQKLISEED) was inserted at the 3′-end of the ORF to encode a C-terminal 9xMyc-tagged Mth1 repressor. Both plasmids were obtained from Mark Johnston, Washington University Medical School.

Inactivation protocol:

The standard maltose permease inactivation assay protocol (Medintz et al. 1996) was used for these studies with a few variations. Briefly, cells were grown at 30° to early log phase (OD600 0.1–0.3) in selective media containing 2% maltose, harvested by filtration, and resuspended in nitrogen starvation media plus 2% glucose, referred to as YNSG. Cycloheximide (CHX) (30 μg/ml) was added to the cell suspension at time zero to inhibit protein synthesis. Three aliquots were taken at time zero and every hour to 3 hr. Cells of aliquot 1 were harvested by filtration and frozen immediately at −80° to be used for Western analysis. Cells of aliquot 2 were used to assay maltose transport activity. Cells of aliquot 3 were used to determine “growth dilution,” which is calculated as the OD600 at time zero divided by OD600 at time x.

Maltose transport assay:

Cells were harvested by filtration and resuspended in 0.1 m tartaric acid, pH 4.2. Maltose transport is measured as the uptake of 1 mm 14C-labeled maltose as described by Cheng and Michels (1991) and Medintz et al. (1996). Assays were done in duplicate on at least three independent transformants. The standard error was <15%.

Enzyme assays:

Maltase activity was assayed as described in Dubin et al. (1985). Activity is expressed as nanomoles of p-nitrophenyl β-d-glucopyranoside (PNPG) hydrolyzed per milligram of total protein per minute. Assays were performed using total cell extracts prepared from at least duplicate cultures. The values reported are the average of duplicate assays and varied by ~15%.

β-Galactosidase was assayed in at least three independent transformants using total cell extracts (Hu et al. 1999). Activity is expressed as nanomoles of o-nitrophenyl β-d-galactopyranoside hydrolyzed per milligram of total protein per minute. The values reported are the average of duplicate assays of at least duplicate cultures and varied by ~20%.

Western blotting:

At each time point 15 optical density (OD) units of cells (grown to an OD600 0.3–0.5) were harvested by filtration on nitrocellulose filters (0.45 μm), washed with KPO4 plus 2% NaAzide pH 7.4, and frozen immediately at −80° until used for preparation of protein extracts. Total cell extracts were prepared by thawing cells in HEPES buffer pH 7.5 supplemented with a protease inhibitor cocktail that contains AEBSF, pepstatinA, and E-64 and 1,10-phenanthroline (Sigma-Aldrich P8215) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich P5726 and P2850). The cells were harvested by centrifugation and resuspended in extraction buffer (40 mm TrisCl, pH 6.8, 8 m urea, 0.1 mm EDTA, 1% β-mercaptoethanol, and 5% SDS) plus the protease inhibitor and phosphatase inhibitor cocktails according to the manufacturer's recommendations. The cell suspension was vortexed with glass beads (425–600 μm) for 15 min at 4° and solubilized for 15 min at 37° followed by 2 min of vortexing. Cell debris was removed by centrifugation for 5 min and the total protein extract was boiled for 3 min. Protein levels were assayed with a protein assay kit from Sigma-Aldrich (P5656). Equal amounts of protein were loaded per lane of 10% SDS–PAGE gels for monitoring protein levels or 7.5% SDS–PAGE gels for separation of differentially phosphorylated species. Mal61/HA protein was detected using anti-HA antibody (Boehringer Mannheim, Indianapolis), Mal61-GFP protein was detected using anti-GFP antibody (Santa Cruz), and Mth1-9xMyc protein was detected using anti-Myc antibody (Roche Diagnostic). Each membrane was also probed with anti-phosphoglycerate kinase (anti-PGK) antibody (Molecular Probes, Eugene, OR) as a loading control. Protein levels were visualized using the Vistra-ECF kit (Amersham, Buckinghamshire, UK) and a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Signal was quantified using software provided by the manufacturer. Loading variations were corrected for by normalizing to the PGK signal. Values presented are the average of results from experiments carried out on at least three independent transformants and the standard deviation varied from 5 to 10%. The immunoblots shown in Figures 1, 3, 4, 6, and 7 are representative of typical results.

The percentage of phosphorylated Mal61 protein was determined from the results of the Western blots quantified using the Storm 860 PhosphorImager. The percentage was calculated as the ratio of the amount of signal in the slowly migrating (highly phosphorylated) species divided by the total Mal61/HAp signal including the full range of migrating species. The results reported are from cell extracts prepared from at least duplicate cultures of three independent transformants.

Fluorescence microscopy:

A Meridian/Olympus IMT-2 confocal microscope equipped with a ×100 oil, NA 1.40 lens, FITC filter set, and phase optics was used to visualize the GFP fluorescent signal. The imaging parameters were constant for all images, with the exception of yckts cells, for which the PMT value was increased by twofold. Images were processed using Adobe Photoshop. All images shown are representative of experiments done on three independent transformants.

RESULTS

Yck1,2 casein kinase 1 activity is required for maltose permease transport activity and glucose-induced inactivation:

We first tested whether Yck1,2 kinase activity is required for any aspect of maltose permease expression or glucose-induced inactivation. HA-tagged Mal61/HA maltose permease was expressed in LRB906 (YCK1 YCK2) and LRB756 (yck1Δ yck2ts), referred to here as yckts, by growth in maltose-induced conditions and subjected to the glucose inactivation protocol described in materials and methods. The inactivation protocol used here differs slightly from that used previously by our laboratory (Medintz et al. 1996, 1998, 2000; Jiang et al. 1997, 2000b) in that CHX is added when the cells are transferred to YNSG. In addition, the buffer used to prepare total cell extracts contains phosphatase inhibitors, which, in conjunction with the use of 7.5% PAGE gels, allowed us to evaluate maltose permease phosphorylation.

Figure 1A shows glucose-induced changes in both maltose transport activity and maltose permease protein levels in cells incubated at 24°, the permissive temperature for the yckts strain. In the parental strain, as was shown for other strain backgrounds (Medintz et al. 1996), glucose induces a very rapid loss of maltose transport activity and a somewhat slower rate of Mal61/HA proteolysis. This difference is observed for most but not all strains and we believe that it results from a more rapid rate of permease internalization than vesicle delivery to the vacuole for degradation. While approximately the same level of maltose permease protein is detected in both the YCK1 YCK2 and yckts strains at the start of the experiment (Figure 1, B and C), the level of maltose transport activity expressed by the yckts strain is only ~15% of that expressed in the wild-type strain (Figure 2). Glucose-induced proteolysis of maltose permease is essentially blocked in the yckts strain, such that Mal61/HA protein levels remain constant over the 3 hr following glucose addition (Figure 1A). However, the rapid glucose-induced loss of maltose transport activity is not observed in the yckts strain. Instead, glucose induces an increase of approximately sevenfold in maltose transport activity. Finally, consistent with previous reports that the yckts mutant shows defects even at the permissive temperature (Panek et al. 1997), there was no obvious difference in the results shown here carried out at 24° and those obtained when the cells were shifted to 35° prior to glucose inactivation (data not shown).

Figure 1.
Glucose-induced inactivation of maltose permease in yck1Δ yck2ts and YCK1,2 overexpressing strains. (A) Strains LRB906 (YCK1 YCK2) and LRB756 (yck1Δ yck2ts), referred to as yckts, were transformed with plasmids pMAL61/HA, pMAL63, and YEp352, ...
Figure 2.
Comparison of maltose transport activity of yckts and reg1Δ mutant strains. Strains LRB906 (YCK1 YCK2 REG1), LRB756 (yckts), and CMY7000 (reg1Δ) were transformed with plasmids pMAL61/HA and pMAL63 and plasmids YEp352, YEp352-YCK1, YEp352-YCK2, ...

The low level of maltose activity in the yckts strain could result from defective localization to the cell surface. We examined the localization of a functional GFP-tagged Mal61 protein in maltose-grown wild-type and yckts strains by confocal microscropy. Mal61/HA-GFP protein is expressed in wild-type and yckts cells at comparable levels (data not shown) and is present mainly in the plasma membrane in both strains (Figure 1B). In wild-type cells, a significant amount of fluorescence is observed in the vacuole, but only a faint vacuolar signal is present in the yckts cells. Thus, Yck1,2 kinase activity does not affect synthesis or plasma membrane localization of maltose permease protein but is required for its transport activity.

The dramatic glucose-induced increase in transport activity in the yckts strain, which occurs in the absence of new maltose permease synthesis, could result from changes in permease localization and/or specific activity. To distinguish between these possibilities, we followed localization of Mal61/HA-GFP after the addition of glucose. In the wild-type strain, the level of maltose permease at the cell surface is dramatically reduced by 1 hr after glucose addition, while fluorescence in the vacuole appears to be enhanced (Figure 1B). Over the course of 3 hr the vacuolar signal gradually declines, presumably due to degradation of the GFP-tagged protein. The results are quite different for the yckts strain. Both cell surface and vacuolar fluorescence remain constant during the 3-hr duration of the experiment. These results are consistent with the complete lack of glucose-induced proteolysis observed for Mal61-HA permease in the yckts strain, but fail to explain the dramatic glucose-induced increase in maltose transport activity observed for the yckts strain. We propose that this increased transport activity results from a glucose-induced stimulation of preexisting plasma-membrane-localized permease. It is interesting to note that glucose addition increases maltose transport activity to approximately the wild-type level, suggesting that the defect in permease activity in the yckts strain can be overcome by a glucose-stimulated event.

We have observed previously that Mal61/HA maltose permease migrates as a number of different mobility species on SDS–PAGE, of which two generally appear to predominate, and determined that these bands correspond to differentially phosphorylated species (Medintz et al. 1996). The slower-migrating (top band in Figure 1C) band is the phosphorylated species, since treatment with acid phosphatase causes the protein to migrate only at the position of the bottom band, presumably the hypophosphorylated species. We tested whether phosphorylation of Mal61p requires Yck1,2 kinase activity by comparing the migration of Mal61/HA protein isolated from wild-type and yckts strains. Approximately 80% of the Mal61/HA protein from the wild-type strain runs more slowly in the phosphorylated state, while from yckts cells only ~30% of the permease protein is phosphorylated (Figure 1C; compare lanes 1 and 2). Yck1p and Yck2p overproduction in the wild-type strain caused much higher relative amounts of the hyperphosphorylated species of maltose permease (Figure 1C, lanes 3 and 4) but no concomitant increase in maltose transport activity (Figure 2). The rate of glucose-induced loss of maltose transport activity does not change significantly upon Yck1,2 kinase overproduction, but the rate of Mal61/HA permease proteolysis increases dramatically to match the rate of rapid loss in transport activity (Figure 1A). These results are consistent with previous reports that protein phosphorylation, particularly by the Yck2 and Pkc1 kinases, regulates trafficking of plasma membrane proteins to the vacuole (D'Hondt et al. 2000; Friant et al. 2000; Marchal et al. 2000, 2002).

The Yck1,2 kinases require palmitoylation of a C-terminal di-cysteine motif for their plasma membrane localization and essential cellular function (Roth et al. 2002; Babu et al. 2004). Akr1p is the palmitoyl transferase responsible for this modification (Roth et al. 2002). AKR1 mutants exhibit reduced phosphorylation of Ste3p, which presumably reflects at least in part the lack of membrane-associated Yck1,2 protein (Feng and Davis 2000). We found that the phenotype of an akr1Δ strain is nearly identical to that of the yckts (data not shown).

Together, these results indicate that plasma-membrane-associated casein kinase 1 encoded by YCK1 and YCK2 regulates the transport activity of Mal61 maltose permease, is required for an early step in the glucose-induced internalization of the permease, and regulates the rate of permease-containing vesicle trafficking to the vacuole and/or permease vacuolar degradation.

Yck1,2 kinase activity acts upstream of DOA4-mediated ubiquitination:

Glucose-regulated degradation of maltose permease requires ubiquitin conjugation (Medintz et al. 1998). Glucose stimulates the ubiquitination of Mal61/HA maltose permease and mutations in RSP5, encoding a HECT domain ubiquitin ligase, and in DOA4, encoding a ubiquitin hydrolase whose loss causes depletion of intracellular ubiquitin levels, leading to defects in glucose-induced proteolysis of Mal61/HAp. Ste2p and Ste3p require Yck1,2-dependent phosphorylation for the ubiquitination that stimulates internalization (Hicke et al. 1998; Feng and Davis 2000). Since Yck1,2 activity is required for Mal61 permease internalization, we tested genetically whether Yck1,2 kinase function is upstream of the ubiquitination step.

MAL61/HA-GFP was expressed in the doa4 null strain CMY1025 with or without a high-copy plasmid carrying YCK2. Transformants were grown in maltose-induced conditions and subjected to the standard inactivation protocol and Mal61/HA-GFP localization was followed for 3 hr by confocal microscropy. The results are shown in Figure 3. Abundant Mal61/HA-GFP protein is observed at the cell surface of the doa4Δ mutant cells but little or no signal is visible in the vacuole, as expected. Functional levels of cell-surface-localized permease are not significantly altered in the doa4Δ mutant since maltose transport activity is only slightly lower than that observed in a congenic DOA4 strain (4.6 vs. 3.6 nmol maltose/mg dry wt of cells/min). Glucose addition has little effect on maltose permease in doa4Δ cells. Maltose transport activity and cell surface localization remain approximately constant up to 3 hr after the addition of glucose and no proteolysis of maltose permease protein or vacuolar accumulation is evident after 3 hr.

Figure 3.
Multicopy YCK2 does not suppress the block in glucose-induced inactivation of maltose permease observed in a doa4Δ mutant. Strain CMY1025 (MAL1 doa4Δ::HIS3) was transformed with pUN30-MAL61/HA-GFP and the empty vector YEp352 or ...

Overexpression of YCK2 causes a significant increase of the rate of maltose transport in the doa4Δ strain (6.0 vs. 3.6 nmol maltose/mg dry wt of cells/min) with little or no apparent increase in cell surface permease protein (Figure 3). Most important, multicopy YCK2 does not suppress the block in glucose-induced inactivation in a doa4Δ mutant. Maltose transport activity is stimulated ~10% following glucose addition and remains above initial levels for up to 3 hr. It is important to note that despite higher levels of phosphorylated Mal61/HA-GFP protein in the doa4Δ [pYCK2] strain, no glucose-induced Mal61/HA-GFP permease proteolysis is observed and Mal61/HA-GFP permease remains at the plasma membrane. Thus, elevated levels of Yck2 kinase activity do not suppress the doa4Δ defects in maltose permease glucose-induced inactivation as predicted if Yck1,2-dependent phosphorylation is the upstream event.

Loss of REG1 causes defects in maltose permease localization similar to those of the yckts mutant:

We previously reported that deletion of REG1 causes resistance to glucose-induced proteolysis of maltose permease, reduced rather than increased levels of maltose permease phosphorylation, and increased maltose transport activity upon glucose addition (Jiang et al. 2000b). Given the phenotypic similarity between the reg1Δ and yckts mutants, we investigated maltose permease localization in a reg1Δ strain. MAL61/HA-GFP was expressed in strain CMY7000 (reg1Δ) and localization of Mal61/HA-GFP permease was followed during the 3-hr course of glucose-induced inactivation. At the outset of the experiment, most GFP fluorescence is seen at the cell surface, with no significant accumulation in the vacuole (Figure 4A). This localization pattern is virtually identical to that observed for the otherwise isogenic yckts strain shown in Figure 1B. Moreover, addition of glucose to reg1Δ cells does not alter the pattern of localization. Mal61/HA-GFP fluorescence remains at the cell surface, with little or no localization to the vacuole. Thus, like yckts, reg1Δ does not appear to affect maltose permease protein synthesis or plasma membrane localization but does block vacuolar localization and glucose-induced internalization.

Figure 4.
Effects of reg1 deletion and REG1 overexpression on glucose-induced inactivation of maltose permease. (A) Strain CMY7000 (reg1Δ) was transformed with plasmids pUN70-MAL61/HA-GFP and pUN90-MAL63. Transformants were grown at 30° to midlog ...

Since previous studies (Jiang et al. 2000b) were carried out using strains of a different genetic background, we examined the effect of reg1Δ on glucose inactivation of maltose permease in the background used in this study, comparing these effects with those of yckts. The results are reported in Figures 2 and and44.

The level of transport activity in the reg1Δ strain on maltose is about eightfold lower than that observed in the parental strain and similar to that of yckts. This reduced transport activity is not reflected either in the level of maltose permease protein detected by immunoblot (Figure 4C) or in our estimate of the fluorescent signal in the reg1Δ and parental strains (Figures 1B and and4A).4A). Therefore, the reduced transport activity observed in the reg1Δ mutant does not result from reduced permease protein expression or mislocalization, suggesting that maltose transport activity of maltose permease is dependent either directly or indirectly on REG1. Jiang et al. (2000b) reported only about a 40% decrease in maltose transport activity in a reg1Δ mutant of different genetic background. Nonetheless, the impact of reg1Δ on the maltose permease phenotypes reported here is qualitatively comparable in both strain backgrounds.

Approximately 70–75% of maltose permease protein is in the hypophosphorylated form in the reg1Δ strain (Figure 4C). This decrease in bulk phosphorylation is similar to that reported previously (Jiang et al. 2000b) for a reg1Δ mutant, as well as to the decrease shown here for the yckts strain. Other aspects of the inactivation phenotype of the reg1Δ strain also are similar to that of the yckts mutant. Glucose fails to stimulate proteolysis of maltose permease, but maltose transport activity increases slightly but reproducibly during the 3 hr following transfer to YNSG, although not to the same extent observed for yckts cells (Figures 1A and and4B4B).

In contrast, overexpression of REG1 in an otherwise wild-type strain increases the rate of maltose permease proteolysis such that it now matches the rapid rate of loss of maltose transport activity (Figure 4B). This is similar to the effects of overexpressing YCK1 or YCK2. Moreover, overexpression of REG1 leads to accumulation of nearly all of the Mal61/HA protein as the phosphorylated species. These results are consistent with the idea that phosphorylation of the permease is positively regulated by Reg1p.

The requirement for Reg1p in glucose-induced inactivation of maltose permease likely reflects a requirement for PP1 activity:

Reg1p is a regulatory subunit of protein phosphatase type 1, for which Glc7p is the catalytic component in yeast. Binding of Reg1p to Glc7p is enhanced in the presence of glucose and is required for glucose repression (Tu and Carlson 1995; Sanz et al. 2000). We used a group of previously characterized glc7 mutant alleles (Baker et al. 1997) to test whether Reg1 function in this pathway is as a Glc7p regulatory subunit by determining whether alleles conferring insensitivity to glucose repression, which correlates with loss of Reg1p–Glc7p binding, also cause resistance to glucose-induced inactivation of maltose permease. We selected glc7-109, glc7-127, glc7-132, and glc7-133 strains (Baker et al. 1997). Strains carrying glc7-127 and glc7-133 are resistant to 2-deoxyglucose, a nonmetabolized glucose analog that induces glucose repression (Neigeborn and Carlson 1987). We also tested glc7-256 in these assays. This allele alters a residue in the hydrophobic cleft on Glc7p, which is essential for binding to a common regulatory subunit motif, V/IXF (Wu and Tatchell 2001). Reg1p fails to interact with Glc7-256 mutant protein in the two-hybrid assay (Wu and Tatchell 2001).

The wild-type GLC7 strain and strains carrying glc7 mutant alleles were transformed with plasmid-borne MAL61/HA and MAL63, and transformants were assayed for glucose repression of maltase expression and glucose-induced inactivation of maltose permease. As shown in Table 1, maltose induction of maltase expression is blocked by the presence of glucose in strains carrying glc7-109 and glc7-132. These mutants are sensitive to repression by 2-deoxyglucose and are presumed to carry alterations in binding sites of Glc7p-targeting subunits other than Reg1p (Baker et al. 1997). On the other hand, strains carrying glc7-127, glc7-133, and glc7-256, those alleles shown to be resistant to 2-deoxyglucose or to prevent Reg1p binding (Baker et al. 1997; Wu and Tatchell 2001), are resistant to glucose repression of maltase expression (Table 1).

TABLE 1
Glucose-repression sensitivity of maltase expression in GLC7 mutants

Glucose effects on maltose permease in this same group of glc7 mutant strains are presented in Figure 5. Strains carrying GLC7 or mutant alleles glc7-109 and glc7-132, which are sensitive to glucose repression, undergo similar glucose-induced inactivation of maltose permease. However, glucose-repression-insensitive glc7-127, glc7-133, and glc7-256 mutants fail to exhibit glucose-induced inactivation of maltose permease. Thus, insensitivity to glucose-induced inactivation of maltose permease correlates with the glucose repression insensitivity of GLC7 mutants. Consistent with the studies reported above for the reg1Δ strain, these results suggest that Glc7p–Reg1p interaction, and thus Reg1p-targeted PP1 phosphatase activity, is required for glucose to stimulate maltose permease endocytosis and proteolysis.

Figure 5.
Glucose-induced inactivation of maltose permease in glc7 mutants encoding the catalytic subunit of protein phosphatase type 1. Strains KT1112 (GLC7), KT1636 (glc7-133), KT1639 (glc7-132), KT1967 (glc7-127), KT1638 (glc7-109), and TW267 (glc7-256) were ...

Epistasis analysis places YCK activity downstream of REG1:

With the exception of the difference in the extent of the glucose-induced increase in maltose transport activity, yckts and reg1Δ have very similar effects on maltose permease subcellular localization, transport activity, phosphorylation, and resistance to glucose-induced inactivation. Therefore, we propose that the Yck1,2 kinase and Glc7–Reg1 phosphatase are components of a common glucose-signaling pathway and explore the relationship between these two regulators.

We constructed a reg1Δ yckts mutant, which is viable and grows on maltose with a doubling time comparable to that of the yckts mutant (data not shown). This strain was transformed with plasmid-borne MAL61/HA and glucose-induced inactivation was assayed. The phenotype of the reg1Δ yckts double mutant strain is similar to that of either single mutant with regard to maltose transport activity, maltose permease phosphorylation, and insensitivity to glucose-induced inactivation of maltose permease (Figure 6). Thus, the reg1Δ yckts mutant does not exhibit enhancement of the phenotype of the single-mutant strains, supporting our hypothesis that Yck1,2 kinase and Reg1–Glc7 phosphatase act in a common pathway. However, the sevenfold glucose-induced increase in maltose transport activity of the yckts strain is not observed in the reg1Δ yckts double mutant. Rather, we observed a slight increase similar to that of the reg1Δ mutant, suggesting that reg1Δ is epistatic to yckts in this glucose-signaling pathway.

Figure 6.
Epistasis analysis places GLC7-REG1 upstream of YCK1 in glucose-induced inactivation of maltose permease. Strains CMY7000 (reg1Δ) and LRB756 (yckts) were transformed with pRS315-MAL61/HA, pUN90-MAL63, and YEp352, pYCK1, or DF041 (REG1). LRB1082 ...

To determine the order of function of these two activities, we examined the effect of high-copy YCK1 on glucose-induced Mal61 inactivation in a reg1Δ strain and of high-copy REG1 in the yckts strain. Overexpression of REG1 in the yckts strain has little effect on the yckts phenotype (Figure 6A). Following glucose addition to the yckts [pREG1] strain, a modest increase in the rate of glucose-induced proteolysis of maltose permease is observed and maltose transport rates increase gradually and only about twofold. In contrast, overexpression of YCK1 in the reg1Δ strain fully rescues the reg1Δ phenotype (Figure 6A). The rapid glucose-induced loss of maltose transport activity is restored and the rate of maltose permease degradation is significantly faster than that observed in the parental strain (Figure 1A). The rate of permease degradation is comparable to that observed in the wild-type strain carrying multi-copy YCK1 (Figure 1A) and parallels the rate of loss of transport activity.

We compared the initial rates of maltose transport for these strains to those obtained for the wild-type and parental mutant strains (Figure 6B). The reg1Δ, yckts, and reg1Δ yckts strains all show a similar very low rate of transport activity, and this rate is not enhanced when REG1 is overexpressed in the yckts strain. However, overexpression of YCK1 in the reg1Δ strain rescues maltose transport activity to almost wild-type levels. Together, these results are consistent with Reg1p acting upstream of the Yck1,2 kinases.

Figure 6C compares the morphology of wild-type, reg1Δ, yckts, reg1Δ yckts, reg1Δ [pYCK1], and yckts [pREG1] strains grown to midlog in 2% maltose at room temperature. A predominance of large-budded cells in the reg1Δ strain is clearly observed. Jiang et al. (2000b) reported that this is associated with a G2 delay in a reg1Δ strain. Overexpression of YCK1 restores an apparently normal bud-size distribution. The elongated bud morphology previously observed in yckts strains (Robinson et al. 1993) is evident under these growth conditions and is not significantly enhanced in the reg1Δ yckts double-mutant strain, although multiple-budded cells are slightly more frequent in this strain. Interestingly, although overexpression of REG1 has no effect on the temperature-sensitive growth of the yckts strain (data not shown), it suppresses the morphology phenotype significantly. Dramatically fewer elongated buds and no multiple-budded cells are seen.

Yck1,2 kinase is inactive in a reg1Δ strain:

The simplest explanation of our results so far is that Reg1–Glc7 phosphatase has a positive effect on Yck1,2 protein levels, localization to the plasma membrane, or activity. We did not observe any change in Yck2 protein levels or plasma membrane localization in the reg1Δ strain (L. C. Robinson, unpublished results). Therefore, we tested whether Yck1,2 kinase activity is compromised in the reg1Δ mutant. Mth1p and Std1p are repressors of HXT1 expression (Schmidt et al. 1999; Flick et al. 2003; Lakshmanan et al. 2003; Kaniak et al. 2004; Moriya and Johnston 2004) that are inactivated by degradation in the presence of glucose (Moriya and Johnston 2004). Moriya and Johnston (2004) report that glucose-induced degradation of Mth1 and Std1 repressors requires phosphorylation by the Yck1,2 kinases. As predicted from these data, they also report that glucose-stimulated HXT1 expression and Mth1p and Std1p degradation are defective in the yckts strain. On the basis of these results, we used glucose-induced Mth1p degradation and HXT1 expression to monitor Yck1,2 kinase activity in vivo in the reg1Δ strain.

The parental YCK1,2 REG1 strain and the yckts REG1 and YCK1,2 reg1Δ mutant strains were transformed with an HXT1 promoter-lacZ reporter plasmid and β-galactosidase expression was assayed following growth in galactose and after 5 hr of growth in glucose (Figure 7A). Glucose induction of HXT1 is clearly blocked in the reg1Δ mutant strain, indicating that the Yck1,2 kinases are inactive in the absence of this Glc7 phosphatase-targeting subunit. We note that glucose induction of HXT1 is partially defective in the yckts strain at the permissive temperature of 25° but a more serious defect is observed at 30°.

Figure 7.
reg1Δ blocks glucose-induced HXT1 expression and Mth1 repressor degradation. Strains LRB906 (YCK1 YCK2 REG1), LRB756 (yckts), and CMY7000 (reg1Δ) were transformed with the HXT1-lacZ reporter plasmid pBM3212 (A) or plasmid pBM4560 carrying ...

These results are consistent with the difference in Mth1p stability shown in Figure 7B. Following 45 min of exposure to glucose, Mth1 protein levels are dramatically decreased in the wild-type strain but are relatively unchanged in the reg1Δ mutant strain and the yckts strain at 30°. In the yckts strain at the permissive temperature of 25°, we observe a very significant decrease in Mth1p levels, although a slight amount of protein remains. On the basis of these findings, we conclude that the activity of the Yck1,2 kinases cannot be stimulated by growth on glucose in a reg1Δ mutant.

DISCUSSION

Our results indicate that Yck1,2 kinase activity is a key player in multiple steps regulating maltose permease activity and trafficking. While not required for permease synthesis or cell surface localization, Yck1,2 kinase activity is required for activation of maltose transport activity. Further, decreased Yck1,2 kinase activity or lack of association of the Yck1,2 proteins with the plasma membrane blocks glucose-induced inactivation of maltose permease. The effects of yckts on maltose permease resemble those of reg1Δ (Jiang et al. 2000b) and those observed for glucose-repression-insensitive glc7 mutants. On the basis of our results, we propose that the Yck1,2 kinases and Glc7–Reg1 PP1 phosphatase are components of a common signaling pathway in which Reg1-Glc7 phosphatase activates Yck1,2 kinase and modulates its activity level in response to carbon source availability.

The yckts mutant exhibits multiple defects specific to maltose permease activation and internalization:

Despite the apparent normal levels of cell surface localization of maltose permease in the yckts strain, maltose transport activity is reduced approximately sevenfold. The basis for this low specific activity is unclear. It may be related to the significantly reduced level of permease phosphorylation observed in the yckts and reg1Δ mutants but some of our results argue against this possibility. For example, the unexpected increase in maltose transport activity observed upon addition of glucose to the yckts strain (Figure 1A) is not associated with a parallel increase in phosphorylated maltose permease protein. On the other hand, overexpression of REG1 in the yckts strain (Figure 6) causes a significant increase in the level of phosphorylated permease with no concomitant increase in maltose transport activity. However, we are monitoring bulk phosphorylation of the permease, so we cannot exclude the possibility that activation of maltose permease is regulated by phosphorylation of specific sites.

The very robust glucose-induced activation of maltose permease in the yckts strain is likely to provide clues as to the molecular basis of transport regulation by Yck1,2 kinase. We suggest that most of the cell-surface-localized maltose permease protein expressed in the yckts strain is inactive but is very rapidly activated by this glucose-stimulated event. Since it does not require de novo protein synthesis, the simplest explanation is a glucose-stimulated modification of the permease protein or its association with a preexisting regulatory cofactor that takes place via a pathway that does not require Yck1,2 kinase activity. It is possible that the membrane topology of maltose permease is altered in the yckts strain, but this seems unlikely to be so quickly remediated by glucose addition. The effects of this alternate pathway are normally obscured in the wild-type strain due to the rapid glucose-induced internalization of maltose permease. Medintz et al. (2000) reported a similar glucose-induced increase in maltose transport activity in cells expressing a mutant allele of Mal61 maltose permease lacking residues 49–78 of the N-terminal cytoplasmic domain. In this case, the stimulation was dependent on the presence of a wild-type allele of RGT2 and occurred constitutively in cells expressing the dominant constitutive RGT2-1 allele.

The yckts strain exhibits severe defects in glucose-induced internalization of maltose permease. It is unlikely that these internalization defects result from a generalized endocytic defect. Fluid-phase endocytosis, as assayed by Lucifer yellow uptake, does not require Yck1,2 casein kinase 1 activity (Friant et al. 2000). We observed that the rate of movement of the lipophilic dye FM4-64 (Fischer-Parton et al. 2000) from the plasma membrane to the vacuolar membrane occurs with approximately the same kinetics in wild-type and yckts strains during growth on maltose at permissive and nonpermissive temperatures (data not shown). Thus, the defects in maltose permease internalization and degradation are specific to glucose-induced turnover of Mal61 permease.

A requirement for phosphorylation for regulated internalization of plasma membrane proteins has been noted previously. Phosphorylation in these cases acts to target proteins for ubiquitination, which is required for recognition by endocytic machinery as well as for sorting in downstream compartments (Bonifacino and Traub 2003; Umebayashi 2003). The results reported here indicate that Yck1,2 kinase activity is upstream of maltose permease ubiquitination. Most likely, as has been shown for Ste2p (Hicke et al. 1998), Ste3p (Feng and Davis 2000), and Fur4p (Marchal et al. 1998), Yck1,2 kinase activity is required for the phosphorylation of maltose permease that stimulates permease ubiquitination. Several putative Ser/Thr kinase target sites are present in the predicted cytoplasmic domains of Mal61p, four of which fit the casein kinase 1 target-site consensus (Kennelly and Krebs 1991). Additional studies will be required to determine whether maltose permease is the direct target of Yck1,2 kinase activity for these functions.

In summary, the Yck1,2 kinases play an important role in activating the maltose transport activity of maltose permease and in regulating the rate of movement of permease-containing transport vesicles and are essential for internalizing maltose permease from the plasma membrane in response to glucose. Which step or steps result from the direct phosphorylation of maltose permease by the Yck1,2 kinases remains to be determined.

The Yck1,2 kinases and Glc7–Reg1 phosphatase act in a common glucose-signaling pathway:

Strains defective for Yck1,2 kinase activity or Glc7–Reg1 phosphatase activity share a strikingly similar maltose permease phenotype and the yckts reg1Δ double mutant resembles each single-mutant strain, suggesting that the Yck1,2 kinases and Glc7–Reg1 phosphatase act in a common pathway. Moreover, epistasis analysis places Yck1,2 kinase activity downstream of Glc7–Reg1 phosphatase in this pathway. These results are consistent with the hypothesis that Reg1–Gcl7 phosphatase is an activator of Yck1,2 kinase activity. Although there is no evidence that the Yck1,2 kinases are regulated by phosphorylation and dephosphorylation, at least one casein kinase 1 isoform is thought to be regulated by the phosphorylation state of a sequence in the C-terminal domain. For casein kinase 1δ, auto-phosphorylation of its carboxy-terminal domain was reported to be inhibitory, and its kinase activity is activated in vitro by phosphatase treatment (Graves and Roach 1995). In this regard, it is interesting to note that deficiency of protein phosphatase 2A or of the Yck1,2 kinases results in similar defects in bud morphogenesis and cytokinesis (Healy et al. 1991; Robinson et al. 1993), possibly due to similar effects on septin function at the bud neck. This similarity is not consistent with a relationship where opposing phosphatase and kinase activities act on a shared substrate, but is consistent with a scenario in which protein phosphatase 2A activates Yck1,2 kinase activity.

It should also be noted that while the observed defects in maltose permease activity and phosphorylation in the yckts and reg1Δ mutant strains grown in maltose are strikingly similar, their growth and morphology phenotypes are not (Figure 6C). Loss of REG1 does not produce the elongated bud phenotype exhibited by yckts even at permissive temperatures nor does it cause a temperature-sensitive growth defect (data not shown). On the other hand, multicopy REG1 does restore near-wild-type morphology to maltose-grown yckts cells and multicopy YCK1 suppresses the large-budded phenotype of reg1Δ mutant strains (Figure 6C; Jiang et al. 2000b). These findings are consistent with our proposed Glc7–Reg1 phosphatase/Yck1,2 kinase regulatory pathway but suggest a nonessential role for Glc7–Reg1 phosphatase as a modulator of Yck1,2 kinase activity, perhaps coordinating Yck1,2p activity with carbon source availability. We envision Glc7–Reg1 phosphatase participating in a priming event that increases Yck1,2p activity or responsiveness to signals. The Yck1,2p priming event could include changes in phosphorylation level, membrane association, localization to membrane subdomains, or some combination of these. Moreover, our results suggest that the variety of Yck1,2p cellular functions has differential requirements for this priming event.

Yck1,2 kinase is the keystone of two independent regulatory pathways controlling both glucose-induced inactivation and glucose induction:

Moriya and Johnston (2004) recently added Yck1,2 kinase activity to a model for the Rgt2p-dependent high-glucose sensing pathway that regulates HXT1 expression. They used the split-ubiquitin system to demonstrate that Rgt2p and Yck1p interact, showed that Yck1,2 kinase activity increases upon glucose stimulation, and demonstrated that activated Yck1,2 kinases phosphorylate Mth1p and Std1p when the latter proteins are tethered to the Rgt2p C-terminal cytoplasmic tail. Further, Yck1,2p-dependent phosphorylation marks these proteins for recognition and ubiquitination by SCFGRR1. They propose that Rgt2p serves as the activator of Yck1,2 kinase in the presence of high extracellular glucose concentrations, although this is not demonstrated experimentally.

Our results extend the findings of Moriya and Johnston (2004) by proposing that a second signaling pathway, of which Glc7–Reg1 phosphatase is an essential component, contributes to the high-glucose signal at the plasma membrane and that both pathways are required to achieve the full high-glucose response. These pathways parallel those described by Jiang et al. (1997, 2000b) who demonstrated that two glucose-signaling pathways are involved in the glucose induction of maltose permease inactivation: pathway 1, the Rgt2p-dependent pathway, and pathway 2, the glucose transport-dependent pathway, of which Glc7–Reg1 phosphatase is an essential component. Glc7–Reg1 phosphatase is activated by growth on high concentrations of glucose via signals generated by the same pathway that inhibits Snf1 kinase in response to rapid glucose utilization (Tu and Carlson 1995; Sanz et al. 2000), but the nature of this glucose signal is unknown. Jiang et al. (1997, 2000b) showed that loss of REG1 blocks glucose-induced inactivation of maltose permease (see also Figure 5) and that the constitutive RGT2-1 allele causes glucose-induced proteolysis of maltose permease even in the absence of glucose (Jiang et al. 1997).

Our conclusion, on the basis of results reported in Figures 6 and and7,7, indicates that both pathways are required for these two glucose-induced processes. Glucose signaling via Rgt2p alone is not sufficient for glucose induction of HXT1 expression and Mth1p degradation since these functions are defective in a reg1Δ mutant. Rgt2p-dependent signaling is also not sufficient to induce maltose permease inactivation in a reg1Δ mutant but is sufficient when YCK1 is overexpressed, suggesting that higher levels of Yck1 kinase can compensate for signaling only via the Rgt2p pathway. Thus it appears that activation of Yck1,2 kinase to very high levels is required to induced maltose permease inactivation and HXT1 expression and that this is achieved normally only by coordinated activation of both the Rgt2p-dependent and Glc7–Reg1 phosphatase glucose-signaling pathways, pathways 1 and 2, respectively. Once activated by both glucose-stimulated Rgt2p and Reg1–Glc7 phosphatase, Yck1,2 kinase then stimulates selective phosphorylation of targets such as Mth1p, Std1p, and possibly maltose permease, thereby marking them for ubiquitination and proteolysis.

In summary, our results connect two glucose-signaling pathways previously believed to represent independent responses to carbon source availability: the Rgt2 extracellular glucose sensor pathway and the Reg1–Glc7 phosphatase/Snf1 kinase pathway that sense intracellular glucose utilization rates (Carlson 1999; Johnston 1999; Sanz et al. 2000) and define a novel role for Reg1–Glc7 phosphatase as an activator of the Yck1,2 kinases.

Acknowledgments

We are grateful to Mark Johnston for providing plasmids and to Kelly Tatchell for providing glc7 mutant strains and plasmids, for valuable discussions, and for critical reading of the manuscript. We thank Tara Williams-Hart for sharing her results prior to publication. This study utilized equipment of the Core Facility for Cell and Molecular Biology and we thank Areti Tsiola for her valuable assistance. This work was supported by grants to C.A.M. from the National Institutes of Health (GM28216) and to L.C.R. from the National Science Foundation (MCB-9974459). The results reported here were obtained in partial fulfillment of the requirements for the Ph.D. degree from the Graduate School of CUNY (N.G.).

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