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Mol Cell Biol. Oct 2004; 24(20): 8994–9005.
PMCID: PMC517892

Regulation and Recognition of SCFGrr1 Targets in the Glucose and Amino Acid Signaling Pathways

Abstract

SCFGrr1, one of several members of the SCF family of E3 ubiquitin ligases in budding Saccharomyces cerevisiae, is required for both regulation of the cell cycle and nutritionally controlled transcription. In addition to its role in degradation of Gic2 and the CDK targets Cln1 and Cln2, Grr1 is also required for induction of glucose- and amino acid-regulated genes. Induction of HXT genes by glucose requires the Grr1-dependent degradation of Mth1. We show that Mth1 is ubiquitinated in vivo and degraded via the proteasome. Furthermore, phosphorylated Mth1, targeted by the casein kinases Yck1/2, binds to Grr1. That binding depends upon the Grr1 leucine-rich repeat (LRR) domain but not upon the F-box or basic residues within the LRR that are required for recognition of Cln2 and Gic2. Those observations extend to a large number of Grr1-dependent genes, some targets of the amino acid-regulated SPS signaling system, which are properly regulated in the absence of those basic LRR residues. Finally, we show that regulation of the SPS targets requires the Yck1/2 casein kinases. We propose that casein kinase I plays a similar role in both nutritional signaling pathways by phosphorylating pathway components and targeting them for ubiquitination by SCFGrr1.

Protein ubiquitylation has emerged as a central regulatory mechanism in eukaryotic cells. A broad array of cellular processes and responses utilize ubiquitylation either to inactivate the target protein via proteolysis or to modify its function or localization. The transfer of free ubiquitin to a protein substrate occurs in several steps, including ATP-dependent activation of ubiquitin via a ubiquitin-activating enzyme (E1), transfer of activated ubiquitin to an ubiquitin-conjugating enzyme (E2) and, finally, transfer of ubiquitin from the E2 enzyme to the substrate, often in collaboration with a ubiquitin ligase (E3) (15).

Typically, the ubiquitin ligase plays the critical role in determining the specificity of substrate recognition and positioning the substrate for ubiquitylation. One of the largest families of E3 ubiquitin ligases is the evolutionarily conserved SCF complex family. This family of enzymes forms a stable complex with an E2 enzyme, most commonly Cdc34, and contains several common components: a scaffold protein Cdc53 (also called cullin), a RING-finger protein Hrt1 (also named Roc1 or Rbx1), and an adaptor protein Skp1 (8, 21, 25, 38, 44, 49). In addition, they contain a variable component, the F-box protein, which confers substrate specificity to the SCF complex (1, 50). The F-box components are exchangeable subunits that facilitate the capacity of SCF to specifically target a large number of structurally and functionally diverse substrates. Typically, F-box proteins have a bipartite structure, with the F-box domain of 40 amino acids interacting with SCF via Skp1 and a substrate recognition motif, such as a leucine-rich repeat (LRR) domain or WD40 repeat domain, which appears to participate in substrate binding (1, 50). The budding Saccharomyces cerevisiae genome encodes a large family of proteins containing F-box domains (at least 17) but, as yet, only four, Cdc4, Grr1, Ufo1, and Met30, have been shown to participate in SCF complexes (6, 8, 22, 44, 51).

SCF was first described based upon its role in the degradation of cell cycle regulatory elements. Cell cycle targets include CDK inhibitors Sic1 and Far1, both of which are ubiquitylated by SCFCdc4 (8, 14), and the G1 cyclins Cln1 and Cln2, which are ubiquitylated by SCFGrr1 (51, 56). In each case, phosphorylation of the substrate is an essential aspect of the recognition signal. Whereas Cdc4 interacts with phosphorylated substrates via its WD40 repeat domain, Grr1 contains a large substrate binding domain built on 12 LRRs (9, 17, 23, 32). Four basic residues predicted to lie on the concave surface of that structure are critical for the binding and degradation of phosphorylated Cln2 (17). Strikingly, four CDK phosphorylation sites on Cln2 contribute to its efficient degradation (4). However, it is unclear whether the one-to-one correspondence is relevant. It is clear that properties of the Cln2 degron in addition to phosphorylation and regions of Grr1 outside of the LRR also contribute to Cln2 recognition (17).

In addition to its role in degradation of G1 cyclins (51), the list of cellular functions controlled by SCFGrr1 has now expanded to include the response to environmental change and morphogenesis (2, 3, 19, 33). This broad array of roles is apparent in the phenotype of cells lacking Grr1, which exhibit multiple abnormalities including cell elongation, slow growth on glucose, increased sensitivity to osmotic stress and nitrogen starvation, decreased divalent cation transport, enhanced filamentous growth, defects in sporulation, and slow growth or inviability when combined with amino acid biosynthetic mutants (5, 9, 33, 43, 57, 58). Although the requirement for Grr1 in those processes has been well documented, its targets in most of those pathways are poorly characterized.

Among the nutritional roles for Grr1 is its essential role in both the transcriptional activation of the genes encoding hexose transporters (HXT1 to HXT4) in response to glucose and the activation of numerous genes encoding amino acid permeases (including AGP1, BAP3, TAT1, and PTR2) in response to external amino acids via the SPS (Ssy1-Ptr3-Ssy5) signaling system (3, 18). Each of those pathways is activated through sensors at the plasma membrane and terminates in the transcriptional activation of specific promoters (11, 41). Grr1 is thought to regulate the stability or activity of components of those pathways.

Regulation of the HXT genes occurs in response to glucose acting via the cell surface sensors Snf3 and Rgt2. Activation of those sensors generates a Grr1-dependent signal that leads to inactivation of the transcriptional repressor Rgt1 (40, 41, 54). Hyperphosphorylated Rgt1 is then released from the HXT1-HXT4 promoters, resulting in transcriptional activation (10, 36). The paralogs Std1 and Mth1 are required to maintain Rgt1 in its hypophosphorylated, promoter-bound state in the absence of glucose (10, 47, 48). Grr1 is required for destabilization of Mth1 in response to glucose (10). Evidence suggests that Std1 and Mth1 interact with the carboxy-terminal tails of plasma membrane-associated Snf3 and Rgt2 (29, 47), respectively, as well as with Rgt1 (30). These findings have led to the suggestion that they transmit the glucose signal from the nucleus to the cytoplasm (10, 30, 47, 53).

Support for the direct involvement of SCFGrr1 in the recognition and ubiquitination of Mth1 and, perhaps, Std1 is provided by the recent finding that the type 1 casein kinases Yck1 and Yck2 are required for their degradation as well as for HXT gene transcriptional induction (35). These kinases interact with the cytoplasmic tails of Rgt2 and Snf3 in vivo and can phosphorylate Std1 and Mth1 in vitro. Furthermore, mutation of putative casein kinase phosphorylation sites in those proteins prevents their degradation in response to glucose (35). Together, these observations suggest a model in which glucose binding to the sensors activates Yck1/2, leading to the phosphorylation of Mth1. Phosphorylated Mth1 is then recognized by SCFGrr1, ubiquitinated, and targeted for degradation via the proteasome.

In this paper, we address several aspects of this model. First, we show that inactivation of Mth1 involves ubiquitylation and proteasomal degradation. Next, degradation of Mth1 is dependent upon both Skp1 and the F-box of Grr1, and mutants affecting their interaction accumulate phosphorylated forms of the Mth1 protein. We find that the degradation of Mth1 and activation of HXT genes requires the LRR domain of Grr1. However, in contrast to the previously characterized Grr1 targets Cln2 and Gic2, regulation of the expression of HXT genes and other genes, including targets of the amino acid-inducible SPS pathway, is largely unperturbed by mutations within the LRR and the carboxy-terminal domain of Grr1. This suggests that distinct properties of Grr1 are required for the recognition of these two classes of targets. Finally, we show that, like for the induction of HXT genes, induction of the SPS signaling pathway by amino acids requires the type 1 casein kinases Yck1 and Yck2. Together, these observations broaden our understanding of the involvement of Grr1 in the glucose induction of HXT gene expression and suggest a conservation of that regulatory motif in the induction of amino acid-induced genes via the SPS signaling pathway.

MATERIALS AND METHODS

Yeast strains.

Yeast strains are described in Table Table1.1. All strains are in the W303a background (ade2-1 can 1-100 his3-1.15 leu2-3.112 trp1-1 ura3), with the exception of LRB341, LRB346, Y20000, Y36902, and CWY1208, which are in the S288c background. Cells were grown in standard culture media, and standard yeast genetic methods were used.

TABLE 1.
Yeast strains used in this study

The carboxy- and amino-terminal epitope-tagged proteins were generated via chromosomal integration of PCR-amplified fragments as described by Longtine et al. (34) or by integration in the pKan vector (S. Haase, M. Wolff, and S. Reed, unpublished data). Deletion mutants were constructed using PCR-based methods (34, 55).

RNA and protein preparation.

Cells were grown to middle-logarithmic phase in 2% galactose, and then cultures were split and half shifted to 4% glucose for 30 min, unless mentioned otherwise. Cells were harvested by filtration, and the pellets were stored at −80°C.

The RNA isolation, cDNA synthesis, reverse transcriptase reactions, and PCRs were performed as described previously (17). Oligonucleotides are available upon request.

Protein extracts were prepared either in urea buffer as described previously (10) or in lysis buffer (50 mM Tris HCl [pH 7.5], 0.1% NP-40, 250 mM NaCl) containing phosphatase inhibitors (10 mM NaPPi, 5 mM EDTA, 5 mM EGTA, 0.1 mM orthovanadate) and protease inhibitors (100 μM phenylmethylsulfonyl fluoride, 1 μg of leupeptin/ml, 1 μg of aprotinin/ml). Lysis buffer extraction was performed by lysing cells at 4°C with glass beads (four times for 40 s) in a FastPrep FP120 apparatus. The protein extracts were collected after 15 min of centrifugation at 10,000 × g at 4°C.

Coimmunoprecipitation assays.

Protein extracts were obtained using glass beads in the immunoprecipitation buffer (50 mM Tris HCl [pH 7.5], 1% Triton X-100, 250 mM NaCl) containing protease and phosphatase inhibitors. The mouse 12C5 antihemagglutinin (anti-HA) monoclonal antibodies (kindly provided by I. Wilson, The Scripps Research Institute, La Jolla, Calif.) covalently conjugated to protein A-Sepharose and mouse anti-myc monoclonal antibodies (Santa Cruz) were used to perform immunoprecipitations. The coimmunoprecipitations were carried out using 1 mg of total protein extract incubated with antibody for 1 h at 4°C and washed three times with the same buffer before being prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Rat anti-HA monoclonal antibodies (Roche) and goat anti-rat secondary antibodies (Roche) were used on immunoblots to detect Mth1-HA protein and avoid cross-reaction with murine immunoglobulin G.

Phosphatase treatments.

Proteins were extracted in the lysis buffer, which contained phosphatase inhibitors and protease inhibitors, as described above. For each strain, 3 mg of whole-cell extract was immunoprecipitated with anti-HA monoclonal antibodies covalently bound to protein A-Sepharose for 1 h at 4°C. The protein A-Sepharose pellets were washed three times with lysis buffer minus phosphatase inhibitors and divided into three samples. All the samples were resuspended in phosphatase buffer (50 mM Tris HCl [pH 7.5], 5 mM dithiothreitol, 0.1 mM EDTA, 0.01% Brij 35, 2 mM MnCl2) and incubated for 30 min at 30°C under the following conditions: one sample from each strain was treated with 1,200 U of phosphatase λ (New England Biolabs), another sample was treated with phosphatase λ in the presence of phosphatase inhibitors, and one sample remained untreated. Proteins were analyzed by SDS-PAGE and immunoblotting.

Microarray experiments.

The strains Y20000, Y36902, and CWY1208 were grown continuously in rich medium to middle-log phase, and RNA was isolated by using an RNeasy mini kit (QIAGEN). Five micrograms of total RNA was used for cDNA synthesis and cDNA amplification, and chips were hybridized to Affymetrix yeast genome S98 arrays (Affymetrix, Santa Clara, Calif.) according to standard Affymetrix protocols (Affymetrix expression analysis technical manual [http://www.affymetrix.com/support/technical/manuals.affx]). Arrays were washed with a custom GNF chip washer and stained with standard Affymetrix reagents. Arrays were scanned with an Affymetrix scanner (model GA 2500). Data were analyzed with Rosetta Resolver's (Kirkland, Wash.) ratio analysis of variance (ANOVA) function. Resolver ANOVA analysis is similar to standard ANOVA, but instead uses two inputs, expression measurement quantity and estimated error of measurement quantity. This additional input provides more reliable variance measurements, a necessity when the number of replicates is small (45). This error estimate also brings extra degrees of freedom to the analysis, allowing for fewer false positives and false negatives (see http://www.rosettabio.com/publications/default.htm for additional references).

In vivo ubiquitylation assays.

Ubiquitylation assays were performed as described elsewhere (56). Cells were grown in 2% galactose to early logarithmic phase, and the CUP1 promoter was induced for 5 h by the addition of CuSO4 to 250 μM. Cells were harvested and washed twice in ice-cold water, pelleted, and frozen at −80°C. Proteins were prepared by glass bead lysis in buffer G (6 M guanidium hydrochloride, 100 mM NaH2PO4, 20 mM Tris HCl [pH 8.0] adjusted to pH 8.0). After centrifugation (15 min at 10,000 × g; 4°C), the supernatant containing 8 mg of proteins was incubated with Ni2+-nitrilotriacetic acid (NTA) Sepharose (QIAGEN) for 60 min at 25°C. The beads were washed three times with buffer G and three times with buffer C (50 mM Tris HCl [pH 8.0], 500 mM NaCl). Bound proteins were analyzed by SDS-PAGE and immunoblotting.

MG132 experiments.

Cultures were grown to early logarithmic phase in 2% galactose. The proteasome inhibitor MG132 (Sigma) was solubilized in dimethyl sulfoxide (DMSO; 10 mg/ml) and added to half of each culture to a final concentration of 50 μg/ml for 90 min. DMSO was added to another portion as a control. Half of each culture was then shifted to 4% glucose for 30 min, after which cells were harvested for protein and RNA preparation.

RESULTS

Skp1 protein is essential for Mth1 degradation and transcriptional activation of HXT3.

The finding that Grr1 is required for the degradation of Mth1 suggests a role for the SCFGrr1 complex (10). Because Skp1 mediates the binding of F-box proteins to the enzymatic components of the ubiquitin ligase (1, 32), we analyzed the degradation of Mth1 protein and the transcriptional activation of HXT genes in skp1 mutants. Two conditional alleles of SKP1, skp1-11 and skp1-12, have been reported to be deficient in SCF complex activities (1). The skp1-11 mutant specifically arrests in G1 phase due to a failure to degrade Sic1 and is deficient in the degradation of the F-box protein Cdc4 (1, 13), consistent with a specific defect in SCFCdc4 activity. In contrast, the skp1-12 mutant is defective in mitotic entry and impaired for the degradation of G1 cyclins, Gic2 and the F-box protein Grr1 (1, 13), consistent with a deficiency in SCFGrr1 activity.

Analysis of the transcriptional activation of the HXT3 gene after glucose induction in wild-type, skp1-11, skp1-12, and cdc4-1 strains revealed that HXT3 transcription failed to be induced in the skp1-12 mutant (Fig. (Fig.1A)1A) but was induced normally in the other strains. This finding is consistent with a role for SCFGrr1, but not SCFCdc4, in HXT gene induction.

FIG. 1.
The skp1-12 conditional allele is defective in HXT3 transcriptional activation and Mth1 protein degradation after glucose induction. Cells were grown at 25°C in 2% galactose to middle-log phase, shifted to 37°C for 30 min, and split in ...

We have established that induction of HXT gene expression is associated with Grr1-dependent degradation of Mth1 (10). Consistent with that finding, the failure of glucose to induce HXT3 in the skp1-12 mutant was associated with a defect of Mth1 degradation (Fig. (Fig.1B).1B). In contrast, Mth1 protein was degraded as efficiently in the skp1-11 sample, or the cdc4-1 sample, as it was in wild-type cells (data not shown). In addition, a slower-migrating form of Mth1 was observed in skp1-12 mutants, consistent with the accumulation of phosphorylated protein. That form appeared to accumulate in glucose (Fig. (Fig.1B1B).

Because the skp1-12 conditional allele was shown to specifically arrest in G2 phase (1), we evaluated whether the stabilization of Mth1 protein was a consequence of cell cycle arrest rather than a direct effect of the SCF deficiency in skp1-12. Mth1 was analyzed in either asynchronous or G1-arrested skp1-12 cells following a shift to the restrictive temperature. In both cases, we observed stabilization of Mth1 protein and the appearance of the slower-migrating form (data not shown). We conclude that Mth1 degradation is dependent on functional Skp1 protein and does not exhibit a cell cycle dependency.

Mutations in the F-box domain of Grr1 affect degradation of Mth1 in response to glucose.

To further evaluate the importance of the interaction between Grr1 and the other components of SCF complexes, we evaluated the F-box mutant Grr1-AAA, in which three highly conserved F-box domain residues are replaced by alanine (13). Although Skp1 still binds to SCFGrr1-AAA in vivo, the degradation of both Grr1-AAA and the putative SCFGrr1 substrate Gic2 appear defective in this mutant (13). Surprisingly, transcriptional activation of HXT3 by glucose in grr1-AAA mutants was comparable to that in wild-type cells (Fig. (Fig.2A).2A). This unexpected observation suggested that this perturbation in the F-box-dependent interaction between Grr1 and Skp1 does not modify the regulation of HXT gene expression. Yet, in the same experiment, the Mth1 protein in the grr1-AAA mutant behaved similarly to that in the skp1-12 mutant, with a slower-migrating form of Mth1 accumulating after glucose induction (Fig. (Fig.2A,2A, right panel). We also noticed that, although the abundance of Mth1 was not substantially reduced within 30 min following addition of glucose in the grr1-AAA mutant, the abundance of Mth1 was significantly decreased relative to that in wild-type cells under noninducing conditions (Fig. (Fig.2A,2A, right panel) despite the lack of an effect on level of MTH1 RNA (Fig. (Fig.2A,2A, left panel) (see below). We conclude that, although Grr1-AAA is compromised in its capacity to target Mth1 for degradation, it retains the capacity to induce HXT gene expression. To explain these contradictory observations, we propose that Grr1-AAA can efficiently inactivate Mth1 by binding to it and restricting its activity as a repressor of HXT3 transcription. Moreover, because Grr1-AAA hyperaccumulates relative to the wild-type Grr1 protein (Fig. (Fig.2A,2A, right panel), it is likely to be more efficient than wild-type Grr1 at sequestering the slower-migrating form of Mth1.

FIG. 2.
Analysis of the role of the F-box domain of Grr1 for glucose induction of HXT3 gene expression. Cells were grown in 2% galactose to middle-log phase. Half of the culture was induced with 4% glucose for 30 min, and the other half remained untreated. (A) ...

If Grr1-AAA inactivates Mth1 by direct binding, we predicted that its capacity to block Mth1 function might be overcome when Mth1 is overexpressed. That hypothesis was tested by overexpressing MTH1 under the GAL1 promoter in wild-type and grr1-AAA mutant cells and then analyzing its abundance following repression of GAL1-MTH1 expression by glucose. Glucose led to repression of MTH1 expression from the GAL1 promoter in both strains (Fig. (Fig.2B,2B, left panel). However, whereas in the wild-type strain, the Mth1 protein was degraded (Fig. (Fig.2B,2B, right panel) and HXT3 gene expression was induced within 30 min following addition of glucose (Fig. (Fig.2B,2B, left panel), in the grr1-AAA mutant Mth1 protein was stabilized (Fig. (Fig.2B,2B, right panel) and HXT3 transcription remained repressed (Fig. (Fig.2B,2B, left panel). We conclude that, unlike the cells expressing Mth1 at the wild-type level, cells overexpressing Mth1 from the GAL promoter accumulate sufficient protein to overcome even the increased abundance of Grr1 in the grr1-AAA mutant, such that induction of HXT gene transcription is not observed within the time frame of this experiment.

A phosphorylated form of Mth1 interacts with the F-box-defective form of Grr1.

It is known that SCF complexes interact with phosphorylated substrates (50, 56). Our data suggest that the F-box-defective Grr1-AAA protein binds Mth1 to inactivate it. Consistent with that interpretation, a slower-migrating form of Mth1 accumulated in grr1-AAA and skp1-12 mutants and became more abundant when MTH1 was overexpressed (Fig. (Fig.2B,2B, right panel). We analyzed the phosphorylation state of Mth1 by protein phosphatase treatment of immune complexes containing Mth1-HA prepared under native conditions from the GAL1-MTH1-3xHA grr1-AAA-myc strain (Fig. (Fig.3A).3A). Like Cln2 (4, 31), the slower-migrating form of Mth1 accumulating in the grr1-AAA cells was lost following treatment with phosphatase. We conclude that the slower-migrating form that accumulated in those mutants, and probably that in skp1-12 mutants, represented a phosphorylated species of Mth1. This form may be stabilized and protected from phosphatases by virtue of its stabilized interaction with Grr1. Consistently, neither wild-type cells nor Grr1-deficient cells overexpressing Mth1 under the GAL1 promoter accumulated this phosphorylated form of Mth1 (Fig. (Fig.4A),4A), suggesting that it is rapidly degraded or rapidly dephosphorylated, or both.

FIG. 3.
Mth1 is a phosphoprotein that is stabilized by interaction with Grr1-AAA. grr1-AAA-myc GAL1-MTH1-3xHA (CWY1376) cells were grown in 2% galactose to middle-log phase and shifted to 4% glucose for 15 min. (A) Phosphatase treatments proceeded on Mth1-HA ...
FIG. 4.
Degradation of Mth1 and induction of HXT gene expression are compromised in grr1-AAA mutants. Cells were grown in 2% galactose to middle-log phase. A sample of each strain was taken at time zero (no glucose), and after glucose addition to 4% (final concentration) ...

To test the hypothesis that Mth1 interacts stably with Grr1-AAA, we analyzed the interaction by immunoprecipitation using extracts prepared from the GAL1-MTH1-3xHA grr1-AAA-myc strain. Anti-HA immune complexes prepared from those extracts contained Grr1-AAA-myc, confirming that the two proteins interacted (Fig. (Fig.3B).3B). Similarly, Mth1-HA coimmunoprecipitated with Grr1-AAA-myc. However, in the latter case, we observed only the phosphorylated form of Mth1 (Fig. (Fig.3B).3B). This experiment demonstrated that Grr1-AAA binds specifically to the phosphorylated form of Mth1. Again, we have been unable to demonstrate an interaction between wild-type Grr1 and Mth1, as the phosphorylated form of Mth1 is undetectable in wild-type cells (see below).

The rate of Mth1 degradation is dramatically reduced in grr1-AAA mutants.

To investigate the extent to which the F-box mutations affect Mth1 degradation, we compared the abundance of Mth1 and the induction of HXT3 in wild-type cells (GRR1), grr1-AAA-myc cells, and cells deficient in GRR1 (grr1Δ). Mth1 was overexpressed under the GAL1 promoter to ensure a similar regulation of its mRNA (Fig. (Fig.4B)4B) and to facilitate detection of the protein (Fig. (Fig.4A4A).

As previously described for wild-type cells, degradation of Mth1 and induction of HXT3 expression was observed within 30 min following the addition of glucose (Fig. (Fig.4).4). In contrast, Mth1 protein remained stable for 2 h following glucose induction in grr1Δ cells, and no induction of HXT3 was observed. The repression of MTH1 mRNA was similar to that in wild-type cells. Finally, as in cells expressing wild-type MTH1, the grr1-AAA-myc cells expressing GAL1-MTH1-3xHA accumulated the phosphorylated form of Mth1 protein (Fig. (Fig.4A).4A). The Mth1 protein was more stable in that strain than in wild-type cells but less stable than in the grr1Δ mutants, decreasing slowly over the 2-h time course. Thus, although the activity of Grr1-AAA appeared compromised, it remained sufficient to promote Mth1 degradation (Fig. (Fig.4A).4A). Consistent with that observation, HXT3 expression was weakly activated after 1 h and achieved a wild-type level of induction after 2 h of glucose induction (Fig. (Fig.4B).4B). Based upon these observations, we conclude that the Grr1-AAA protein retains the capacity to bind to phosphorylated substrates and promote their degradation, although at a much slower rate than wild-type Grr1 protein. The retention and accumulation of phosphorylated Mth1 by Grr1-AAA suggest that the reduced efficiency of Mth1 degradation is a consequence of a reduced rate of ubiquitylation and release of the phosphorylated substrate rather than a failure to bind that substrate.

The hyperaccumulation of the Grr1-AAA protein and the finding that it retains a portion of the wild-type activity, along with the observation that Mth1 is degraded in a Grr1-dependent manner in the absence of glucose, provide an explanation for the reduced abundance of Mth1 observed under noninducing conditions. We suggest that a portion of Mth1 is phosphorylated under noninducing conditions and can either be trapped by Grr1 or dephosphorylated. We found that the phosphorylated form of Mth1 accumulates in the grr1-AAA mutant, consistent with the ability of Grr1-AAA to bind and stabilize the phosphorylated substrate. We suggest that because Grr1-AAA is dramatically increased in abundance, more of the phosphorylated form is captured and shuttled into the degradation pathway, thereby decreasing the abundance of the Mth1 protein.

Distinct properties of the LRR domain of Grr1 are required for Mth1 and Cln2 degradation.

The previous analysis revealed that a functional F-box domain is not required for the interaction between phosphorylated Mth1 and Grr1 but is required for Mth1 degradation. It has previously been shown that the LRR domain of Grr1, an established protein-protein interaction domain, is required for binding and degradation of phosphorylated Cln2 (17, 24). To determine whether the interaction between Mth1 and Grr1 requires the LRR domain, the glucose-induced degradation of Mth1 was compared in cells either expressing wild-type Grr1, expressing Grr1 with a deletion of the LRR domain, or lacking Grr1. The structural domains of Grr1 and the positions of Grr1 mutations used in this study are represented in Fig. Fig.5A.5A. As in the grr1Δ strain, Mth1 protein failed to be degraded in response to glucose in the absence of the LRR domain (grr1ΔL) (Fig. (Fig.5B).5B). In parallel to the defect in Mth1 degradation, a lack of transcriptional activation of HXT3 was detected in grr1Δ and grr1ΔL mutants (Fig. (Fig.5C).5C). As in the case of two other substrates of SCFGrr1, Cln2 and Gic2, the LRR domain is essential for the recognition of Mth1 (17, 24).

FIG. 5.
Characterization of Grr1 domains necessary for Mth1 degradation and HXT3 transcription in response to glucose. (A) Domain structure of Grr1 and positions of deletion mutations and point mutations. Domains are indicated across the top of the diagram with ...

To further investigate the requirements for recognition of Mth1 by Grr1, we studied Mth1 degradation in cells expressing the grr1-B4Q allele, in which four basic residues in the LRR domain are converted to neutral residues (Fig. (Fig.5A).5A). The Grr1-B4Q protein fails to bind phosphorylated Cln2 and is impaired in Cln2 and Gic2 degradation, suggesting that the four residues are essential for the recognition of phosphorylated substrates by Grr1 (17). However, the same four point mutations had little impact on Mth1 abundance (Fig. (Fig.5B)5B) or HXT3 transcriptional activation (Fig. (Fig.5C)5C) (17). This striking result demonstrates that recognition of Mth1 involves residues in the LRR separate from, or in addition to, those shown to be important for the recognition of other characterized targets.

Finally, we analyzed the importance of the carboxy terminus of Grr1 in the degradation of Mth1. Grr1-ΔC lacks the last 234 amino acids and, like Grr1-B4Q, is impaired in Cln2 and Gic2 degradation (Fig. (Fig.5A)5A) (17). The grr1-ΔC mutant retained the capacity to degrade Mth1 and, consequently, to induce HXT3 transcription (Fig. 5B and C), consistent with a difference in the requirements for the recognition of Mth1 from those for recognition of Cln2 and Gic2 by Grr1. Thus, SCF complexes containing Grr1-B4Q and Grr1-ΔC can mediate the degradation of, at least, some substrates.

Inactivation of Grr1 affects the expression of many genes (2, 43) via targets that are, as yet, unknown. To determine whether the retention of HXT gene regulation observed in the grr1-B4Q mutant is common to all transcriptional regulons affected by grr1Δ, we performed global analysis of gene expression by comparing RNA microarrays for grr1Δ and grr1-B4Q mutants grown in rich glucose medium to those for wild-type cells grown under the same conditions. The behaviors of 18 of the most highly induced or highly repressed genes in the grr1Δ mutant relative to the expression level in wild-type cells are presented in Fig. Fig.5D.5D. In parallel, the effect of grr1-B4Q on the same genes is also presented. Many of those genes have either been previously shown to be affected by grr1Δ or can be understood based upon the effect of grr1Δ on amino acid uptake. For instance, the failure of cells to uptake amino acids results from a defect in the induction of amino acid permeases via the SPS pathway (18). This likely leads to starvation for methionine and the associated induction of genes in the MET regulon. The magnitude of repression or induction of this group of genes in grr1Δ mutants relative to that in wild-type cells was on the order of 15- to 80-fold. Strikingly, grr1-B4Q had relatively little effect on most of the genes, which were induced or repressed approximately two- to threefold relative to the wild-type level (Fig. (Fig.5D).5D). The exception was HXT1, which was reduced approximately eightfold. We conclude that these pathways are impacted by GRR1 mutations in a manner more similar to Mth1 than to Cln2, suggesting that the regulation of these nutrient signaling pathways may occur via a common mechanism.

Mth1 is ubiquitylated in vivo and degraded via the proteasome.

We supposed that like Cln2, Mth1 is ubiquitylated by SCFGrr1. To evaluate Mth1 ubiquitylation in vivo, we utilized UbiK48R,G76A, a mutated form of ubiquitin that generates only monoubiquitinated substrates and is resistant to deubiquitinating enzymes (56). The mutant ubiquitin is tagged with both the six-His and myc epitopes (UbiHIS-MYC-RA) to allow purification on a Ni2+-NTA-Sepharose matrix and immunodetection of ubiquitylated proteins, respectively. This construct was expressed from the CUP1 promoter along with HA-tagged Mth1, which was expressed from the GAL1 promoter to facilitate immunodetection. Total UbiHIS-MYC-RA-conjugated protein was purified on a Ni2+-NTA-Sepharose matrix, and modified forms of Mth1 were detected by anti-HA immunoblotting (Fig. (Fig.6A).6A). Mth1 detected in the ubiquitylated fraction migrated at a size consistent with monoubiquitylated protein, suggesting that Mth1 is ubiquitylated in vivo. The degradation of Mth1 is known to be constitutive but accelerated after glucose induction (10). Indeed, this ubiquitylation assay was performed under noninducing conditions. However, we also compared the rate of loss of Mth1 in cells induced with glucose and found that the rate of Mth1 degradation was slower in the presence of the UBIHIS-MYC-RA mutant than in its absence (data not shown). Consequently, we conclude that interfering with polyubiquitination of Mth1 slows its rate of degradation and, therefore, that ubiquitylation is involved both in constitutive and glucose-induced proteolysis.

FIG. 6.
In vivo ubiquitylation of Mth1. Cells were grown in 2% galactose to middle-log phase to induce the GAL1 promoter, and the CUP1 promoter was induced for 5 h with 250 μM CuSO4. The total extract (ex) was incubated with Ni2+-NTA agarose. ...

We then determined whether the appearance of ubiquitylated forms of Mth1 was Grr1 dependent. Using the same assay, we failed to detect any UbiHIS-MYC-RA-conjugated forms of Mth1, whereas total ubiquitylated proteins bound to the Ni2+-NTA-Sepharose matrix were easily detected (Fig. (Fig.6B).6B). This suggests that Mth1 ubiquitylation is Grr1 dependent. However, because the level of Mth1 expression was much lower in the grr1Δ mutant and because the cells stopped proliferating when Mth1 expression was induced, the relevance of this observation remains unclear. This problem was restricted to grr1Δ cells coexpressing CUP1-UBIHIS-MYC-RA and GAL-MTH1-3xHA.

Polyubiquitinated SCF substrates are typically targeted to the proteasome (16). To detect an involvement of the proteasome in Mth1 degradation, we compared the degradation of Mth1 in cells treated with the proteasome inhibitor MG132 or untreated. In untreated cells, Mth1 protein was strongly reduced within 30 min after glucose induction. In MG132-treated cells, Mth1 protein was stabilized such that its abundance was unchanged relative to that in untreated cells (Fig. (Fig.7A).7A). Degradation of Cln2, a known target of the proteasome (2), was dramatically reduced under the same conditions (Fig. (Fig.7A).7A). Together, these experiments suggest that Mth1 is ubiquitylated by SCFGrr1 which, in turn, targets it to the proteasome.

FIG. 7.
Mth1 degradation is proteasomal dependent and is not required for the transcriptional activation of HXT3. Cells were grown in 2% galactose to middle-log phase and split in two, and one half was treated with MG132 in DMSO or DMSO alone for 90 min. The ...

We also evaluated the regulation of HXT3 transcription in MG132-treated and untreated cells. Surprisingly, we observed that although the degradation of Mth1 was inefficient in cells treated with MG132, the transcriptional activation of HXT3 by glucose still occurred (Fig. (Fig.7B).7B). This is consistent with other observations that Mth1 degradation is not a requirement for HXT gene transcriptional activation and suggests that binding to Grr1 or ubiquitylation of Mth1 might be sufficient to activate the glucose induction pathways.

Grr1-dependent activation of the SPS system does not require the LRR domain of Grr1 protein but requires the Yck1/2 casein kinases.

Grr1 is required for activation of a family of amino acid permeases via the SPS signaling system (3, 18) in addition to its involvement in the transcriptional activation of HXT genes by glucose. In fact, several of the genes activated via the SPS system (BAP3, PTR2, and TAT1) are among those highly affected by inactivation of Grr1 (Fig. (Fig.5D)5D) (27). As reported previously for AGP1 (17), a transcriptional target of the SPS system, the regulation of BAP3, PTR2, and TAT1 was largely intact in grr1-B4Q mutants (Fig. (Fig.5D).5D). However, we found that, unlike HXT gene induction, domains of Grr1 outside of the LRR domain were sufficient for BAP3 induction on rich medium (Fig. (Fig.8A).8A). This suggests that the requirements for recognition of the Grr1 target participating in regulation of the SPS system are, at least in part, distinct from those required for the recognition of Mth1 or Cln2. Consistent with that conclusion, Mth1 does not appear to be involved in the induction of BAP3 (Fig. (Fig.8A8A).

FIG. 8.
The transcriptional activation of BAP3 via the SPS system can bypass the requirement of the Grr1 LRR domain but is dependent on functional casein kinases I. (A) Cells were grown in rich medium at 30°C. The transcriptional induction of the amino ...

Both the SPS pathway and the pathway leading to HXT gene induction involve plasma membrane receptors. Glucose induction of HXT genes requires Rgt2 and Snf3 (42), and induction of SPS targets by amino acids requires Ssy1 (7, 18, 26). The known components of each pathway do not appear to overlap, with the exception of Grr1. However, Moriya et al. recently demonstrated that the casein kinases Yck1/2 act in concert with Rgt2 at the plasma membrane to target Mth1 and Std1 inactivation, presumably via SCFGrr1-dependent proteolysis (35). We hypothesized that, perhaps as in the HXT regulatory system, Yck1/2 interact with the cytoplasmic domain of Ssy1 and phosphorylate a substrate of SCFGrr1 that is involved in SPS signaling to target it for degradation. To test this hypothesis, we compared the induction of expression of BAP3 and HXT3 in wild-type cells and casein kinase-deficient cells grown in rich medium (Fig. (Fig.8B).8B). At permissive temperature, HXT3 and BAP3 induction was comparable in wild-type cells and yck1Δ yck2ts mutants. However, after incubating cells for 2 h at restrictive temperature, HXT3 and BAP3 expression was inhibited to the same extent as in the grr1Δ mutant. A similar result was observed for TAT1 and PTR2 expression (data not shown). We conclude that casein kinase I is necessary for the transduction of glucose and amino acid signals, suggesting that, as it occurs in stimulation of the Snf3/Rgt2 receptor, stimulation of the Ssy1 receptor results in the activation of casein kinase I. Analysis of another gene, GAP1, which encodes a general amino acid permease that is neither a target of the SPS system nor dependent upon Grr1 (27, 52) revealed that its activation is Yck1/2 independent (Fig. (Fig.8B).8B). We propose that activation of the SPS system activates casein kinase I, leading to the phosphorylation of an as-yet-unknown target and directing it for degradation by SCFGrr1.

DISCUSSION

The F-box protein Grr1 has been shown to be required for proper regulation of HXT gene expression via the glucose signaling pathway (10, 36). We have presented evidence that it exerts its function via Mth1 ubiquitylation by SCFGrr1 and subsequent degradation by the proteasome. Several independent lines of evidence support that conclusion. First, like Cln2 and Gic2, Mth1 is degraded in a Grr1-dependent manner and its degradation requires the LRR domain of Grr1. Similarly, the skp1-12 mutant, which is deficient in functions associated with SCFGrr1, is impaired in Mth1 degradation. Second, the phosphorylated form of Mth1 accumulates in the grr1-AAA and skp1-12 mutants, both of which are compromised for SCFGrr1activity. Grr1-AAA protein binds only to the phosphorylated form of Mth1 in vivo. Finally, with the caveat that the level of Mth1 expression was much lower in the grr1Δ mutant, we have shown that the appearance of ubiquitylated forms of Mth1 is dependent upon Grr1.

Using the conditional allele skp1-12 and the grr1-AAA mutant, we have detected the accumulation of a phosphorylated form of Mth1. We suggest that stabilization of phosphorylated Mth1 is a consequence of trapping by the LRR domain of Grr1 when SCFGrr1 is compromised for its ubiquitylating function. Moriya et al. recently reported that degradation of Mth1 is dependent upon phosphorylation by casein kinase, and they identified eight potential casein kinase sites on Mth1 (35). Although the precise number and identity of the sites required for signaling has not been established, at least one of those sites is required to promote transcription of HXT1 in vivo (35). It is noteworthy that phosphorylated Mth1 was not detected in a grr1Δ strain, even if Mth1 was overexpressed under the GAL1 promoter (Fig. (Fig.4A),4A), and that in a grr1Δ strain HXT3 transcription was uninducible despite the presence of functional Yck1/2 (Fig. (Fig.4B).4B). These two results support the idea that phosphorylation of Mth1 by Yck1/2 is readily reversible, perhaps due to the activity of an unknown protein phosphatase, and that phosphorylated Mth1 only accumulates when protected by its interaction with Grr1. This is consistent with our proposal that phosphorylated Mth1 binds efficiently to the LRR domain of Grr1-AAA and that binding of Grr1, either alone or coupled with a slow rate of degradation, is sufficient to activate HXT gene transcription.

Grr1 mutated at three highly conserved residues in the F-box retains the capacity to direct the degradation of SCF targets, albeit slowly. Unexpectedly, the Grr1-AAA protein can form a complex with Cdc53 (reference 13 and unpublished results). It is not clear whether the interaction of Grr1-AAA with the other SCF subunits occurs via remaining contacts between the F-box and Skp1 or whether interaction between the cullin and Grr1 is sufficient (44). Nevertheless, that complex was thought to be incapable of directing protein ubiquitination. Although we have not established that this complex directs ubiquitination, Mth1 is clearly degraded at a much greater rate than in the absence of the Grr1 protein. Thus, whatever the nature of the remaining interactions, they are sufficient to position the substrate for ubiquitination by the E2-SCF.

We have shown, using the proteasome inhibitor MG132, that the degradation of Mth1, like Cln2, is proteasome dependent. However, we also found that degradation of Mth1 is not required for activation of HXT3 transcription. It is possible that, although degradation via the proteasome is its natural fate, ubiquitylation of Mth1 by SCFGrr1 is sufficient to irreversibly inactivate it. A similar mechanism has been proposed for the ubiquitin-dependent, but proteolysis-independent, inhibition of the transcription factor Met4 during transcriptional induction of MET genes (20; see reference 28 for a differing view). Nevertheless, it seems likely that inactivation of Mth1 by ubiquitin-dependent proteolysis is the natural mode of regulation in untreated wild-type cells.

One of the key steps in ubiquitin-mediated degradation is substrate recognition. As Cln2 phosphorylation is necessary for binding to Grr1 (17, 51, 56), the dissection of Cln2 has defined a phosphodegron motif necessary and sufficient for recognition by Grr1 (4). That motif contains four CDK phosphorylation sites that contribute to the Grr1-Cln2 interaction. Similarly, a phosphodegron containing at least six phosphorylated residues is known to be necessary for the recognition of Sic1 by SCFCdc4 (37). Based upon that work, it was suggested that multiple low-affinity sites create a concentration threshold and a kinetic lag that contributes to the regulation of the length of G1 phase. More recently, the involvement of equilibrium binding between a single receptor site in Cdc4 and multiple low-affinity sites in Sic1 was suggested (39). It is not clear to what extent these models apply to other F-box protein-substrate interactions. However, the requirements for multiple basic residues in the Grr1 LRR domain and for multiple phosphorylation sites on Cln2 are consistent with a similar mechanism for Cln2 ubiquitination (4, 17).

Our analysis of grr1ΔL, grr1-B4Q, and grr1-ΔC mutants indicates that the specific recognition of substrates by SCFGrr1 differs between Cln2, Mth1, and the putative target in the SPS signaling system, despite the apparent importance of protein phosphorylation for all of these interactions. One interpretation of our results is that distinct domains and residues within domains are involved in recognition of each of these three substrates, or that the importance of each domain or residue may differ depending on the substrate. It is clear that the residues relevant for the interaction of Cln2 with Grr1 are much less significant for the recognition of Mth1 by Grr1. There may also be significant differences in the affinity of Grr1 for each of these targets. It seems therefore probable that the affinity of Grr1 for phosphorylated Mth1 is high, because Mth1 proteolysis is rapid following glucose induction (t1/2 < 5 min) despite the low abundance of Mth1 protein. However, the relative affinity of Grr1 for the various substrates is unknown.

In addition to its role in the glucose induction of HXT genes, Grr1 also plays a role in the regulation of genes by external amino acids via the SPS system (3, 18). This suggests that there is at least a partial conservation of mechanism between these distinct signaling pathways. Rgt2, a permease-like glucose sensor, interacts with Yck1/2, which promote the phosphorylation of Mth1 and Std1, thereby targeting them for ubiquitylation by SCFGrr1 (35). We found that, like Grr1, either Yck1 or Yck2, or both, are important for the activation of BAP3, TAT1, and PTR2. Based upon that observation, we propose that Ssy1, a permease-like amino acid sensor, interacts with those casein kinases, promoting phosphorylation of an as-yet-unknown substrate, thereby targeting it for ubiquitylation by SCFGrr1. It is intriguing that domains other than the LRR domain of Grr1 can efficiently participate in the recognition of this substrate. The target could be either a negative component of the signaling pathway that is inactivated by ubiquitination or a positive component that needs to undergo ubiquitin-dependent processing to be activated in response to amino acids. Ssy5 or Ptr3 are potential targets of casein kinase phosphorylation and Grr1-dependent ubiquitylation, as both have been reported to be modified in response to external amino acids (12).

It is tempting to suggest, based upon the apparent coupling of SCFGrr1 and Yck1/2 in signal transduction, that recognition of signaling pathway components by Grr1 is dependent upon a specific kinase signature inscribed on the substrate. In addition to their roles in these nutrient signaling pathways, both Yck1/2 and SCFGrr1 have been implicated in other pathways, including those leading to sporulation and pseudohyphal growth, suggesting that this coupling may extend to other systems (17, 33). However, establishing whether the effects of Yck1/2 and SCFGrr1in those pathways are due to the same collaboration between Yck1/2 and Grr1, as observed for the degradation of Mth1, will require the identification and characterization of the relevant targets of those enzymes.

Acknowledgments

We thank M. Peter, L. Robinson, and D. Wolf for strains used in this study. We appreciate the technical support of M. Guaderrama (TSRI) and R. Soden (GNF). We thank J.-M. Galan, F. van Drogen, R. de Bruin, and G. Schoch for helpful comments on the manuscript and members of the TSRI cell cycle group for helpful discussion and advice.

J.R.W. was supported by the Novartis Research Foundation. This work was supported by funding to C.W. from U.S. Public Health Service grants GM43487 and GM59441.

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