INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Ubiquitin-mediated proteolysis is now widely recognized as an important regulatory process in eukaryotes. Its usefulness in biological regulation is due to the tremendous flexibility and specificity of the ubiquitination machinery. That machinery comprises an elaborate assemblage of components that includes the E3 ubiquitin ligases. Two classes of E3 complexes, the anaphase promoting complex and the Skp1/cullin/F-box protein (SCF) complex have come under intense scrutiny due largely to their involvement in regulation of the cell division cycle. Both factors are now understood to comprise modular sets of adapters that confer specificity on relatively nonspecific E2 ubiquitin-conjugating enzymes. Furthermore, both classes of regulatory complexes are highly conserved among eukaryotes (reviewed in reference 14). One key to understanding the regulation conferred by these factors is to understand the basis for substrate discrimination.

SCF complexes comprised of Skp1, Cdc53/cullin, Rbx1/Roc1, and an F-box protein associate with ubiquitin-conjugating enzymes (Ubcs) to mediate the interaction of SCF with specific substrates (21, 24, 48, 50). SCF comprises a family of protein complexes, each with a different F-box protein component. The nature of the F-box protein determines the specificity of the interaction between Ubc-SCF and its targets. Related complexes have been demonstrated in yeast, plant, and animal cells. Although 11 F-box proteins have been identified in the budding yeast genome most have yet to be assigned to a specific function. Three (Cdc4, Grr1, and Met30) have been shown clearly to associate with SCF (7, 42). SCF complexes containing each of those proteins have been shown to interact with a distinct group of ubiquitination targets. For example, SCF^Cdc4 is specifically required for the ubiquitination of the CDK inhibitors Sic1 (9, 49) and Far1 (13) and the replication protein Cdc6 (8). SCF^Grr1 targets the G₁ cyclins Cln1 and Cln2 (2, 42) and the putative Cdc42 effector Gic2 (17) for ubiquitination, and SCF^Met30 has been shown to be involved in recognition of both the morphogenesis checkpoint kinase Swe1 (19) and transcription factor Met4 (18, 45). Involvement of F-box proteins as specificity factors for ubiquitination is not restricted to yeasts but appears to be a common theme among eukaryotes. Among the best-characterized mammalian F-box proteins are -TRCP (reviewed in reference 22) and Skp2 (6, 33, 36, 52), which play roles in the recognition of IB and of p27 and E2F1, respectively. Thus, the E2-SCF ubiquitinating complexes comprise an evolutionarily conserved system that is widely exploited in the regulation of diverse biological processes.

Although there does not seem to be any consistent feature in the proteins targeted by SCF for ubiquitination, many of the targets that have been studied to date are phosphorylated. In some cases those proteins have been shown to be phosphorylated and to depend on phosphorylation for proteolysis. In several cases, the target proteins were shown to bind to SCF only in their phosphorylated state. Phosphorylation-dependent binding, ubiquitination, and degradation of Cln1 and Cln2 by SCF^Grr1 (30, 42, 49, 55), of Sic1 by SCF^Cdc4 (9, 49), and of IB by SCF^TcRP (reviewed in reference 22) have been demonstrated using a combination of in vivo mutational analysis and in vitro reconstituted ubiquitination systems. In each case, targeting of the proteins for ubiquitination has been shown to depend on a specific F-box protein and the phosphorylation state of the protein. Thus, ubiquitination of specific targets not only depends on the identity of that target but also on its state.

In addition to their involvement in targeting proteins for ubiquitin-mediated proteolysis, there are a number of examples of the involvement of F-box proteins in transcriptional regulation. Although in some cases SCF-dependent ubiquitination and even proteolysis may be involved, in most cases the mechanism has yet to be established. Met30 has been shown to be important for repression of MET gene expression (51), and Grr1 is required for induction of the expression of hexose and amino acid permeases encoded by the HXT genes (reviewed in reference 38) and AGP1 (15), respectively. Although Met30 clearly regulates MET gene expression via SCF-dependent ubiquitination of the transcription factor Met4, ubiquitin-mediated proteolysis appears to be unimportant for that regulation (18; but see reference 45). The role of Grr1 in nutrient-regulated transcription in yeasts is even less clear. Induction of the HXT1 gene in response to glucose is defective in grr1 mutants. That defect is suppressed by inactivation of the transcriptional repressor, Rgt1 (39-41, 53). This has led to the hypothesis that Grr1 inactivates Rgt1 or a coregulator in response to glucose (10, 31), perhaps via ubiquitin-mediated proteolysis. Grr1 has also been shown to be required for transcriptional induction of the amino acid permease gene AGP1 in the presence of exogenous amino acids. However, because the relevant transcription factors have not been identified, neither the targets nor the precise role of Grr1 in that process has been established.

These and other observations clearly define a role for F-box proteins in determining the specificity of SCF-target interactions. However, the basis for that specificity and the nature of the protein-protein interaction sites remain to be elucidated. This class of proteins is characterized by the presence of an F box. Although their other features are less conserved, many contain motifs recognized as protein-protein interaction domains conserved throughout biological systems. The leucine-rich repeat (LRR) is one such motif (reviewed in reference 26). An LRR domain is comprised of multiple LRRs, each consisting of a beta strand and an alpha helix separated by a variable region, which all fold into a horseshoe structure that forms a parallel beta sheet on the concave surface with helices on the convex surface. These domains are found in a variety of proteins of disparate function, including the F-box proteins Grr1 and Skp2.

We investigated the LRR domain of Grr1 as a potential site for target recognition. Grr1 contains 12 complete LRRs and 1 partial LRR belonging to the LRR cysteine-containing subfamily. We investigated whether the LRR domain of Grr1 interacts with its substrates and characterized the basis of the specificity of Grr1 for phosphorylated substrates. As was previously shown (23, 31), we found that the LRR region is essential for the functioning of Grr1 and its ability to bind target proteins. Molecular modeling of the Grr1 LRR revealed an unusually high density of cationic charges on the concave surface of the horseshoe. Based on that model, we showed that those positively charged residues are important for binding phosphorylated G₁ cyclin. In contrast, the same residues were shown not to be important for assembly of the SCF^Grr1 complex. The inability of Grr1 to bind phosphorylated targets resulted in their stabilization and in phenotypes consistent with inactivation of SCF^Grr1. However, the same mutations had no effect on some of the other Grr1-dependent functions. We concluded that the positively charged surface of the LRR is critical for the recognition of at least one class of phosphorylated SCF^Grr1 targets.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Yeast strains and culture. Yeast strains used are listed in Table 1. All strains were isogenic with 15 Daub, W303a, or 1278b, as indicated. Culture conditions and medium were as indicated and were prepared by standard methods.

Plasmids. Plasmids used in this study are listed in Table 2. Full-length GRR1 was cloned by adding an NcoI-NdeI fragment encompassing the first 840 nucleotides generated by PCR into pADH1-grr1N (31). All pADH1-derived plasmids contained a single hemagglutinin (HA) epitope at the amino-terminal end of the GRR1 open reading frame. The grr1 point mutants were constructed by site-directed mutagenesis with the pALTER mutagenesis system (Promega). Fragments encompassing the mutated site or sites were subcloned into pADH1-GRR1 for use in interaction studies and into the pKAN-6His vector (modified from pKHA3) (Kan^r) (35; S. B. Haase, M. Wolff, and S. I. Reed, unpublished results) for targeted integration into the GRR1 locus. grr1L was constructed by deleting the EcoRV-StuI fragment encoding amino acids 447 to 754 from the open reading frame, whereas grr1C was constructed by adding the first 840 nucleotides of the open reading frame of GRR1 to a grr1NC construct that was generated by PCR (nucleotides 840 to 2700, amino acids 280 to 900). grr1NCF was generated by PCR and included nucleotides 1171 to 2700, which encode amino acids 391 to 900. All DNA fragments generated by PCR were sequenced to confirm fidelity prior to use.

Antibodies. The 12CA5 anti-HA monoclonal antibody was derived from ascites fluid provided by I. Wilson, The Scripps Research Institute, La Jolla, Calif. The antibody was conjugated to protein A-Sepharose for use in immunoprecipitation experiments. Affinity-purified polyclonal anti-Cln2 (57) and anti-Cdc28 (56) antibodies were prepared as previously described. Anti-6His (Quiagen), anti-myc (Santa Cruz Biotechnology) and anti-HA (Babco) were obtained commercially. Polyclonal anti-glutathione S-transferase (anti-GST) was a generous gift from S. Reed (The Scripps Research Institute).

Preparation of yeast cell extracts. Exponentially growing yeast cells were pelleted, washed with cold 1× phosphate-buffered saline, and resuspended in yeast lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% NP-40, 10% glycerol) containing 50 mM sodium fluoride, protease inhibitors (0.4 mM phenylmethylsulfonyl fluoride, 1 µg of pepstatin per ml, 1 µg of leupeptin per ml, and 1 µg of aprotinin per ml), and phosphatase inhibitors (0.1 mM sodium orthovanadate, 5 mM EDTA, 5 mM EGTA, and 10 mM sodium pyrophosphate). Cells were lysed with glass beads by using seven cycles of vortexing for 1 min followed by a 1-min incubation on ice. Extracts were collected after centrifugation for 15 min at 14,000 rpm in an Eppendorf S417R microcentrifuge. The protein concentrations of the lysate were determined by the Bio-Rad protein assay (Bio-Rad Laboratories).

Immunoprecipitation and immunoblotting. Immunoprecipitation was carried out by incubating 750 to 1,000 µl of yeast extract containing 0.5 to 1.0 mg of total yeast protein with anti-HA (12CA5) antibody conjugated on protein A-Sepharose beads (Sigma) for 1 h at 4°C with gentle rocking. The beads were then washed three times with 1 ml of yeast lysis buffer, resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and resolved by SDS-PAGE. For immunoblotting, proteins were transferred to Imobilon membranes (Millipore Corp.) by semidry transfer at 200 mA for 2 to 6 h. The proteins were detected using Super Signal (Pierce). Dilutions of the antibodies for Western blots were 1:1,000 for anti-Cln2 and anti-6His antibodies (Quiagen), 1:2,000 for anti-HA (Babco) and anti-myc antibodies (Santa Cruz Biotechnology), 1:5,000 for anti-GST antibody, and 1:10,000 for anti-Cdc28 antibody.

Determination of the stability of Cln2 and Gic2. For determination of Cln2 and Gic2-green fluorescent protein (GFP) half-life by promoter shut-off, cell strains were grown in 2% raffinose until optical density at 600 nm (OD₆₀₀) was 0.5 to 0.8. Galactose was added to a concentration of 2% to induce expression driven by the GAL1 promoter for 45 min in the case of CLN2 and for 3 h in the case of GIC2. Glucose was added to a concentration of 2% to repress the expression. Samples were taken before the induction and at the indicated time following repression by glucose. Cln2 abundance was assayed by immunoblotting with anti-Cln2 antibody (57), and Gic2-GFP abundance was assayed using an anti-GFP antibody (Boehringer-Mannheim).

Structural modeling of the LRR of Grr1. Coordinates used for three repeats of the LRR from Grr1 were those from the previous model of Kajava (20). Those repeats contain three -strands covered on one face by two -helices. Side chains of the three repeats were replaced by those of the Grr1 molecule using XFIT (34), and their positions were energy minimized using X-PLOR 3.8 (5) with the CHARMM22 all-atoms force field (4). To assemble the 12 repeats of Grr1, we superimposed the Grr1 repeats on the crystal structure of the RNase inhibitor (27) using InsightII (MSI, Inc., San Diego, Calif.). Side chains were replaced by XFIT and subsequently energy minimized in X-PLOR. Finally, two insertions required in the 6th and 12th repeats and a deletion in the 11th repeat were carried out in TURBO-FRODO (46). Insertions increased the length of both C-terminal helices by almost a turn (three residues). This model was optimized by several rounds of refinement using TURBO-FRODO and X-PLOR.

RT-PCR. Grr1 mutant strains were grown in 2% galactose in rich medium to mid-log phase. Glucose was added to half of the culture to a 4% concentration, and cells were collected 90 min after induction of glucose. For AGP1 reverse transcriptase PCR (RT-PCR), only the glucose-induced cells were used.

Filamentous assay conditions. To evaluate pseudohyphal development, cells were pregrown for 2 days on synthetic minimal (SD) medium at 30°C and then transferred to SLAD. To avoid disturbing the agar surface and the colony density-dependent inhibition of pseudohypha formation (58), single unbudded cells were carefully placed 1 cm from each other by using the needle of a dissecting microscope. Between 20 and 100 colonies, each derived from a single unbudded cell, were analyzed for each strain. Pseudohyphal growth was evaluated by multiple criteria. First, colony and cell morphologies were monitored after 1 day of growth on SLAD plates (an assay of the extent of early morphological differentiation). Next, the cell and colony morphologies were evaluated after 5 days of growth on SLAD before (total growth) and after mechanically washing the noninvasive cells from the plate surface (invasive growth).

Microscopy and imaging. Cells from suspension cultures were imaged with differential interference contrast (DIC) optics on a Nikon Eclipse E800 microscope using IPLAB Spectrum software and a Photmometrix Quantix charge-coupled device camera. Images were processed for publication with Adobe Photoshop software.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Protein-protein interaction domain containing LRR and additional domains in the C terminus are required for Cln2 binding. The interaction between the E2-E3 complex Cdc34-SCF and its substrates depends on both the nature of the F-box protein component of the complex and the identity and phosphorylation state of the substrate. The interaction of Grr1 with phosphorylated G₁ cyclins Cln1 and Cln2 can be demonstrated in vivo (23, 55) (Fig. 1B) as well as in vitro (49, 50). Those complexes also contain SCF components including Cdc53 and Skp1 (1, 9, 42, 49, 55) (Fig. 1C). We have evaluated the importance of various Grr1 domains (Fig. 1A) for the interaction between Grr1 and Cln2 by analyzing the abundance of Cln2 present in immune complexes prepared from extracts of yeast cells expressing wild-type and mutant forms of epitope-tagged Grr1 (Grr1-HA) from the ADH1 promoter. In agreement with previous analyses of these same deletion mutants with the yeast two-hybrid assay (31), we found that deletion of the amino-terminal 280 amino acids of Grr1 (Grr1N) caused only a modest defect in the capacity to specifically interact with Cln2 protein in vivo (Fig. 1C). In contrast, a more dramatic effect was caused by deletion of either the C-terminal 234 amino acids (Grr1C) or the 308-amino-acid domain that comprises the majority of the LRRs (Grr1L). Deletion of the carboxy terminus results in a dramatic reduction in binding to Cln2 compared to that observed with wild-type Grr1. However, the small amount of Cln2 protein that forms a complex with Grr1C, like the protein binding to wild-type Grr1, is primarily in the phosphorylated form. Finally, Grr1 lacking the predicted LRR region was completely deficient in Cln2 binding (Fig. 1B), consistent with a previous report (23). Based on this analysis, it appears that both the LRR and, to a somewhat lesser extent, the carboxy-terminal sequences of Grr1 are important in the interaction of Grr1 with its substrates.

View larger version (45K): [in this window] [in a new window]   FIG. 1.   (A) Domain structure of Grr1 and positions of deletion mutations. Domains are indicated across the top of the diagram, and positions of the deletion mutations are indicated across the bottom of the diagram. Residue numbers are indicated for the end points of the deletions. (B) Comparison of Cln2 binding to full-length and domain deletion mutations of Grr1. Anti-HA immunoprecipitates were prepared using extracts (WCE) from 15Daub strains expressing epitope-tagged CLN2 from the GAL1 promoter and wild-type or deletion mutation forms of GRR1-HA from the ADH1 promoter. Components of the extracts and immune complexes were evaluated using either anti-HA (12CA5), anti-Cln2, or anti-Cdc28 antibodies. (C) Interaction of Grr1 derivatives with Cln2 and the SCF components, Skp1 and Cdc53. Anti-HA immunoprecipitates were prepared using extracts (WCE) from 15Daub strains carrying CDC53-myc, SKP1-GST expressed from the CUP1 promoter, GAL-CLN2-FLAG, and wild-type or deletion mutation forms of GRR1-HA from the ADH1 promoter. Components of the extracts and immune complexes were visualized using either anti-HA (12CA5), anti-Cln2, anti-myc, or anti-GST antibodies.

Predicted structure of LRR of Grr1 reveals a potential binding site for phosphorylated substrates. The apparent importance of the LRR for the interaction between Grr1 and phosphorylated Cln2 as well as the impact of LRR deletion on formation of SCF^Grr1 prompted us to analyze this motif in greater detail. Consistent with the observations described above, LRRs in a number of proteins have been shown to be important for protein-protein interactions. Structural models for a number of the subfamilies of LRRs have been proposed based on the conservation of the motif between members of those subfamilies (20, 26) and the crystal structure of the porcine ribonuclease inhibitor (RI) (25-27). The basic repeat units of a number of distinguishable classes of LRRs have been defined. The Grr1 LRRs fall into the cysteine-containing LRR family (20). Using that repeat, we derived an alignment for the 12 LRRs of Grr1 (Fig. 2A). This repeat structure differed somewhat from previously suggested repeat alignments for Grr1, but it was most consistent with the repeat unit derived from structural modeling by Kajava (20) for the cysteine-containing family.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   (A) Alignment of LRRs. The LRRs of Grr1 used to construct the structural model are shown. Residues indicated in green are those that are generally conserved between repeats. The residues that have been altered in the mutations presented in this study are depicted in red letters. Structural properties (-sheet by the arrow; -helix by the helix) of the repeats are indicated by the diagram at the top. (B) Space-filling model. Structural prediction for the LRR of Grr1. A tube diagram is overlaid on a space-filling model derived for the LRR domain comprised of the residues presented in panel A. Amino acid side chains are shown only for those residues that have been altered by mutation. Red and blue surfaces of the space-filling model represent negatively and positively charged regions of the surface of that domain, respectively.

Basic residues localized on the putative binding surface but not the opposite surface affect binding of SCF^Grr1 to Cln2. The structure of the Grr1 LRR derived from computational modeling indicates an abundance of basic residues on the concave surface. In contrast, a relatively random distribution of charged residues was observed on the convex surface predicted by the model. This is particularly striking when considered in the context of the role of Grr1 as an adapter for recognition of phosphorylated targets by SCF. At least in the case of Cln2, those phosphorylated residues appear to be clustered (30; S. Lanker, unpublished data). Based on the predicted structure, we hypothesized that either the basic character of the concave surface or the specific distribution of basic residues on that surface determines the capacity of SCF^Grr1 to recognize phosphorylated Cln2.

View larger version (46K): [in this window] [in a new window]   FIG. 3.   Analysis of Cln2 binding by Grr1 with alterations in the basic residues of the LRR. (A) Cln2 binding by Grr1 with basic residues of the LRR replaced by glutamine (Q). Anti-HA immunoprecipitates were prepared from extracts (WCE) of strains expressing wild-type or mutant GRR1-HA expressed from the ADH1 promoter and CLN2 expressed from the GAL1 promoter. Immunoblots were probed with the anti-HA or anti-Cln2 as indicated. Grr1-B4Q represents the protein with all four basic residues on the concave surface of the LRR converted to glutamine. (B) Interaction of Grr1 point mutants with glutamate replacement of basic residues with Cln2. This experiment was similar to that shown in panel A, except that the basic residues in the LRR of Grr1 were replaced by glutamate (E) instead of glutamine. Grr1-B4E represents the protein with all four basic residues on the concave surface of the LRR converted to glutamate.

grr1LRR

grr1C

View larger version (82K): [in this window] [in a new window]   FIG. 4.   Interaction of Grr1 point mutant proteins with Skp1, Cdc53, and Cln2. Anti-HA immunoprecipitates were prepared from strains expressing the wild type or the indicated mutation of GRR1-HA from the ADH1 promoter along with SKP1-GST expressed from the CUP1 promoter, CDC53-myc, and GAL-CLN2, as described in the legend to Fig. 1C.

grr1 mutants deficient in Cln2 binding are also defective in proteolysis of Cln2. To obviate problems associated with the use of overexpressed proteins, single copies of each of the deletion mutant constructs as well as several of the point mutant constructs were introduced into cells under control of the native GRR1 promoter by targeted integration. Each of the point mutant products accumulated to levels equivalent to or modestly higher than that of the wild-type protein. The grr1L product accumulated to a level that is approximately two- to threefold higher than that of the wild-type protein when expressed from that promoter (Fig. 5A). A similar increase relative to the wild-type protein was observed when grr1L was expressed in multiple copies from the ADH1 promoter (Fig. 1C). In contrast, the Grr1C protein accumulated to approximately one-half the level of the wild-type Grr1 (Fig. 5A; data not shown). It is likely that the altered accumulation of these proteins is a consequence of their altered stability, which is known to be dependent on SCF function (59). However, we have not evaluated the stability of these Grr1 derivatives.

View larger version (40K): [in this window] [in a new window]   FIG. 5.   (A) Accumulation of Grr1-6His and Cln2 proteins in various point and deletion mutations of GRR1. Twenty-five micrograms of whole-cell extract from asynchronously growing cells expressing the wild type or the indicated GRR1-6His form from its endogenous promoter were analyzed by immunoblotting using anti-His, anti-Cln2, and anti-Cdc28 antibodies, respectively. (B) Stability of Cln2 in cells having point and deletion mutations in Grr1. Cells carrying CLN2 under the inducible GAL1 promoter and wild-type GRR1 or its derivatives (grr1, grr1L, grr1-B4Q, and grr1-B4E) under its native promoter were pregrown under noninducing conditions (2% raffinose). GAL1-CLN2 was expressed for 45 min by addition of 2% galactose and then repressed by addition of 2% glucose. Extracts prepared from samples taken at the times indicated were analyzed by immunoblotting using anti-Cln2 and anti-Cdc28 antibodies. UI, uninduced.

grr1

grr1L

Morphological consequences of grr1 mutations. We have analyzed the morphological phenotypes associated with the various grr1 mutants (Fig. 6). The mutants have been analyzed both when growing in rich liquid medium (yeast extract-peptone-dextrose [YEPD] liquid; Fig. 6A) or on rich solid medium (YEPD plate; Fig. 6B). Consistent with their effect on Cln2 stability, both the grr1L and grr1C mutants caused cells to become elongated and to bud in a unipolar fashion. This was particularly apparent in cells growing on solid medium (Fig. 6B). The grr1 point mutants exhibited a gradation of severity that was roughly consistent with their effect on Cln2 proteolysis. Mutations resulting in neutralization of basic residues on the predicted convex surface of the LRR had no phenotypic effect, whereas those neutralizing basic residues on the predicted concave surface had a pronounced effect on cell polarity, bud site selection, and, to a lesser extent, abscission (Fig. 6 and data not shown). Whereas single- and double-point mutations had a relatively modest effect on cell morphology, the effect of the quadruple-point mutations (grr1-B4Q and grr1-B4E) on polarized growth and bud site selection became more pronounced. Finally, replacement with an acidic residue generally resulted in more severe phenotypes than replacement with a neutral residue at the same position. Nevertheless, whereas grr1-B4E mutations are similar to the grr1 mutations in the severity of their effects on morphological phenotypes, they have less severe effects on growth rate (data not shown).

View larger version (103K): [in this window] [in a new window]   FIG. 6.   (A) Morphological phenotypes of point and deletion mutations of GRR1 growing in rich liquid medium. Cells grown to late log phase were analyzed by DIC microscopy and digital imaging. (B) Morphological phenotypes of point and deletion mutations of GRR1 growing on rich solid medium. Cells were grown on rich glucose medium (YEPD) for 4 h prior to photomicrography.

Mutations in GRR1 affecting Cln2 binding and stability also result in stabilization of Gic2, a target for SCF^Grr1-dependent proteolysis. The fact that mutations on the putative concave surface of the Grr1 LRR affected the capacity of SCF^Grr1 to bind to Cln2 and resulted in its stabilization suggested that the same mutations would also result in the stabilization of other SCF^Grr1 targets. The stability of the putative Cdc42 effectors Gic1 and Gic2 has been shown to be phosphorylation dependent and to be mediated by SCF^Grr1 (17). To determine whether the basic residues on the concave surface of the Grr1 LRR were required for the degradation of the Gic2 protein, we analyzed the stability of Gic2-GFP expressed from the repressible GAL1 promoter in cells having the grr1-B4Q allele at the chromosomal locus as the only source of Grr1. The Gic2-GFP fusion protein was rapidly degraded when the GAL1 promoter was repressed by the addition of glucose (Fig. 7). However, the same protein was dramatically stabilized in grr1L mutants, as well as in cells carrying grr1-B4Q (Fig. 7) and grr1-B4E (not shown) mutations. Thus, mutations that either eliminate the LRR domain of Grr1 or neutralize the charge on its concave surface result in stabilization of the Gic2 protein, presumably due to the failure of Grr1 to bind to Gic2 and promote its ubiquitination. We conclude that the concave surface of the Grr1 LRR, by virtue of its basic nature, participates in binding to phosphorylated targets of the SCF^Grr1 ubiquitin ligase.

View larger version (57K): [in this window] [in a new window]   FIG. 7.   Stability of Gic2 in cells having point and deletion mutations in Grr1. Cells carrying GIC2-GFP under the inducible GAL1 promoter and wild-type GRR1 or its derivatives (grr1L and grr1-B4Q) under its native promoter were pregrown under noninducing conditions (2% raffinose). GAL1-GIC2-GFP was expressed for 3 h by addition of 2% galactose and then repressed by addition of 2% glucose. Extracts prepared from samples taken at the times indicated were analyzed by immunoblotting using anti-GFP and anti-Cdc28 antibodies. UI, uninduced.

Mutations in GRR1 have differential effects on Cln2 stability and other established functions of Grr1. Grr1 has been implicated in a number of cellular functions in addition to proteolysis of G₁ cyclins and Gic1/2. Genetic analysis has implicated GRR1 as a regulator of the expression of several nutrient responsive genes. Of those, the best characterized are the systems governing expression of the HXT genes, which encode a family of hexose permeases in response to glucose (reviewed in reference 38) and which govern the expression of the broad-specificity amino acid permease gene AGP1 in response to amino acids (15). We have investigated the effect of specific grr1 mutations on these functions (Fig. 8). A summary of the properties of a representative set of mutants is presented in Table 3.

View larger version (54K): [in this window] [in a new window]   FIG. 8.   Nutrient-regulated transcription is not affected by mutations in LRR of Grr1. (A) The capacity of wild-type cells or cells carrying the indicated grr1 mutation to induce HXT1 gene expression was evaluated by analysis of the HXT1 mRNA. Total RNA was isolated from strains carrying the indicated GRR1 allele either prior to (G, galactose) or following 90 min of induction with 4% glucose (D, dextrose). The HXT1 mRNA level in each strain was determined using RT-PCR. PCRs for ACT1 were carried out using an equal amount of RNA to ensure equal loading. Samples were performed without reverse transcriptase (-RT) to evaluate the extent of DNA contamination in each RNA preparation. (B) The expression of AGP1 in wild-type cells and cells carrying the indicated grr1 allele under inducing conditions in rich medium. AGP1 expression was analyzed (left panel) by RT-PCR, and (right panel) Northern hybridization. AGP1 and ACT1 transcript levels were determined by RT-PCR (as described for panel A) and Northern hybridization using RNA isolated from glucose-grown cells.

grr1L

grr1C

grr1

grr1

grr1

grr1L

grr1C

grr1L, grr1C

grr1

grr1C

grr1L

grr1

grr1L

View larger version (82K): [in this window] [in a new window]   FIG. 9.   Pseudohyphal differentiation is enhanced by mutations in the LRR of GRR1. (A) 1278b strains carrying either grr1, grr1L, grr1C, or grr1-B4E mutations were grown on SLAD plates for 5 days at 30°C. Photomicrographs were taken after 1 day and 5 days of growth. (B) Effect of inactivation of CLN1 and CLN2 on pseudohyphal differentiation in grr1 strains. 1278b strains carrying the indicated mutations were treated as described for panel A.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

SCF formation and Cln2 binding by grr1 mutants. Grr1 plays a central role in the regulation of SCF-dependent ubiquitination of a number of proteins including Cln2. SCF binding and Cln2 ubiquitination are dependent on phosphorylation of Cln2. We have shown here that the LRR is important for binding phosphorylated Cln2 and for its subsequent proteolysis. Furthermore, we showed that basic residues, predicted by our three-dimensional model to lie on the concave surface of the Grr1 LRR, are critical for the interaction between Cln2 and Grr1. In contrast, basic residues on the convex surface are not important for that interaction nor do they affect Cln2 proteolysis. These results reveal the domain of Grr1 required for the Grr1-Cln2 interaction as well as defining the probable binding surface as predicted by our model. We suggest that electrostatic interactions between basic residues on the concave surface of the LRR and the phosphoryl groups on Cln2 provide the binding energy for this interaction. Similar electrostatic interactions are characteristic of the contacts observed by the X-ray crystal structure of RI and RNase A (25) on which our structural model for the Grr1 LRR was based. Although the charged residues of RI are not as clearly concentrated on the concave surface of the LRR, about a third of the contacts between the LRR of RI and the RNase A molecule are between acidic and basic residues. In that case, primarily acidic residues are contributed by the LRR whereas the binding partner contributes predominantly basic residues to the interaction (27). The importance of electrostatic interactions was further supported by our demonstration that Cln2 phosphorylation is required for recognition by SCF^Grr1 (30, 55). Thus, Grr1 serves as target-specific adapter for ubiquitination by providing a specific binding pocket for interaction with the target.

Differences between requirements for the Grr1-Cln2 interaction and the regulation of other putative targets of Grr1. The role of Grr1 in SCF-dependent ubiquitination extends beyond Cln2 to other protein targets including the Cdc42 interactor Gic2 (17). We show that basic residues on the concave surface of the LRR are required for the instability of Gic2. In addition, Grr1 also plays a role in several systems of nutrient-regulated transcription. The direct targets of Grr1 in those systems are unknown. In fact, it has not been established in either of these cases that the role of Grr1 is in the context of protein ubiquitination. We have shown here that, although disruption of Grr1 results in failure to induce either the HXT1 or AGP1 transcripts, the other deletion mutations or point mutations we have constructed result in little or no defect in the induction of those genes. In contrast, all of those mutations interfere with the ubiquitin-dependent degradation of Cln2. Thus, we consider it likely that the protein domains of Grr1 involved in recognition of phosphorylated Cln2 are unimportant for recognition of the targets required for the regulation of these transcription systems. Alternatively, the capacity of these mutant proteins to function in those pathways may be a reflection of differences in the efficiency of the interactions required for transcriptional activation and G₁ cyclin proteolysis. The differences observed in the requirements for recognition of the relevant targets in these transcriptional pathways may reflect differences in the basis for recognition. For example, recognition of those targets may not involve protein phosphorylation. Resolution of these issues awaits the identification of the relevant targets of Grr1 in those pathways.

LRR as recognition domain for phosphorylated proteins. The capacity of proteins to interact specifically with each other has long been recognized as a fundamental property of living systems and is the basis for the assembly of the macromolecular complexes central to many biological processes. Regulated protein-protein interaction is a critical component of many biological regulatory mechanisms. Protein phosphorylation is one of the most widely studied and perhaps most common mechanisms governing regulated protein recognition. Despite that fact, the basis for recognition is only poorly understood. This may be a consequence of the wide array of solutions to this specific recognition problem.

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