The ubiquitin ligase SCFGrr1 is required for Gal2p degradation in the yeast Saccharomyces cerevisiae

BBRC Biochemical and Biophysical Research Communications 335 (2005) 1185–1190 www.elsevier.com/locate/ybbrc

The ubiquitin ligase SCFGrr1 is required for Gal2p degradation in the yeast Saccharomyces cerevisiae J. Horak a, D.H. Wolf b,* a

Institute of Physiology, Department of Membrane Transport, Academy of Science of the Czech Republic, 142 20 Prague, Czech Republic b Institut fur Biochemie der Universita¨t Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany Received 18 July 2005 Available online 11 August 2005

Abstract F-box proteins represent the substrate-specificity determinants of the SCF ubiquitin ligase complex. We previously reported that the F-box protein Grr1p is one of the proteins involved in the transmission of glucose-generated signal for proteolysis of the galactose transporter Gal2p and fructose-1,6-bisphosphatase. In this study, we show that the other components of SCFGrr1, including Skp1, Rbx1p, and the ubiquitin-conjugating enzyme Cdc34, are also necessary for glucose-induced Gal2p degradation. This suggests that transmission of the glucose signal involves an SCFGrr1-mediated ubiquitination step. However, almost superimposable ubiquitination patterns of Gal2p observed in wild-type and grr1D mutant cells imply that Gal2p is not the primary target of SCFGrr1 ubiquitin ligase. In addition, we demonstrate here that glucose-induced Gal2p proteolysis is a cell-cycle-independent event.
2005 Elsevier Inc. All rights reserved. Keywords: SCFGrr1 ubiquitin ligase; Gal2 transporter; Catabolite degradation; Yeast

Like other eukaryotic microorganisms, the budding yeast Saccharomyces cerevisiae must be able to respond rapidly to varying environments in its natural habitat in order to grow and proliferate appropriately. To achieve optimal exploitation of available nutrients, the yeast developed a complex regulatory network which commonly operates both at the level of gene transcription and/or at the posttranslational level. A well-known example of posttranslational regulation of activities of some key gluconeogenic enzymes and some sugar-specific transporters in response to an easily fermentable carbon source is a mechanism known as catabolite degradation or catabolite inactivation [1,2]. The heart of the mechanism lies in a glucose-induced ubiquitination of the target proteins which, in turn, triggers their degradation through the 26S proteasome (in the case of key enzymes of gluconeogenesis) or by the action of vacuolar *

Corresponding author. Fax: +49 711 685 4392. E-mail address: [email protected] (D.H. Wolf).

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2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.08.008

proteolysis (in the case of sugar-specific transport proteins). We have previously reported that glucose addition to the galactose-grown cells triggers monoubiquitination of the galactose transporter Gal2p at several lysine residues, followed by its release from the plasma membrane and transfer via the endocytotic system to the vacuole for proteolysis [3,4]. In contrast, upon glucose addition to cells grown on media containing a non-fermentable carbon source like ethanol or glycerol, polyubiquitination of fructose-1,6-bisphosphatase (FBPase), the key enzyme of gluconeogenesis, leads to its degradation by the 26S proteasome [5–8]. Furthermore, examination of the mechanism that determines the different fates of Gal2p and FBPase observed during their catabolite degradation uncovered that the systems responsible for their proteolysis, share protein components of the same glucose signaling pathway [9]. Indeed, initiation of both, Gal2p and FBPase proteolysis appears to require the rapid transport of glucose and/or those glucose-related sugars that are at least partly

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metabolized, e.g., phosphorylated, by hexokinase Hxk2p. The yet unknown metabolite signal generated in this way is subsequently transmitted to the targets through downstream components of the glucose signaling pathway, including Reg1 and Grr1p [9]. In this study, we demonstrate that Grr1p plays a role in Gal2p catabolite degradation in the form of the SCFGrr1 complex. We show strong stabilization of Gal2p in cdc34, skp1, and rbx1 (but not in cdc53) mutant cells under the conditions of catabolite degradation, indicating that the Cdc34, Skp1, and Rbx1 proteins are required for this process as is Grr1p. However, similarity-if not identity—of ubiquitination patterns of Gal2p observed in wild-type and grr1D mutant cells suggests that the role of SCFGrr1 in Gal2p degradation is most likely indirect. We also show that Gal2p degradation is an event which proceeds independently of cell-cycle progression. Materials and methods Strains, growth conditions, and methods. The genotype and source of the S. cerevisiae strains used in this study are given in Table 1. Strains are derived from W303: MATa,ade2-1, ura3, leu2-3,112, his31,15, trp1-1, and can1-100, or BY4743: Mata/a,his3D1/his3D1, leu2D0/ leu2D0, MET15/met15D0, lys2D0/LYS2, and ura3D0/ura3D0. In strains used for detection of Gal2p ubiquitination, wild-type ubiquitin (Ub) and Myc-tagged ubiquitin (Myc-Ub) were expressed from the PCUP1 promoter in high-copy plasmids YEp96 and YEp105, respectively. Both plasmids were a kind gift from Hochstrasser [10]. Yeast cells were cultured in rich media (1% yeast extract, 2% bacto peptone, and 50 lM/L adenine) or synthetic media (0.67% yeast nitrogen base without amino acids supplemented with auxotrophic requirements) in the presence of the appropriate 2% carbon source. Except for temperature-sensitive strains, all yeast strains were grown at 30 C to an early exponential phase and further treated at the same temperature. Strains carrying temperature-sensitive alleles were grown at 25 C and thereafter incubated at 37 C for different time intervals, depending on the strain used. To induce Gal2p inactivation, the cells were washed with water and resuspended in 0.17% yeast nitrogen base without ammonium sulfate and amino acids plus 2% glucose. After inactiva-

tion, the cells were collected by centrifugation, washed with water, and assays of Gal2p degradation were performed as previously described [4,9]. Crude cell extracts were prepared either by a TCA procedure or by glass bead lysis. The proteins were resolved by SDS–PAGE, and Western blot analyses of Gal2p were carried out as described previously [4,9]. Gal2p blotted onto nitrocellulose sheets was detected using polyclonal anti-Gal2p antibody (1:2000) and peroxidase-coupled goat anti-rabbit antibody (1:10,000, Medac, Germany). Antibodies were detected using enhanced chemiluminescence (ECL) with ECL or ECL Plus reagents (Amersham Biosciences, Germany). The relative intensities of Gal2p bands at each time point were quantified by scanning densitometry of ECL-films. Synchronization of cells. Exponentially grown wild-type cells were synchronized in early G1 phase with the mating pheromone a-factor (3 lM final concentration), in early S-phase with hydroxyurea (130 mM final concentration), and in metaphase with nocodazole (50 lM). Incubation at OD600 of 0.4–0.5 and 30 C was performed until more than 90% of cells were unbudded (for a-factor) or budded (for hydroxyurea and nocodazole arrest). Synchronization of cdc28-4 and cdc15-2 mutant strains in G1 phase and telophase, respectively, was achieved by shifting 25 C grown cells to 37 C for 4 h at an OD600 of 0.4–0.5. Detection of ubiquitinated Gal2p. This method was adopted from Jarosch et al. [11]. Wild-type and grr1D mutant cells transformed with a plasmid expressing Ub and/or Myc-Ub were grown on synthetic medium containing 2% raffinose until an OD600 of 0.5–1.0 was reached. After harvesting and washing, the cells were incubated in the same medium in the presence of 2% galactose and 100 lM CuSO4 at 25 C for 4 h and then shifted to 37 C for additional 2 h. The cells were harvested by centrifugation, and samples (20 OD600/ml) were taken before and 30 min after addition of inactivation medium. The cell sediments were washed once with ice-cold 20 mM sodium azide containing 2 mM PMSF and 20 mM N-ethylmaleimide (NEM) and once with sorbitol buffer (SB buffer; 0.7 M sorbitol, 50 mM Tris–HCl, pH 7.5) with 2 mM PMSF and 20 mM NEM. Subsequently, all material was kept on ice. Cells were mechanically lysed using glass beads in ice-cold SB buffer containing 2 mM PMF, and 20 mM NEM and protease inhibitors (Complete, Roche Diagnostics, Germany) for 20 min at 4 C on a multivortexer. Lysates were cleared by centrifugation at 3500 rpm for 5 min and the crude membrane fraction was then prepared by centrifugation of the supernatant at 14,000 rpm for 1 h and 4 C. The pellet was resuspended in 100 ll of solubilization mixture (50 mM Tris–HCl, pH 6.8, containing 8 M urea, 5% SDS, and 0.1 mM EDTA) and 3% dithiothreitol. The samples were incubated at 37 C for 30 min and diluted in 1 ml of cold immunoprecipitation buffer (IP buffer; 50 mM Tris–HCl, pH 7.5, 190 mM NaCl, 6 mM

Table 1 Yeast strains used in this study Strain name

Relevant genotype

Background

Source

K699 YMT670 YMT871 Y80 Y552 Y554 rbx1-1 YHY284 H192

Wild-type cdc34-2 cdc53-1 Wild-type skp1-11 skp1-12 rbx1::HIS3 · PDK102 (rbx1-1 in pRS314) grr1::LEU2 gal2::HIS3

W303 W303 W303 W303 W303 W303 W303 W303 W303

M. Tyers M. Tyers M. Tyers S. Elledge S. Elledge S. Elledge S. Elledge C. Wittenberg H. Ronne

Y34633(cul3D) Y31376(cul8D) Y38902(grr1D) Y32692(gal2D)

ygr003w::KANMX4/ygr003w::KANMX4 yjl047c::KANMX4/yjl047c::KANMX4 yjr090c::KANMX4/yjr090c::KANMX4 ylr081w::KANMX4/ylr081w::KANMX4

BY4743 BY4743 BY4743 BY4743

Euroscarf Euroscarf Euroscarf Euroscarf

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EDTA, and 1.25% Triton X-100) containing protease inhibitors, 5 mM PMSF and 10 mM NEM. Insoluble material was removed by centrifugation for 10 min at 14,000 rpm. Anti-Gal2p antibody (10 ll) was added to the supernatant, and the mixture was incubated on a rotary shaker overnight at 4 C. The immunocomplexes were collected by binding to 30 ll of 50% protein A–Sepharose CL-4B slurry (Amersham Biosciences, Sweden) for 1 h at 4 C. After two washes with IP buffer containing 2 M urea, and two washes with IP, the proteins were released from the beads by the addition of the solubilization mixture and incubation at 37 C for 15 min prior to electrophoresis. The proteins were analyzed by SDS–PAGE (7.5%) and immunoblotting with a mouse monoclonal anti-c-Myc (Ab-1) antibody (1.5 lg/ml; Oncogene Research Products). Immunoreactive species were visualized using peroxidase-coupled anti-mouse secondary antibody (1:10,000 dilution, Bio-Rad) and ECL system (Amersham Biosciences, Germany).

Results Protein components of the SCFGrr1 ubiquitin ligase complex are required for Gal2p degradation Grr1p, one of the 21 F-box proteins identified in S. cerevisiae, is a subunit of the SCFGrr1(Skp1-CullinF-box) complex, the member of the large and versatile class of E3 ubiquitin ligases that target proteins for degradation by polyubiquitination [12,13]. Typically, the SCF complexes are comprised of several common, evolutionary conserved components, referred to as a RING-finger protein Rbx1p (also named Hrt1p or Roc1p), a cullin Cdc53p/Cul1p, and a substrate adaptor protein Skp1 in S. cerevisiae. They form a stable complex with a ubiquitin-conjugating enzyme, most commonly Cdc34p [12,13]. In addition, they contain a variable component, the F-box protein, which directly recruits the substrate and is therefore responsible for the substrate specificity of the SCF. With the observation of a strong stabilization of Gal2p in grr1D mutant cells either derived from wild-type strain BY4743 [9] or W303 (Fig. 1A) under the conditions of catabolite degradation, we considered whether Grr1p participates in this process as a component of the SCFGrr1 complex. Because all the other genes encoding for the proteins of the SCFGrr1 complex are essential for growth, we used a set of isogenic temperature-sensitive strains bearing rbx1-1, cdc53-1, skp1-11, skp1-12, and cdc34-2 mutant alleles, respectively. All strains were grown at the permissive temperature of 25 C, then shifted to the restrictive temperature of 37 C for 2 h and the fate of Gal2p was determined under our standard conditions of catabolite degradation at 37 C. Except for cdc53-1 we uncovered a strong impairment of Gal2p degradation in all examined mutants when compared to wild-type (Figs. 1B and C). In the cdc53-1 strain, the Gal2p degradation appears to be almost unaffected and, in addition, its expression is 6–10 times reduced as compared to the other strains tested. Interestingly, we also found that none of the two other cullin family members in S. cerevisiae, which are

Fig. 1. Gal2p degradation depends on SCFGrr1 components. Analysis of Gal2p degradation (A–D) was performed in two sets of isogenic strains. The first set consisted of K699 (wild-type), YHY284 (grr1D), and temperature-sensitive MTY670 (cdc34-2), rbx1 (rbx1-1), Y552 (skp1-11), Y554 (skp1-12), and MT871 (cdc53-1) mutant strains (A–C). The other set consisted of strains BY4743 (wt) and cul3 and cul8 single deletion strains (D). The strains in (B,C) were grown on rich medium at 25 C in the presence of 2% galactose to an early exponential phase and shifted to 37 C for 2 h, while the strains in (A,D) were grown and analyzed at 30 C. For controls, the corresponding gal2D strains (D) were grown on rich medium with 2% raffinose, instead of galactose. The standard inactivation protocol was used as described under Materials and methods. At the indicated times, cell extracts were prepared, proteins were separated by SDS–PAGE and subsequently subjected to Western blot analysis using Gal2p-specific antibodies. Times are given in hours after the addition of inactivation medium to turn off Gal2p expression and initiate its degradation.

encoded by the non-essential CUL3 and CUL8 genes [14], is involved in Gal2p proteolysis (Fig. 1D). Whether the Cdc53p does not participate in Gal2p degradation, or whether the cdc53-1 point mutation does not affect the role of Cdc53p in this process, remains to be determined using other cdc53 alleles. Taken together, these findings however indicate that Grr1p is required for Gal2p degradation in the form of the SCFGrr1 complex. One of the best characterized functions of SCFGrr1 in S. cerevisiae is to target the phosphorylated G1 cyclins Cln1p and Cln2p, and the bud emergence protein Gic2 for ubiquitin-mediated degradation by the 26S proteasome, an event, which is necessary for G1-to-S phase transition [15–17]. We wanted to know whether the role of SCFGrr1 in the control of Gal2p transport activity shares at least some features with those of the above proteins. Therefore, we analyzed the ubiquitination

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pattern of Gal2p and followed its fate in cells synchronized at different cell-cycle stages under the conditions of catabolite degradation. The role of SCFGrr1 ubiquitin ligase in Gal2p is probably indirect

Myc-Ub exhibit no ubiquitinated Gal2p species prior to initiation of catabolite degradation in response to glucose (Fig. 2, lanes 3 and 6), while a complex pattern of Myc-Ub-Gal2p conjugates can be seen after shifting the cells to glucose for 30 min (Fig. 2, lanes 4 and 7). The absence of higher-molecular-mass bands in cells expressing wild-type ubiquitin (Fig. 2, lanes 2 and 5) and in gal2D mutant cells expressing Myc-Ub (Fig. 2, lane 1) after 30 min of glucose addition confirms that the bands observed in Fig. 2 represent ubiquitinated forms of Gal2p.

We previously demonstrated that Gal2p is monoubiquitinated at several lysine residues under the conditions of catabolite degradation through the Ubc1p–Ubc4p–Ubc5p triad of ubiquitin-conjugating enzymes and the Rsp5p ubiquitin ligase [4]. Moreover, it appeared that such a Gal2p modification is necessary and sufficient for its proteolysis by vacuolar proteases, and that the 26S proteasome is not involved in proteolysis [3,4]. The involvement of yet another ubiquitin ligase in Gal2p proteolysis, the SCFGrr1 complex, raised the question, if Gal2p is a direct target of SCFGrr1. We therefore followed the fate of Gal2p in two yeast strains transformed with a plasmid bearing a Myc-tagged version of ubiquitin under the control of the CUP1 promoter. Cultures of wild-type and grr1D mutant cells co-expressing Gal2p with a Myc-Ub and/or Ub were grown and inactivated as described under Materials and methods. Gal2p was immunoprecipitated from the cell extracts and the immunocomplexes containing Gal2p were probed with monoclonal Myc-antibodies for detection of putative ubiquitin-Gal2p conjugates. To minimize the activity of deubiquitinating enzymes that could destroy ubiquitin–protein conjugates during sample preparation, we included NEM, an inhibitor of these enzymes, in all lysis buffers. As can be seen in Fig. 2, extracts from wild-type cells, and grr1D mutants expressing

To decide whether the Gal2p degradation is associated with cell-cycle progression, or not, we synchronized the galactose-grown wild-type cells in G1 phase by a-mating pheromone treatment at 30 C, and followed the fate of Gal2p under the conditions of catabolite degradation. We also applied the same protocol to cells synchronized at early S-phase with hydroxyurea and cells synchronized in metaphase with the spindle toxin nocodazole. Asynchronous cells were followed as a control. Immunoblot analysis shown in Figs. 3A–D clearly demonstrates that the rates of Gal2p degradation are almost identical, irrespective of the point of cell-cycle arrest. Similar results were obtained when Gal2p degradation was tested in the temperature-sensitive mutants cdc28-1 and cdc15-1 with blocked cell division at late G1 and telophase, respectively, at the restrictive temperature of 37 C (data not shown). Based on these experiments we conclude that glucose-induced Gal2p proteolysis is not associated with the regulation of cellcycle progression.

Fig. 2. Glucose-induced ubiquitination patterns of Gal2p are not affected in a grr1D mutant. Extracts prepared from wild-type (K699) and YHY284 (grr1D) cells induced for Gal2p synthesis and overexpressing wild-type ubiquitin (wt-Ub) or Myc-ubiquitin (Myc-Ub) for 4 h were immunoprecipitated with Gal2p antibodies, separated by SDS–PAGE, and probed with monoclonal Myc-antibodies. Lanes 2 and 5: control cells expressing wt-Ub; lanes 3, 4, 6, and 7: cells expressing Myc-Ub- before (lanes 3 and 6) and 30 min after (lanes 4 and 7) glucose addition; lane 1: gal2D mutant cells expressing Myc-Ub, 30 min after glucose addition.

Fig. 3. Cell-cycle-independent degradation of Gal2p. Wild-type cells (K699) were grown to an early exponential phase on rich medium with 2% galactose at 30 C, and were either not treated (A), or arrested in G1 phase by a-factor (B), in early exponential phase with hydroxyurea (C), or in metaphase with nocodazole (D). The standard inactivation protocol at 30 C was applied as described under Materials and methods. Cell extracts were analyzed for Gal2p by immunoblotting as described in the legend to Fig. 1. Times are given in hours after the addition of inactivation medium.

Gal2p degradation is cell-cycle progression-independent

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Discussion We have shown previously that Grr1p is required for proper regulation of glucose-induced proteolysis of Gal2p and FBPase via the glucose-signaling pathway [9]. We here present evidence that Grr1p exerts its function as a component of the SCFGrr1 complex and the ubiquitin-conjugating enzyme Cdc34p. Whether the Cdc53p is not necessary, can be substituted by some other protein, or the point mutation in cdc53-1 does not affect the respective function of the SCFGrr1 complex in Gal2p degradation, has to be shown in the future. The same point mutation in the CDC53 gene also appears not to affect the induction of the APG1 gene encoding a broad-specificity amino acid transporter [18], as well as induction of the MET3 and MET16 genes, whose products are involved in the sulfate assimilation pathway [19], despite the fact that both systems require all other components of the corresponding SCF complexes. Several lines of evidence strongly suggest that the SCFGrr1 is involved in a broad array of cellular functions [12,13]. One of these roles is the regulation of cell-cycle progression via targeting proteins, such as G1 cyclins [15,16], the bud emergence protein Gic2 [17], or the Hop1 protein which is important for actomyosin contraction during cytokinesis [20], by ubiquitination for proteolysis via the 26S proteasome. Here, we present two pieces of evidence showing that SCFGrr1 function in Gal2p proteolysis is indirect. First, ubiquitination patterns of Gal2p are almost identical in wild-type and grr1D mutant cells, suggesting that Gal2p is not a direct target of SCFGrr1 ubiquitination. This idea is consistent with our previously reported data that showed monoubiquitination of Gal2p exerted by the ubiquitin conjugating enzymes Ubc1 and Ubc4/Ubc5 as well as the ubiquitin ligase Rsp5p, a process which is required for its proteolysis, is independent of 26S proteasome function [4]. Second, the rates of Gal2p degradation are the same in cells arrested in distinct stages of cell-cycle, irrespective of the position of the cell-cycle block. In addition to its role in regulation of the cell-cycle, Grr1p is also required for the function of at least two nutrient-sensing pathways, which lead to induced transcription of either some glucose transporter genes HXT ([21,22], for reviews) or a variety of amino acid transporter genes in response to their substrates in the extracellular medium (SPS pathway) [18,21,23]. Each of those pathways is activated via plasma membrane sensors and terminates in the transcription activation of specific promoters. Grr1p is an essential part of those pathways and its exact role begins to emerge slowly. According to the current model, the glucose sensor Rgt2p involved in transcription of glucose transporter genes interacts with the type 1 casein kinases Yck1p and Yck2p in a manner, which results in a phosphorylation of Mth1p and Std1p [24], two proteins, which are

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required to maintain the transcriptional repressor Rgt1p in its hypophosphorylated, promoter-bound form in the absence of glucose [25,26]. When phosphorylated in response to glucose, at least Mtp1p is recognized by Grr1p, ubiquitinated, and targeted for degradation via the proteasome [25,27], leading to dissociation of Rgt1p from HXT promoters due to its hyperphosphorylation, finally leading to the transcriptional induction of HXT genes [25,28]. As for the induction of HXT genes, induction of the SPS pathway requires the plasma membrane sensor Ssy1p, which is thought to form the membraneassociated signaling complex together with the Ssy5 and Ptr3 proteins [21]. Its key role is to activate at least two transcriptional factors, Stp1p and Stp2p, whose binding to the specific promoters leads to increased transcription of the responsive amino acid transporter genes [29,30]. Activation of Stp1p and Stp2p involves their processing [31], possibly via Ssy5p [32]. Some data are consistent with the view that Grr1p-mediated modification is essential for Stp1p cleavage reaction, which might be preceded by its phosphorylation via Yck1p and Yck2p [27,32]. We believe that Gal2p proteolysis in response to the glucose signal is prevented when some yet unknown factor is not degraded via SCFGrr1-mediated ubiquitination. The factor might be a component negatively or positively regulating the signaling pathway, which undergoes ubiquitin-dependent modification for activation in response to glucose as suggested by Bernard and Andre [18]. Alternatively, the SCFGrr1-mediated ubiquitination might not result in degradation of its target, but might be involved in a change of cellular localization and/or activity of a target, as shown recently for Grr1p-dependent ubiquitination of Gis4p, the protein thought to connect the glucose-repression and derepression pathways [33]. At present we are performing a genomic screen to identify other components of the glucose-signaling pathway, including the protein target(s) of the SCFGrr1 complex.

Acknowledgments We thank S. Elledge, M. Hochstrasser, H. Ronne, M. Tyers, and C. Wittenberg for providing yeast strains and plasmids. This work was supported by the Deutsche Forschungsgemeinschaft, SFB 495. J.H. was supported by Grants 204/05/2578 and IAA5011407 from the grant agency of the Czech Republic and the Czech Academy of Science, respectively.

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