HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 10, 8248-8254, March 8, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/10/8248    most recent M107255200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Horak, J. Articles by Wolf, D. H. Search for Related Content PubMed PubMed Citation Articles by Horak, J. Articles by Wolf, D. H. Social Bookmarking                      What's this?

Two Distinct Proteolytic Systems Responsible for Glucose-induced Degradation of Fructose-1,6-bisphosphatase and the Gal2p Transporter in the Yeast Saccharomyces cerevisiae Share the Same Protein Components of the Glucose Signaling Pathway^*

Jaroslav Horak, Jochen Regelmann^§, and Dieter H. Wolf^§¶

From the  Institute of Physiology, Department of Membrane Transport, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic and ^§ Institut für Biochemie der Universität Stuttgart, D-70569 Stuttgart, Germany

Received for publication, July 31, 2001, and in revised form, December 20, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Addition of glucose to Saccharomyces cerevisiae inactivates the galactose transporter Gal2p and fructose-1,6-bisphosphatase (FBPase) by a mechanism called glucose- or catabolite-induced inactivation, which ultimately results in a degradation of both proteins. It is well established, however, that glucose induces internalization of Gal2p into the endocytotic pathway and its subsequent proteolysis in the vacuole, whereas FBPase is targeted to the 26 S proteasome for proteolysis under similar inactivation conditions. Here we report that two distinct proteolytic systems responsible for specific degradation of two conditionally short-lived protein targets, Gal2p and FBPase, utilize most (if not all) protein components of the same glucose sensing (signaling) pathway. Indeed, initiation of Gal2p and FBPase proteolysis appears to require rapid transport of those substrates of the Hxt transporters that are at least partially metabolized by hexokinase Hxk2p. Also, maltose transported via the maltose-specific transporter(s) generates an appropriate signal that culminates in the degradation of both proteins. In addition, Grr1p and Reg1p were found to play a role in transduction of the glucose signal for glucose-induced proteolysis of Gal2p and FBPase. Thus, one signaling pathway initiates two different proteolytic mechanisms of catabolite degradation, proteasomal proteolysis and endocytosis followed by lysosomal proteolysis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Signal transduction and conversion into different cellular responses are of utmost importance for cell life and adaptation. It is the aim of our work to shed some light on the question of how the nutrient glucose is able to induce two different degradation pathways, the ubiquitin proteasome-linked hydrolysis of FBPase¹ and the lysosomal uptake and elimination of the membrane transporter Gal2p, in the yeast Saccharomyces cerevisiae.

Selective modification by ubiquitin serves as the primary signal, which confers instability on numerous naturally and/or conditionally short-lived proteins in eukaryotic cells. One well-established role for the covalent linkage of ubiquitin is to mark cytosolic and nuclear proteins as well as those proteins that are subjected to endoplasmic reticulum-associated degradation to hydrolysis by the 26 S proteasome (1-7). For most of the 26 S proteasome protein targets, the attachment of a polyubiquitin chain composed of at least four ubiquitin monomer units linked by isopeptide bonds between Lys⁴⁸ of ubiquitin molecules and the C-terminal carboxyl group of the following ubiquitin facilitates their binding to the proteasome (8, 9). However, ubiquitination of many short-lived cell surface nutrient transporters, ion channels, and signal-transducing receptors appears to play no role in proteasomal proteolysis and signals their internalization into the endocytotic pathway followed by subsequent proteolysis in the vacuole instead (1, 10-12). In all known cases of yeast plasma membrane proteins, a single ubiquitin moiety or di-ubiquitin chains in which ubiquitin molecules are linked through Lys⁶³ appear to suffice to trigger their endocytotic uptake and degradation in the vacuole. Most of the short-lived plasma membrane proteins are constitutively internalized into the endocytotic system. The rate of their internalization can be increased by a change in a variety of parameters such as nutrient availability, binding of substrates or other ligands, or stress (13). Sugar transporters that are inducible by their own substrates, including the maltose-specific Mal11p and Mal61p (14-16), the galactose-specific Gal2p (17, 18), and also the hexose-specific Hxt6p and Hxt7p (19), represent a group of proteins that are significantly degraded in the presence of an easily fermentable carbon source such as glucose. Their degradation occurs by a mechanism called glucose- or catabolite-induced inactivation due to apparent analogy with the catabolic inactivation of gluconeogenic enzymes (20, 21). Glucose-induced inactivation is a process that occurs upon arrest of cytosolic protein synthesis by either nitrogen source depletion or the addition of cycloheximide to the growth medium in combination with glucose (in the case of transporters) or simply by the addition of glucose (in the case of gluconeogenic enzymes). Although inactivation conditions and the ultimate fate of the proteins are similar, the gluconeogenic enzymes and the above-mentioned sugar transporters are hydrolyzed by two distinct proteolytic systems: gluconeogenic enzymes are degraded by the 26 S proteasome, whereas transporters are degraded by the vacuolar proteases.

To identify the mechanism(s) that determines the different fates of proteins observed during their inactivation, we wanted to elucidate whether these differences could be due to utilization of distinct glucose sensing/signaling pathways by different proteolytic systems. Two proteolytic target proteins undergoing glucose-induced degradation, FBPase as a substrate of the 26 S proteasome and galactose transporter Gal2p as a substrate of vacuolar proteases, were chosen. Our previous studies (17, 18) had revealed that in response to glucose addition to galactose-grown cells, Gal2p is monoubiquitinated at several lysine residues. This is triggered through the Ubc1p-Ubc4p-Ubc5p triad of ubiquitin-conjugating enzymes and the ubiquitin-protein ligase Npi1p/Rsp5p before transfer of Gal2p to the vacuole, the site of its degradation. In contrast, upon glucose addition to cells growing on a nonfermentable carbon source, polyubiquitination of FBPase occurs. This is also induced by the Ubc1p-Ubc4p-Ubc5p triad of ubiquitin-conjugating enzymes and, in addition, requires Ubc8p. This modification appears to be essential for its proteolysis by the 26 S proteasome (22-25).

Each extracellular signal must be registered by the cell; thereafter, the signal must be transduced and ultimately translated into a biochemical response. For glucose signaling in yeast, a variety of signal transduction pathways exist, and the ultimate response is a change in synthesis or degradation of specific sets of proteins. The main glucose (catabolite) repression pathway (reviewed in Refs. 26-29), the pathway controlling the induction of hexose transporter genes (reviewed in Refs. 28-31), and the cAMP-protein kinase A pathway (reviewed in Refs. 29, 32, and 33) are the three best-characterized pathways that respond to the increased availability of glucose. There is evidence that some protein components of these pathways, Rgt2p, Rgt1p, Grr1p, Reg1p, and Hxk2p, are involved in posttranslational proteolysis of the maltose-specific Mal61p transporter (34-36). We started our analysis with a search for the potential functions of the main protein components of glucose sensing systems in the Gal2p and FBPase proteolysis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Yeast Strains and Growth and Inactivation Conditions-- The S. cerevisiae strains used in this study are listed in Table I. For assays of Gal2p turnover, the yeast cell cultures were grown in either rich medium (1% yeast extract and 2% Bacto peptone) or minimal SD medium (0.67% Difco nitrogen base without amino acids), supplemented with 2% D-galactose plus 0.02% glucose and auxotrophic requirements. The cells were grown at 30 °C on a rotary shaker (200 rpm) to an A₆₀₀ of 0.5-1.0. To induce the Gal2p inactivation, the cells were harvested by centrifugation (2500 × g, 4 min), washed with water, and resuspended in 0.17% yeast nitrogen base without ammonium and amino acids plus 2% glucose or any other "inactivating" sugar as indicated to an A₆₀₀ of 3. In wild type cells, the effect of 2-deoxy-D-glucose, 6-deoxy-D-glucose, and 3-O-methyl-D-glucose on catabolite degradation of Gal2p was also tested in the presence of 2% D-galactose. Similarly, the influence of glucose on the fate of Gal2p in the hxk1hxk2glk1 triple mutant was tested in the presence of 2% D-galactose. The cell samples were taken at the times indicated below over a 6-h period, and for each sample, total cell extracts were prepared for Western analysis as described previously (18). For assays of FBPase turnover, the overnight cell cultures grown on rich medium in the presence of 2% D-glucose were diluted 1:100 in the same medium and grown for additional 6-7 h. Then the cells were washed, resuspended in rich medium containing 2% ethanol to an A₆₀₀ of 0.5, and grown for an additional 16-17 h to de-repress FBPase (final A₆₀₀ = 4-5). To induce FBPase inactivation, D-glucose was added to 2% concentration to the cell suspension at an A₆₀₀ of 3, and cell samples were taken at the times indicated below over 1-2 h. For each sample, total cell extracts were prepared for Western analysis. When other sugars were used for FBPase inactivation, pulse-chase analysis was done (see below).

Metabolic Labeling and Immunoprecipitation of FBPase-- Yeast cultures were grown in complete medium (SD medium with amino acid supplements) without L-methionine (CM-Met) containing 2% D-glucose until an A₆₀₀ of 5-6 was reached. After harvesting and washing with water, the cells were preincubated in CM-Met medium containing 2% ethanol for 2.5 h at an A₆₀₀ of 5. Radiolabeling was done by adding [³⁵S]methionine (Amersham Biosciences, Inc.) to the suspension to a final concentration of 45 µCi/ml. After 3.5 h at 30 °C, the cells were collected by centrifugation and resuspended in complete medium containing 10 mM L-methionine and 2% glucose or another "inactivating" sugar. 1-ml samples were taken at the times indicated, and trichloroacetic acid (final concentration, 5%) was added. The precipitates were washed with 100 µl of acetone and lysed in 100 µl of breaking buffer (50 mM Tris-HCl, pH 7.5, 6 M urea, 1% SDS, and 1 mM EDTA) with glass beads three times for 5 min with intermittent heating for 1 min at 100 °C. After cooling, the lysate was diluted with 1 ml of immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 190 mM NaCl, 6 mM EDTA, and 1.25% Triton X-100) containing complete protease inhibitor mix and 1 mM phenylmethylsulfonyl fluoride and centrifuged, and the supernatants were transferred to new test tubes. FBPase antiserum (3 µl) was added, and the samples were gently agitated for 2 h at room temperature. Immunoprecipitates were collected by adding a protein A-Sepharose CL-4B suspension (Amersham Biosciences, Inc.; 5% (w/v) in immunoprecipitation buffer) and further incubated for 1 h. The Sepharose beads were centrifuged (2 min, 3000 rpm) and washed three times with immunoprecipitation buffer. Proteins were released from Sepharose by boiling in 50 µl of 2× Laemmli sample buffer (200 mM Tris-HCl, pH 6.8, 10% SDS, 20% glycerol, and 0.2% bromphenol blue) for 10 min at 100 °C. Proteins were separated by SDS-PAGE (10%), and protein bands were quantified with a PhosphorImager (Molecular Dynamics).

Western Blotting Analysis-- Western blot analysis of FBPase was carried out as described previously for the Gal2p transporter (20), with one exception; the trichloroacetic acid-precipitated proteins were solubilized for 40 min at 37 °C and then solubilized for another 3-4 min at 95 °C. The Gal2p protein and FBPase in the extracts were detected using anti-Gal2p (1:2000) and anti-FBPase (1:5000) specific antibodies and the ECL Western blotting kit (Amersham Biosciences, Inc.). Antibodies to Gal2p were raised against a synthetic peptide corresponding to amino acids 1-20. The peptide was coupled to keyhole limpet hemocyanin and used for immunization of rabbits (Eurogentec). The relative intensities of the Gal2p and FBPase bands at each time point were quantified by scanning densitometry on ECL-Hyperfilms.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Components of the cAMP-Protein Kinase A Signal Pathway Are Not Involved in the Proteolysis of Gal2p and FBPase-- The addition of rapidly fermentable sugars such as glucose to glucose-depleted cells or cells grown on nonfermentable carbon sources causes a rapid, transient peak in intracellular cAMP concentration. This cAMP signal triggers a cAMP-protein kinase A-mediated protein phosphorylation cascade that affects numerous targets at both the transcriptional and posttranslational levels (29, 32, 33). Components of this cAMP pathway in yeast include the heterotrimeric G protein Gpa2p and, upstream of Gpa2p, the G protein-coupled receptor Gpr1p, which stimulate cAMP synthesis by adenylate cyclase Cyr1p in response to glucose and other easily fermentable carbon sources (37-39). Activation of cAMP production is also dependent on glucose transport via Hxt transporters and hexokinase-mediated intracellular phosphorylation of this sugar, suggesting that glucose is sensed extracellularly by Gpr1p and intracellularly by hexokinases (29). Besides Gpa2p, the G proteins Ras1p and Ras2p regulate basal activity of adenylate cyclase; however, the role of Ras signaling in signal transmission, if any, is unclear at present.

The fact that the conditions used to trigger proteolysis of the Gal2p transporter and FBPase also cause an increase in intracellular cAMP concentration prompted us to examine the fate of these proteins in gpr1 and gpa2 deletion mutants in response to glucose. Our data indicate that the rates of Gal2p (Fig. 1A) and FBPase (Fig. 1B) degradation are not altered in the mutant strains as compared with the wild type. Analogous results were obtained when the fates of Gal2p and FBPase were measured in a strain carrying a deleted RAM1 gene encoding the -subunit of protein farnesyltransferase, which modifies the Ras protein (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Glucose-induced degradation of the Gal2p transporter and FBPase is independent of the Gpr1p-Gpa2p G protein-coupled receptor system. Strains BY4743 (parental strain), gpr1, and gpa2 were grown to exponential phase on rich medium containing 2% galactose plus 0.02% glucose (A) or 2% ethanol (B). For controls, the gal2 strain ( in A) was grown on rich medium containing raffinose (2%) instead of glucose, whereas the fbp1 strain ( in B) was grown as the wild type strain. Standard inactivation protocols were used as described under "Materials and Methods." At the indicated times, total cell extracts were prepared, and the proteins were subjected to Western blot analysis with Gal2p (A)- and FBPase (B)-specific polyclonal antibodies. The intensity of the protein bands present in the immunoblots was quantified by scanning densitometry.

The Glucose Sensors Snf3p and Rgt2p and the Putative Sensor-interacting Proteins Mth1p/Htr1p and Std1p/Msn3p Are Not Involved in Glucose-induced Proteolysis of Gal2p and FBPase-- Because in addition to its regulatory functions, glucose is a nutrient, its presence can be detected by the cells in several ways: (i) by glucose-specific receptors, (ii) by its metabolite(s), or (iii) by the metabolic changes it causes. To decide among these alternatives, we analyzed the fate of Gal2p and FBPase by measuring their turnover rates in yeast strains carrying deletions in two genes, SNF3 and RGT2, encoding putative glucose sensors/receptors using standard inactivation assays. The Snf3p and Rgt2p proteins are unique members of the hexose transporter (Hxt) family comprising about 20 proteins related in sequence (40-42). These two proteins differ from the other Hxt family members in that they are poorly expressed, control rather than mediate glucose transport, and possess unique, extremely long, presumably cytoplasmic C-terminal tails. They are thought to play a role in glucose sensing at low (Snf3p) and high (Rgt2p) extracellular glucose concentrations (31, 43-45). Immunoblot analysis monitoring the fate of Gal2p and FBPase indicated a half-life of about 1 h for Gal2p (Fig. 2A) and about 20 min for FBPase (Fig. 2B) in wild type as well as snf3 and rgt2 mutant cells. Thus, the rate of proteolysis of both proteins in response to glucose is not altered in snf3 and rgt2 deletion mutants, indicating that none of the glucose receptors examined is involved in the signaling pathway for degradation of Gal2p and FBPase.

View larger version (65K): [in this window] [in a new window]   Fig. 2.   Putative glucose sensors/receptors Snf3p and Rgt2p do not play any role in glucose-induced degradation of Gal2p and FBPase. Analysis of Gal2p (A) and FBPase (B) in strains CEN.PK2-1C (parental strain), SK1 (snf3), and SK2 (rgt2) was performed as described in the Fig. 1 legend.

Recent work has discovered two additional proteins, Mth1p/Htr1p and Std1p/Msn3p, that may also be components of the glucose induction mechanism, which regulates HXT gene expression (46-48). Both proteins interact with the C-terminal tails of the glucose sensors Snf3p and Rgt2p and are presumably involved in the transmission of glucose signals in the upper part of the glucose sensing/signaling pathway(s). Although our results suggest that a glucose signaling pathway independent of the glucose sensors Snf3p and Rgt2p is involved in the proteolysis of Gal2p and FBPase, they do not exclude the requirement of Std1p and Mth1p in this process a priori. Therefore, we compared the fates of Gal2p and FBPase in wild type strains with those in std1 and mth1 deletion mutants. The rates of the Gal2p and FBPase degradation are not significantly affected in std1 and mth1 mutants as compared with the wild type strain (data not shown).

Transport and Phosphorylation of Glucose and Certain Related Sugars Are Necessary and Sufficient to Trigger Proteolysis of Gal2p and FBPase-- Because metabolism-independent glucose receptors/sensors Snf3p and Rgt2p are not involved in the detection of extracellular glucose, other obvious candidates for such a function are the general hexose transporters (Hxt) and glucokinases, which mediate the uptake and subsequent phosphorylation of intracellular glucose and related sugars. To decide between glucose itself and its metabolite(s) as the primary signal for Gal2p and FBPase degradation, we first compared the fate of both of these proteins in response to glucose, glucose analogues, and related sugars known or supposed to be the substrates of Hxt transporters. The selected sugars include the nonmetabolizable 6-deoxy-D-glucose and D-fucose, as well as 2-deoxy-D-glucose and 3-O-methyl-D-glucose, which can be phosphorylated but not metabolized further. Other sugars tested are the rapidly fermented D-fructose and D-mannose, which are metabolized like D-glucose but with somewhat lower efficacy. To unravel whether the signal needed for triggering of Gal2p and FBPase proteolysis is specific for the substrates of Hxt transporters or not, the capability of the sugars D-galactose, D-maltose, raffinose, and sucrose, which are substrates of other transporters and/or other metabolic pathways, was examined. Our results are shown in Fig. 3 and are summed up as follows. (i) Gal2p is not degraded when the nonmetabolizable sugars 6-deoxy-D-glucose and D-fucose, a gratuitous inducer of Gal2p,² are present. However, the presence of 2-deoxy-D-glucose and 3-O-methyl-D-glucose is sufficient for a relatively rapid degradation of Gal2p (Fig. 3A). Because degradation of Gal2p is tested under starvation conditions devoid of a carbon source, the effects of nonmetabolizable sugars on Gal2p degradation could be due to the lack of ATP production. The ability of 2-deoxy-D-glucose to deplete intracellular ATP pools has been described previously (49). We therefore examined the fate of Gal2p in the presence of 2-deoxy-D-glucose, 3-O-methyl-D-glucose, and 6-deoxy-D-glucose and also in the presence of the easily usable carbon and energy source D-galactose. No differences in the inactivation patterns were observed (data not shown), indicating that changes in the ATP content, if any, are not responsible for the effects observed. (ii) The easily fermentable sugars D-fructose and D-mannose appear to be comparably as efficient as glucose in triggering the proteolysis of Gal2p (Fig. 3A). This is in contrast to the Mal61p transporter, whose proteolysis is not significantly induced by D-fructose or D-mannose (36). (iii) Selected di- and trisaccharides that are split into their monosaccharide moieties either in the extracellular space (sucrose in most yeast strains, or raffinose) or after uptake by specific transporters are hydrolyzed intracellularly (such as maltose, for instance) serve as "inactivating" sugars to a different extent. Maltose triggers degradation of Gal2p in galactose-grown wild type cells carrying a constitutive MAL2-8^C allele that causes high MAL gene expression even in the absence of maltose (CEN.PK2-1C strain), but not in the wild type W303-1A strain (mal background) This suggests that transport of maltose has to occur for initiation of Gal2p degradation (Fig. 3A). (iv) Galactose itself does not lead to significant proteolysis of the Gal2p transporter. Taken together, our results show that the generation of an intracellular metabolic signal that initiates proteolysis of the Gal2p transporter requires transport and at least phosphorylation of the proper sugar. The signal is not specific for glucose, neither its transport through Hxt transporters nor its metabolism. The rapid glucose-triggered inactivation of FBPase does not occur under the same inactivation conditions used for the Gal2p transporter, whose proteolysis is strongly accelerated by nitrogen starvation (see here) or by the addition of cycloheximide, an inhibitor of cytosolic protein synthesis (17). The presence of cycloheximide or nitrogen starvation prevents glucose-triggered degradation of FBPase, probably due to the absence of an "inactivation factor" whose glucose-induced synthesis is necessary to fulfill this function (50). Moreover, although glucose immediately stops new synthesis of FBPase in cells growing in ethanol, it is not clear whether the same effect also occurs in the presence of other putative "inactivating" sugars. Therefore, to avoid possible misinterpretations due to the interference of new synthesis of FBPase with its simultaneous degradation, we examined the fate of FBPase in response to some glucose analogues and related sugars by pulse-chase analysis. The results shown in Fig. 3B demonstrate that FBPase behaves similarly to the Gal2p transporter in response to different "inactivating" sugars, such as the easily fermentable D-mannose and D-fructose, which are comparably as efficient as D-glucose. In contrast, 6-deoxy-D-glucose and D-galactose do not significantly induce degradation of FBPase. FBPase proteolysis in galactose-grown strains W303-1A and CEN.PK2-1C is similarly affected by maltose, as is Gal2p hydrolysis.

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Proteolysis of Gal2p and FBPase in the presence of different sugars. A, analysis of Gal2p in wild type strain W303-1A was done as described in the Fig. 1 legend. The ability of D-maltose to induce proteolysis of Gal2p (A) was measured in W303-1A (maltose (1); mal background) and CEN.PK2-1C (maltose (2); MAL2-8C background) wild type strains. B, the fate of FBPase in response to the representative group of sugars was followed in the same wild type cells by pulse-chase analysis as described under "Materials and Methods." Gal, D-galactose; Fru, D-fructose; Man, D-mannose; Fuc, D-fucose; 6dGlc, 6-deoxy-D-glucose; 2dGlc, 2-deoxy-D-glucose; 3mGlc, 3-O-methyl-D-glucose; Raf, D-raffinose; Suc, sucrose; Mal, D-maltose.

The HXK2 Gene Product Plays a Primary Role in the Induction of Proteolysis of Gal2p and FBPase-- S. cerevisiaecontains three glucose-phosphorylating enzymes, hexokinase 1, hexokinase 2 (Hxk2p), and glucokinase 1. These enzymes catalyze the first step in the glycolytic pathway, the phosphorylation of glucose to glucose-6-phosphate. Hxk2p is primarily responsible for catalyzing this step of glycolysis when glucose is abundant. It is also required for transcriptional repression of a large group of genes (MAL, GAL, SUC2, and so forth; Refs. 26-28), for generation of a signal for expression of some HXT genes encoding hexose transporters (31), and for the inactivation of Mal61p, the maltose transporter (36). These properties of Hxk2p are unique and are not shared by the other two glucose kinases. To further examine the involvement of the above-mentioned glucokinases in the degradation of Gal2p and FBPase, we compared their fate in mutants carrying single or multiple mutations in these kinases. Glucose-induced proteolysis of Gal2p (Fig. 4A) and FBPase (Fig. 4C) is similar to that of the wild type in strains expressing Hxk2p as the only glucose kinase. Interestingly, the rate of Gal2p degradation was more rapid in the mutant strain expressing only Hxk2p than in the wild type strain. A similar effect has also been observed with Mal61p inactivation and proteolysis (35). Deletion of HXK2 completely abolishes degradation of Gal2p (Fig. 4B) and FBPase (Fig. 4D), whereas deletion of none of the other kinases (glucokinase 1 and hexokinase 1) affects proteolysis of the two proteins. As expected, no degradation of Gal2p and FBPase occurs in the strain missing all three glucokinases. To exclude the possibility that the inability of glucose to induce Gal2p degradation in the glucokinase triple mutant is due to insufficient ATP, we also examined the fate of Gal2p in the presence of glucose and galactose. No alteration in the block of Gal2p degradation was observed (data not shown). These findings strongly support the view that Hxk2p alone is sufficient for full generation of the glucose signal triggering degradation of both enzymes.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Hexokinase Hxk2p plays a dominant role in Gal2p and FBPase degradation. Analysis of Gal2p (A and B) and FBPase (C and D) degradation was done in two sets of isogenic strains. The first set consisted of strains W303-1A (parental strain), YSH7.4-11A (hxk1HXK2glk1), and YSH7.4-3C (hxk1hxk2glk1) (A and B). The other set consisted of strains BY4743 (parental strain) and hxk1, hxk2, and glk1 single deletion strains (B and D). For an analysis of FBPase degradation, the cells were grown on 2% D-galactose before enzyme de-repression in a 2% ethanol medium. Inactivation of Gal2p and FBPase was started by the addition of 2% D-glucose.

The Roles of GRR1, RGT1, and REG1 Gene Products in Glucose-induced Proteolysis of Gal2p and FBPase-- We also examined the fate of Gal2p and FBPase in mutant strains defective in three additional genes, GRR1, RGT1, and REG1, encoding putative downstream effectors of the pathway. Because mutations in the GRR1 gene relieve the repression of numerous glucose-repressed genes (51) and prevent glucose induction of some HXT genes (52), Grr1p was suggested to play a central role in the transcriptional regulation by glucose. Genetic analysis suggested that Grr1p inhibits the function of Rgt1p, a zinc finger containing DNA-binding transcription factor that functions as a transcriptional repressor in the absence of glucose and as a transcriptional activator at high glucose concentrations, thereby causing de-repression of HXT gene expression (31, 53). In addition to its role in regulating Rgt1p, Grr1p has a variety of functions (34, 54-57) that may be unrelated to glucose-induced signal transduction. Reg1p is a regulatory subunit of Glc7p protein phosphatase type 1 that directs the catalytic subunit of the enzyme to proteins involved in transcriptional repression of several genes by glucose (28). It is furthermore involved in the induction of proteolysis of the Mal61p transporter (36). When following the fate of Gal2p (Fig. 5A) and FBPase (Fig. 5B) in grr1, rgt1, and reg1 deletion mutants, we found that their proteolysis is significantly disturbed in grr1 and almost completely blocked in reg1 mutants, whereas the rate of their proteolysis observed in the rgt1 mutant is comparable with the rate measured in the wild type strain. Our results thus indicate that Grr1p and Reg1p are central components of the glucose signal transduction mechanism responsible for glucose-induced proteolysis of both Gal2p and FBPase.

View larger version (51K): [in this window] [in a new window]   Fig. 5.   Glucose-induced proteolysis of Gal2p and FBPase requires the presence of functional GRR1 and REG1 gene products. Analyses of Gal2p (A) and FBPase (B) in BY4743 (parental strain) and grr1, rgt1, and reg1 mutant strains were done as described in the Fig. 1 legend.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Glucose-induced degradation of the plasma membrane-located Gal2p transporter and of cytoplasmic FBPase follows different proteolytic pathways: Gal2p is endocytosed and degraded via vacuolar proteases, whereas FBPase is degraded by the 26 S proteasome. We show that the nature of the pathway selected is not due to different initiating signals generated by distinct glucose sensing/signaling pathways. Recent analyses of proteins involved in glucose-triggered inactivation and proteolysis of the plasma membrane-localized maltose transporter Mal61p in yeast revealed the function of two glucose signaling pathways called pathway 1 and pathway 2 that contribute to this process (34-36). Pathway 1 is a glucose transport-independent pathway in which glucose probably binds to the glucose sensor Rgt2p and generates a signal, which is transmitted through the Grr1p protein followed by proteolysis of Mal61p. In pathway 2, rapid transport and metabolism of glucose and/or some other sugars yield an unknown glucose metabolite(s) sufficient to generate an intracellular signal. This is transmitted via signal transducers, which are composed of regulator subunits Reg1p and Reg2p of the Glc7p protein phosphatase type 1.

The results presented here are consistent with the view that glucose signaling pathway 2 generates the major signals that initiate proteolysis of Gal2p and FBPase. Proteolysis of both model proteins appears to be independent of Snf3p and Rgt2p, the putative glucose sensors/receptors (Fig. 2, A and B). Interestingly, the same result, independence of Snf3p and Rgt2p, was also found for proteolysis of the Hxt6p and Hxt7p glucose transporters in response to high external glucose concentrations (19). Based on our examination of the fate of Gal2p and FBPase in response to glucose, glucose analogues, and related sugars, several conclusions can be drawn. Transport and at least sugar phosphorylation are prerequisites for generation of a signal initiating proteolysis of Gal2p and FBPase. 6-Deoxy-D-glucose and D-fucose are not phosphorylated and do not induce proteolysis (Fig. 3). The strict requirement for phosphorylation of sugars suggests that the sensing mechanism probably does not likely operate at the level of sugar transport. Also consistent with such a view is our finding that maltose, the substrate of maltose-specific transporters, is able to elicit a signal needed for proteolysis of Gal2p in a galactose-grown strain that constitutively synthesizes the maltose transporter. The ability of 2-deoxy-D-glucose and 3-O-methyl-D-glucose, two phosphorylated glucose analogues, to induce degradation of Gal2p, also firmly substantiates the necessity of phosphorylation of the appropriate sugar for the different Gal2p and FBPase degradation pathways. Hxk2p is the most active enzyme of the three yeast glucokinases catalyzing phosphorylation of glucose at C6. In addition, it is rather specifically required for a variety of regulatory effects, including transcriptional repression and/or induction of certain genes as well as posttranslational inactivation of the maltose transporter. Hxk2p exists in a phosphorylated monomer-unphosphorylated dimer equilibrium, and the available data suggest that the state of phosphorylation is essential for at least transcriptional repression and induction (58). Nevertheless, the molecular basis of its exact function in glucose signaling remains unclear. Our knowledge about the role(s) of Hxt2p in glucose sensing stems mainly from an analysis of the main repression pathway in yeast strains carrying different hxk2 mutant alleles. In most of these hxk2 mutants, glucose repression appears to inversely correlate with the phosphorylation activity of Hxk2p. However, in some mutant strains, the catalytic and regulatory functions of Hxk2p are at least partially uncoupled, suggesting that not just the catalytic activity of the enzyme may be important for its role in glucose sensing. Our results shown in Fig. 4, A and B, indicate that Hxk2p plays a major, if not exclusive, role in the pathway whose ultimate result is proteolysis of Gal2p and FBPase. D-Galactose catabolism bypasses the hexokinases in galactose-grown cells: galactose is phosphorylated via galactose kinase, Gal1p. The molecule also finally yields the glucose derivative glucose-6-phosphate, but without catalysis by Hxk2p. This might argue that only hexose-binding, catalytically active Hxk2p can exert a signaling function. This view is supported by the observation that deletion of the two other kinases, hexokinase 1 and glucokinase 1, increases the degradation rate of Gal2p as compared with the wild type (Fig. 4A). Their absence must increase the glucose concentration, which can interact solely with Hxk2p in the mutants. Only actively catalyzing Hxk2p might expose a domain at its surface, which is able to transmit a signal. There is still the possibility that the intracellular glucose-6-phosphate concentration necessary to trigger proteolysis of Gal2p and FBPase is attained in response to glucose but not galactose addition. There is also the possibility that Hxk2p may have some noncatalytic role in sensing and/or signaling required for proteolysis of Gal2p and FBPase. The strong inhibition of glucose-induced degradation of Gal2p and FBPase observed in grr1 and reg1 deletion mutants (Fig. 5, A and B) suggests that the GRR1 and REG1 gene products may function as downstream components of the glucose sensing/signaling pathway. However, grr1 mutations are very pleiotropic (34, 51, 52, 54-56), and we do not know at present whether the contribution of Grr1p to glucose-induced degradation of Gal2p and/or FBPase is direct or indirect (for a summary, see Fig. 6). Studies in this direction, as well as attempts to elucidate mutual interactions between downstream effectors Grr1p, Rgt1p, and Reg1p and their functional consequences, are in progress.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Key protein components of the glucose signaling pathway responsible for glucose-triggered proteolysis of the Gal2p transporter and FBPase. Red script set on a blue tone, proteins excluded from signaling Gal2p and FBPase proteolysis. Blue script set on a yellow tone, proteins necessary for signaling Gal2p and FBPase proteolysis. PM, plasma membrane.

The chemical nature of the signal(s) initiating glucose-induced degradation of Gal2p and FBPase is unknown. The ATP:AMP ratio or its change during repression/de-repression of yeast genes observed in response to the availability of glucose led to a proposal that this ratio might act as a signal for the glucose repression pathway (59). Although the same signal might also serve as a messenger in other glucose-regulated processes, including posttranslational inactivation and/or proteolysis of some proteins, it nevertheless remains possible that the glucose metabolites glucose-6-phosphate, UDP-glucose, or trehalose may fulfill this function.

	ACKNOWLEDGEMENTS

We thank E. Boles and J. M. Thevelein for kindly providing strains and A. Kruckeberg for an initial supply of anti-Gal2p antibodies. We thank Zuzana Syková for technical assistance. We also thank Frank Josupeit and Elisabeth Tosta for expert help with preparation of the manuscript.

	FOOTNOTES

^* This work was supported by Grants 204/01/0272, 204/02/1240, and IAA50 11005 from the Grant Agency of the Czech Republic, the Deutsche Forschungsgemeinschaft, Bonn, SFB 495 and the Fonds der Chemischen Industrie, Frankfurt.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Institut für Biochemie, Universität Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany. Tel.: 49-711-685-4390; Fax: 49-711-685-4392; E-mail: dieter.wolf@po.uni-stuttgart.de.

Published, JBC Papers in Press, December 28, 2001, DOI 10.1074/jbc.M107255200

² J. Horak, J. Regelmann, and D. H. Wolf, unpublished data.

	ABBREVIATIONS

The abbreviations used are: FBPase, fructose-1,6-bisphosphatase; Hxk2p, hexokinase 2.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				G. M. Santangelo Glucose Signaling in Saccharomyces cerevisiae Microbiol. Mol. Biol. Rev., March 1, 2006; 70(1): 253 - 282. [Abstract] [Full Text] [PDF]

				G.-C. Hung, C. R. Brown, A. B. Wolfe, J. Liu, and H.-L. Chiang Degradation of the Gluconeogenic Enzymes Fructose-1,6-bisphosphatase and Malate Dehydrogenase Is Mediated by Distinct Proteolytic Pathways and Signaling Events J. Biol. Chem., November 19, 2004; 279(47): 49138 - 49150. [Abstract] [Full Text] [PDF]

				K. M. Dombek, N. Kacherovsky, and E. T. Young The Reg1-interacting Proteins, Bmh1, Bmh2, Ssb1, and Ssb2, Have Roles in Maintaining Glucose Repression in Saccharomyces cerevisiae J. Biol. Chem., September 10, 2004; 279(37): 39165 - 39174. [Abstract] [Full Text] [PDF]

				D.-Y. Cui, C. R. Brown, and H.-L. Chiang The Type 1 Phosphatase Reg1p-Glc7p Is Required for the Glucose-induced Degradation of Fructose-1,6-bisphosphatase in the Vacuole J. Biol. Chem., March 12, 2004; 279(11): 9713 - 9724. [Abstract] [Full Text] [PDF]