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Hkx2 (hexokinase 2) from Saccharomyces cerevisiae was one of the first metabolic enzymes described as a multifunctional protein. Hxk2 has a double subcellular localization: it functions as a glycolytic enzyme in the cytoplasm and as a regulator of gene transcription of several Mig1-regulated genes in the nucleus. To get more insights into the structure–function relationships of the Hxk2 protein, we followed two different approaches. In the first, we deleted the last eight amino acids of Hxk2 and replaced Ser304 with phenylalanine to generate Hxk2wca. Analysis of this mutant demonstrated that these domains play an essential role in the catalytic activity of yeast Hxk2, but has no effect on the regulatory function of this protein. In the second, we analysed whether amino acids from Lys6 to Met15 of Hxk2 (Hxk2wrf) are essential for the regulatory role of Hxk2 and whether there is an effect on the hexose kinase activity of this protein. In the present paper, we report that the Hxk2wca mutant protein interacts with the Mig1 transcriptional repressor and the Snf1 protein kinase in the nucleus at the level of the SUC2–Mig1 repressor complex. We have demonstrated that Hxk2wca maintained full regulatory function because the glucose-repression signalling of the wild-type machinery is maintained. We also report that the Hxk2wrf mutant allele is incapable of glucose repression signalling because it does not interact with Mig1 at the level of the SUC2–Mig1 repressor complex. The two mutants, Hxk2wca and Hxk2wrf retain single functions, as a transcriptional factor or as an enzyme with hexose-phosphorylating activity, but have lost the original bifunctionality of Hxk2.
There are three glucose-kinase isoenzymes in Saccharomyces cerevisiae : isoenzymes 1 and 2 phosphorylate both aldo- and keto-sugars, whereas glucokinase is specific for aldo-hexoses. Isoenzyme 2 of hexokinase (Hxk2) is the predominant glucose-kinase in S. cerevisiae grown on glucose media  and carries out at least two cellular functions. Classical structural studies of yeast Hxk2 reveal a well-defined catalytic domain that binds ATP and hexose (e.g. glucose), allowing transfer of a phosphoryl group from ATP to the C-6 of the sugar [3–5]. In addition to the well-known catalytic role of Hxk2, in the last few years, a new non-canonical function for this protein has been described. It has been suggested that the Hxk2 protein has special functions in transcriptional regulation . Functional studies suggest that the main regulatory role of Hxk2 is produced by interaction with the transcriptional repressor Mig1 and the Snf1 protein kinase to generate a repressor complex in the nucleus [7,8]. Under high-glucose conditions, Hxk2 stabilizes the repressor complex, blocking Mig1 phosphorylation by Snf1 kinase . Thus the complex is involved in the glucose-repression signalling of several Mig1-regulated genes. A determinant for the Hxk2–Mig1 interaction has been characterized as an internal sequence between Lys6 and Met15 of the Hxk2 protein . Moreover, it has been suggested that a fraction of the Hxk2 protein is sequestered to the nucleus by interacting with Mig1 through this amino acid region.
Despite the large number of studies carried out to identify a correlation between catalytic activity and regulatory function in the Hxk2 protein, little is known about the existence of different domains in the protein that may control each activity. Both point  and null  mutations in the HXK2 gene have been found that blocked glucose repression of certain genes. Since the glucose-phosphorylating activity in the corresponding extracts was reduced, the idea that there was a correlation between the glucose-phosphorylating activity of Hxk2 and glucose repression appeared a very attractive one [1,13]. However, this idea can be challenged if we take into account the following results: (i) when the GLK1 (glucokinase 1) gene is overexpressed in a Δhxk1Δhxk2 double-null mutant the transformed strains are still insensitive to glucose repression, even though a 3-fold increase in phosphorylating activity is achieved , (ii) glucose repression is not linearly reduced with decreasing kinase activity , and (iii) mutant alleles with low catalytic activity are still fully functional in glucose signalling . These results suggest that sugar kinase activity and sugar signalling are mediated at least in part through separate domains of Hxk2 [14–17]. Thus the correlation between the catalytic activity of Hxk2 and its ability to mediate glucose repression appears less likely at present.
In the present study, we have exploited the accumulated knowledge on the structure and transcriptional glucose-repression mechanism of Hxk2, to characterize the catalytic and regulatory domains of Hxk2. For the first time, we describe two mutant Hxk2 alleles, one without sugar kinase activity, but with almost full glucose-repression signalling capacity and the other without regulatory function, but with almost full catalytic activity. Our results offer strong evidence that there is no correlation between the regulatory functions of Hxk2 in glucose repression signalling and its catalytic activity.
The strains used in the present study were derived from W303.1A (MATα ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1) . We created two loss-of-function alleles of HXK2 by mutating the wild-type gene in plasmid YEp352-HXK2 to generate HXK2wca (encodes an Hxk2 protein without catalytic activity) and HXK2wrf (encodes an Hxk2 protein without regulatory function) genes using a PCR-based mutagenesis protocol. The KanR (kanamycin-resistance) gene was inserted into the XbaI site, located in the yeast genome 371 bp from the HXK2 stop codon, of plasmids YEp352-HXK2wca and YEp352-HXK2wrf. Then, by using these constructs, recombination cassettes were obtained by PCR; these cassettes contained the mutated HXK2 locus and the KanR marker together with 515 bp from the 5′ genomic region downstream of the HXK2 gene. These linear DNAs were integrated into the HXK2 locus of strains W303.1A and THG1 (MATα leu2-1 ura3-1 lys1-1 hxk1::LEU2 hxk2::LEU2 glk1::LEU2) . To confirm the correct insertion, we PCR-amplified the HXK2 locus from strains W303.1Awca, W303.1Awrf, THG1wca and THG1wrf to detect the presence of the HXK2wca and HXK2wrf mutant alleles by sequencing analysis. The strain FMY20VP is derived from DBY2052 (MATα hxk1::LEU2 hxk2-202 ura3-52 leu2-3,2-112 lys2-801 gal2) and contains the GLK1 gene promoter replaced by the ADH1 (alcohol dehydrogenase 1) constitutive promoter. Yeast two-hybrid experiments employed strain Y187 (MATα ura3-52 his3-200 ade2-101 trp1-901 leu2-3,112 gal4Δ gal80Δ URA3::GAL1UAS-GAL1TATA-lacZ). Immunoprecipitation experiments employed strain FMY303H2 (MATα ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 hxk2::kanMX4) . This strain has a modification of the SNF1 gene at the chromosomal level and produces a triple-HA (haemagglutinin) epitope-tagged Snf1 protein. As a control, in ChIP (chromatin immunoprecipitation) experiments, strain DBY2052 was used .
To identify and understand the roles of the different domains of Hxk2, we designed various truncation, deletion and amino acid substitution mutants and tested their ability to confer function. The mutant design was based on hexokinase sequence alignments and structural data together with the published data on Hxk2 mutants [3,13,15,21].
To determine the functional role of several amino acids identified previously as involved in glucose and ATP binding to the catalytic site of the enzyme, single amino acid substitution mutants were constructed to yield the following: in Hxk2S157A, Ser157, which is involved in glucose binding and is phosphorylated during γ-phosphate transfer from ATP to glucose [22–24], was replaced with alanine. In Hxk2T233A, Thr233, which may interact with the β-phosphate of ATP , was replaced with alanine. In Hxk2S304F, Ser304, which is localized in the active centre of the enzyme , was replaced with phenylalanine.
To assess the functional role of the C-terminal region of Hxk2, both a deletion mutant and a substitution mutant were constructed. The deletion mutant was constructed by insertion of a stop codon into a specific site within the coding region of the HXK2 gene to yield HXK2ΔK478A485, which encodes an Hxk2 protein without the last eight amino acids of the C-terminal end (Hxk2ΔK478A485). The substitution mutant (Hxk2S479A) was constructed by changing Ser479 to alanine. Finally, from the HXK2ΔK478A485 and HXK2S304F genes we obtained the double mutant HXK2ΔK478A485-S304F (HXK2wca).
To analyse the functional role of the N-terminal region of the Hxk2, a truncation mutant and a substitution mutant were constructed. The truncation mutant was obtained by PCR-deletion of 30 bp, between nucleotides +19 and +48, the resulting gene HXK2ΔK6M15, expressed a truncated Hxk2 protein without the amino acids from Lys6 to Met15 (Hxk2wrf). The substitution mutant (Hxk2S14A) was obtained by changing Ser14 to alanine.
The constructs were cloned into the yeast YEp352 vector under the control of the native HXK2 gene promoter. The resulting plasmids were transformed in DBY2052 (Δhxk1Δhxk2 GLK1), a double-mutant strain in which the two hexokinase genes have been deleted.
Yeast protein extracts were prepared as follows: yeasts were grown in 10–20 ml of synthetic or complex galactose medium [SG−ura (synthetic galactose lacking uracil); YEPG (yeast extract/peptone/galactose)] containing 2% galactose at 28 °C to a D600 of 1.0. Then, half of the culture was shifted to synthetic or complex high-glucose medium [SD−ura (synthetic dextrose lacking uracil); YEPD (yeast extract/peptone/dextrose)] containing 2% glucose for 1 h and the other half of the culture was shifted to synthetic or complex low-glucose medium [SE−ura (synthetic ethanol lacking uracil); YEPE (yeast extract/peptone/ethanol)] containing 0.05% glucose plus 3% ethanol for 1 h. Cells were collected, washed twice with 1 ml of 1 M sorbitol and suspended in 100 μl of 50 mM Tris/HCl (pH 7.5) buffer containing 0.2 mM EDTA, 0.5 mM DTT (dithiothreitol), 0.5 mM PMSF, 0.42 M NaCl and 1.5 mM MgCl2. The cells were broken in the presence of glass beads by one 20 s pulse using a FastPrep (Thermo Electron) and 400 μl of the same buffer were added to the suspension. After centrifugation at 14000 rev./min for 15 min at 4 °C using a Beckman F2402H rotor and Allegra 21R centrifuge (Beckman), the supernatant was used as crude protein extract.
For β-galactosidase activity determinations, crude extracts were prepared with glass beads as described above and 2 mg/ml ONPG (o-nitrophenyl β-D-galactopyranoside) was used as a substrate . Specific activity was calculated in relation to total protein in the crude extract, using BSA as the standard. Invertase activity was assayed in whole cells as described previously  and expressed as μmol of glucose released per min per 100 mg of cells (dry weight). Activities are the average of at least ten assays of at least five independent colonies.
Mutant or wild-type yeast cells were grown to a D600 of 1.0 in selective medium containing galactose (2%) and shifted to high- and low-glucose conditions for 1 h. Cells were collected by centrifugation at 3000 g for 1 min at 4 °C, and crude extracts were prepared as described above. For Western blotting, 20–40 μg of protein was separated by SDS/12% PAGE and transferred by electroblotting on to enhanced chemiluminescence PVDF transfer membrane (Amersham Biosciences), which was then incubated with an anti-Hxk2 or anti-HA antibody. Horseradish-peroxidase-conjugated Protein A was used as the secondary reactant. The complex was detected using the West Pico Chemiluminescent system (Pierce).
The yeast two-hybrid analysis  employed yeast vectors pGADT7 and pGBDKT7 and host strains Y187 (described above), in accordance with the Matchmaker two-hybrid system 3 from Clontech. The transformed yeasts were grown in high-glucose [SD−Leu−Trp (synthetic dextrose lacking leucine and tryptophan)] medium until reaching a D600 of 0.8. Assays for β-galactosidase activity followed protocols described previously . Activities are the average of at least ten assays of at least five independently isolated clones. Expression levels of the GAD (Gal4 activation domain) and GBD (Gal4 DNA-binding domain) fusion proteins were controlled by Western blot analysis. Strains lacking the reporter construct did not exhibit any significant activity.
Immunoprecipitation experiments were performed by using whole-cell extracts, from two wild-type yeast strains with modified MIG1 and SNF1 genes which code for C-terminal Mig1 and Snf1 proteins tagged with 3HA epitopes respectively. Extracts were incubated with anti-Hxk2 antibody (Santa Cruz Biotechnology) or anti-Pho4 antibody for 1 h at 4 °C. Protein A–Sepharose beads (Amersham Biosciences) were then added and incubated for 1 h at 4 °C. After extensive washes with Staph A buffer (150 mM NaCl, 100 mM Na2HPO4, 18 mM NaH2PO4, pH 7.3, 20% Triton X-100, 1% SDS and 5% sodium deoxycholate), immunoprecipitated samples were boiled in SDS-loading buffer. The supernatant was subjected to SDS/12% PAGE and detected by Western blot using anti-HA monoclonal antibody and horseradish-peroxidase-conjugated Protein A by the West Pico Chemiluminescent system.
GST-fusion protein expression vector pGEX-MIG1 was transformed into Escherichia coli strain BL21(DE3) pLysS. Cells were grown to a D600 of 0.5–0.8, induced with 0.5 mM IPTG (isopropyl β-D-thiogalactopyranoside) at 37 °C for 3 h, and collected by centrifugation at 12000 g for 5 min. Cell pellets were resuspended in PBS (150 mM NaCl, 100 mM Na2HPO4 and 18 mM NaH2PO4, pH 7.3) and sonicated. Insoluble material was removed by centrifugation at 17000 rev./min for 20 min at 4 °C using a Beckman F2402H rotor and Allegra 21R centrifuge (Beckman). Soluble extracts were incubated with glutathione–Sepharose 4B (Amersham Biosciences) for 1 h at 4 °C, washed extensively with PBS and resuspended in the same buffer. The GST–Mig1 fusion protein coupled to glutathione–Sepharose was incubated with yeast whole-cell extracts from W303.1A (wild-type) and the W303.1Awca and W303.1Awrf mutant strains. The cell extracts were obtained from yeast cells grown in YEPG and shifted to high- and low-glucose media for 1 h. Beads were gently washed five times with 2.5 ml of PBS, boiled in 25 μl of sample-loading buffer (50 mM Tris/HCl, pH 6.8, 100 mM DTT, 2% SDS, 0.1% Bromophenol Blue and 10% glycerol), and analysed by SDS/12% PAGE followed by Western blotting using anti-Hxk2 antibodies and horseradish-peroxidase-conjugated Protein A. Bound antibodies were detected using the West Pico Chemiluminescent system.
ChIP assays were performed essentially as described previously . Cells were harvested and disrupted by vortex-mixing in the presence of glass beads, and the lysate was sonicated to generate DNA fragments that ranged in size from 200 to 400 bp. To immunoprecipitate HA-tagged proteins, we incubated the extract overnight at 4 °C with anti-Hxk2 antibodies . The sequence of primers used for the PCR amplification of the SUC2 promoter region containing the MIG1 element were 5′-TTATTACTCTGAACAGGA-3′ (sense) and 5′-AAGTCGTCAAATCTTTCT-3′ (antisense). The sequence of primers used for the PCR amplification of the ACT1 gene were 5′-GCCTTCTACGTTTCCATCCA-3′ (sense) and 5′-GGCCAAATCGATTCTCAAAA-3′ (antisense).
To identify the domains of the Hxk2 protein involved in catalytic activity and glucose-signalling regulation, we generated several Hxk2 mutants containing one or more substitutions, truncations or deletions in the amino acid chain (Figure 1A). The different mutations were used to test their effect on fructose and glucose growth. The growth properties of each mutant were tested by spotting serial dilutions of galactose-grown cells on media containing fructose or glucose as the carbon source. As can be seen in Figure 1(A), all of the strains grew normally on both media except the double-mutant Δhxk1Δhxk2 strain which had growth difficulties in fructose medium and the Δhxk1Δhxk2 strain transformed with the YEp352-Hxk2wca plasmid, which had clear growth problems in both fructose and glucose media.
The Hxk2wca protein content of the Δhxk1Δhxk2 strain transformed with the YEp352-Hxk2wca plasmid was evaluated by Western blot analysis after SDS/PAGE, to determine whether impairment of growth in fructose medium could be related to a lower quantity of the Hxk2wca protein. As can be seen in Figure 1(B), the Hxk2wca mutant protein was expressed in this strain and migrated at its predicted molecular mass. Since impairment of Hxk2 function was not due to decreased protein expression, the growth defect in fructose medium suggests a lack of hexokinase activity. Moreover, since this strain has a native GLK1 gene, which is not expressed in cells grown in glucose medium , but is fully expressed in cells grown in glucose medium in the absence of the Hxk2 protein , the absence of growth in glucose medium (Figure 1A) suggests that the Hxk2wca protein maintains normal glucose-repression signalling.
In order to analyse the regulatory activity of the different mutants, we have replaced the GLK1 promoter with the constitutive ADH1 promoter in the Δhxk1Δhxk2 strain, thus allowing the expression of Glk1 under high-glucose conditions in the presence of Hxk2. The strain obtained (Δhxk1Δhxk2 ADH1p::GLK1) grew in glucose medium at the same rate as the corresponding wild-type yeast. This strain was transformed with multicopy plasmids containing different HXK2 mutant genes under the control of the HXK2 promoter and we measured the exocellular invertase activity under high- and low-glucose conditions. The expression of the SUC2 gene for invertase is repressed in the presence of glucose in the growth medium, therefore we used the invertase level as a marker for glucose repression.
Figure 2 clearly shows that the wild-type phenotype (low invertase activity under high-glucose conditions) was restored after transformation of the double-mutant Δhxk1Δhxk2ADH1p::GLK1 strain with the wild-type (HXK2) or the mutant (HXK2wca) genes. When the double-mutant Δhxk1Δhxk2ADH1p::GLK1 strain was transformed with the HXK2wrf gene, the wild-type phenotype was not restored. However, this mutant that harbours a truncation between Lys6 and Met15 (Hxk2wrf)  exhibited normal growth both in fructose and glucose media (Figure 1A), which suggests a normal catalytic activity. These results revealed that the Hxk2wca protein exhibited a complete loss of catalytic function, but maintained a normal regulatory activity and the Hxk2wrf protein exhibited complete loss of regulatory function, while maintaining wild-type catalytic activity.
These consistent results prompted us to obtain, for the first time, stable strains containing the two HXK2 mutant alleles in the HXK2 locus.
To construct the loss-of-catalytic and regulatory function W303.1Awca and W303.1Awrf strains, we replaced the native HXK2 gene with exogenous HXK2wca and HXK2wrf mutant sequences in the wild-type W303.1A strain.
The glucose-phosphorylating activity of the Hxk2wca and Hxk2wrf mutant proteins was first assessed by measuring the ability of strains containing these proteins to grow on medium with glucose as a carbon source (Figure 3A). All of the strains grew normally on galactose and glucose media, except the W303.1Awca strain which had a clear growth problem in glucose medium. This growth problem in may occur because the Hxk2wca protein lacks catalytic activity and the HXK1 and GLK1 genes are not expressed under high-glucose conditions in the presence of a Hxk2 protein with normal regulatory function [2,30]. All of the strains grew well in galactose medium because, in the presence of galactose, the HXK2 gene has a basal expression similar to that observed for non-fermentable carbon sources such as ethanol or glycerol . Thus, under these conditions, the GAL genes are expressed and galactose is used for normal growth.
Although the studies described above measure the growth capacity on glucose medium of W303.1Awca and W303.1Awrf strains, they do not establish the Hkx2 catalytic activity of these strains. To address this issue, we constructed two new strains by integrating the HXK2wca and HXK2wrf genes into the HXK2 locus of a triple mutant strain (THG1; Δhxk1Δhxk2Δglk1). The glucose-phosphorylating activity was determined in crude extracts from strains THG1wca and THG1wrf in a Δhxk1Δhxk2Δglk1 background. As can be seen in Figure 3(B), the glucose-phosphorylating activity of the THG1wca strain was identical with that of the THG1 strain. Interestingly, the pattern of activity for the THG1wrf strain was similar to that of the W303.1A strain. Thus our results indicate that the W303.1Awrf strain maintained a normal (wild-type) Hxk2 catalytic activity, but the W303.1Awca strain had lost its catalytic function.
To investigate the Hxk2-dependent glucose signalling function of the W303.1Awca and W303.1Awrf strains (Figure 3C), the cells were grown in galactose medium to a D600 of 1.0 and then shifted to high- and low-glucose media for 1 h. Finally the exocellular invertase activity was determined in the whole cells. The specific activity of invertase shows strong derepression on both high- and low-glucose media in the presence of the Hxk2wrf mutant protein (W303.1Awrf strain). On high- and low-glucose media, in the presence of the Hxk2wca protein (W303.1Awca strain), wild-type repression and derepression signalling was observed.
These results indicate that we had constructed two new strains of S. cerevisiae with loss-of-catalytic (W303.1Awca) and regulatory (W303.1Awrf) functions.
Current evidence suggests that the main role of S. cerevisiae Hxk2 in the glucose signalling pathway is achieved through its interaction with both Mig1 and Snf1 proteins. It has been proposed that Hxk2 inhibits the phosphorylation of the Mig1 repressor when the cells are growing under high-glucose conditions, maintaining the transcriptional repression of target genes. To investigate the mechanism by which the Hxk2wca mutant protein signals glucose repression in the W303.1Awca strain and the loss-of-repressing function in the W303.1Awrf strain, we performed two-hybrid, co-immunoprecipitation and GST pull-down experiments.
First, we monitored the possible physical interaction between Mig1 and the Hxk2 mutant proteins Hxk2wca and Hxk2wrf using a yeast two-hybrid assay. Plasmid pGBKT7-Mig1 (expressing a GBD fusion to Mig1) was co-transformed with a plasmid expressing a fusion of the GAD with Hxk2, Hxk2wca or Hxk2wrf into an appropriate reporter strain. The interaction between the selected proteins was monitored by measurement of the β-galactosidase activity under high-glucose conditions. As shown in Figure 4(A), the GAD–Hxk2 and GAD–Hxk2wca fusion proteins produced a strong interaction with GBD–Mig1. However, this interaction was absent from a GAD–Hxk2wrf mutant, indicating that the decapeptide between Lys6 and Met15 of Hxk2 is essential for Hxk2 interaction with Mig1, as observed previously . Similar levels of all protein fusions were detected in all cases (Figure 4B). To confirm the interaction, we performed co-immunoprecipitation and GST pull-down assays. First, we examined the ability of Hxk2wca and Hxk2wrf to interact with Mig1 by using an immunoprecipitation assay. Cell extracts from a strain with a modified MIG1 gene which codes for a C-terminal Mig1 protein tagged with 3HA epitopes, were immunoprecipitated with the antibodies indicated in Figure 5(A). The resulting immunoprecipitates were assayed for the presence of Mig1–HA by immunoblot analysis with anti-HA antibodies. As shown in Figure 5(A) (lanes 1 and 3), a strong and specific signal of Mig1 was observed both with samples immunoprecipitated from extracts of wild-type or Hxk2wca strains grown under high-glucose conditions. However, signals were not observed when the experiment was performed using extracts of wild-type or Hxk2wca strains grown under low-glucose conditions, or when the experiment was performed using the strain expressing the Hxk2wrf protein. The lack of interaction cannot be attributed to lack of expression, as the Hxk2wrf protein was detected by Western blotting using an anti-Hxk2 antibody. When an anti-Pho4 antibody or no antibody was used to detect non-specific immunoprecipitation and non-specific protein binding to the anti-HA antibody respectively, no signals were observed. Thus this interaction is dependent on the production of HA-tagged Mig1 as well as an anti-Hxk2 antibody.
In order to determine the phosphorylation status of Mig1 in the Hxk2wca and Hxk2wrf mutant cells, we examined Mig1 phosphorylation in vivo in response to high and low levels of glucose (Figure 5B). For this purpose, we prepared extracts from wild-type, Hxk2wca and Hxk2wrf mutant strains transformed with the Mig1–HA construct. The transformed yeast cells were grown in the presence of high or low glucose. The mobility of Mig1 on SDS/PAGE was examined by Western blotting using an anti-HA antibody. The mobility of Mig1 varies depending on its phosphorylation state . As shown in Figure 5(B), in the presence of either Hxk2 or Hxk2wca proteins, Mig1 from low-glucose grown cells displays a slight shift in molecular mass, resulting in decreased mobility, in comparison with Mig1 from high-glucose-grown cells. However, in the presence of Hxk2wrf, Mig1 from both high- and low-glucose-grown cells had decreased mobility (Figure 5B, lanes 5 and 6) indicating that the protein is phosphorylated. This result suggests that the Snf1 protein kinase complex assumes an active conformation even in the presence of glucose, a similar result to that obtained in the absence of Hxk2 [27,32].
To confirm the interaction of Mig1 with Hxk2wca, we also employed the GST pull-down technique. We used extracts from the Hxk2wca- and Hxk2wrf-producing strains and a bacterially produced GST–Mig1 fusion protein. As shown in Figure 5(C) (lanes 1 and 2), a strong and specific band was observed in extracts from wild-type and Hxk2wca strains grown under high-glucose conditions. No retention was detected with the extracts obtained from low-glucose-grown cells or with extracts from the Hxk2wrf strain grown under both high- and low-glucose conditions. Similar amounts of Hxk2, Hxk2wca and Hxk2wrf proteins were detected in these immunoprecipitates by immunoblot analysis with anti-Hxk2 antibody (Figure 5D).
Previous data indicate that Hxk2, Mig1 and Snf1 form a complex at the nuclear level and that Hxk2 functions as a bridge between Snf1 and Mig1, interacting with Snf1 to inhibit Mig1 phosphorylation at Ser311 under high-glucose conditions . Thus it is tempting to speculate that the loss-of-function mutants of Hxk2 could have differences in their capacity to interact with the Snf1 protein. To address these possibilities, we examined the ability of Hxk2wca and Hxk2wrf proteins to interact with Snf1 by using an immunoprecipitation assay. Cell extracts from a strain with a modified SNF1 gene which codes for a C-terminal Snf1 protein tagged with 3HA epitopes were immunoprecipitated with the antibodies indicated in Figure 6. The resulting immunoprecipitates were assayed for the presence of Snf1–HA by immunoblot analysis with anti-HA antibodies. As shown in Figure 6 (lanes 3 and 4, and 5 and 6), a strong signal of Snf1–HA protein was observed both with samples immunoprecipitated from high- and low-glucose-grown cultures. When an anti-Pho4 antibody or no antibody was used to detect non-specific immunoprecipitation and non-specific protein binding to anti-HA antibody respectively, no signals were observed. Similar amounts of Hxk2, Hxk2wca and Hxk2wrf proteins were detected in these immunoprecipitates by immunoblot analysis with anti-Hxk2 antibody. Thus the interactions between Snf1 and both Hxk2wca and Hxk2wrf are dependent on the production of HA-tagged Snf1 as well as anti-Hxk2 antibody.
These results provide strong evidence that the Hxk2wca mutant protein signals glucose repression in yeast by interacting with the Mig1 repressor. Moreover, the Hxk2wca mutant protein probably plays a role identical with that of Hxk2 acting as a bridge between Snf1 and Mig1, to inhibit Mig1 phosphorylation by Snf1 at Ser311 under high-glucose conditions.
Furthermore, since the interaction between Mig1 and Hxk2wca was detected only under high-glucose conditions, our data suggest that both proteins interact in a glucose-dependent manner. Our results also suggest that Hxk2wrf is incapable of glucose-repression signalling because it does not interact with Mig1 either in vivo or in vitro. This result confirms that the ten-amino-acid motif between Lys6 and Met15, identified previously as essential for Hxk2 translocation to the nucleus , is required for the Hxk2–Mig1 interaction.
Previous reports have demonstrated that Hxk2 participates in the SUC2–Mig1 repressor complex through an interaction with the Mig1 protein and not by direct binding to DNA [6–9]. As Hxk2wca and Mig1 may form an in vivo complex (see above), we investigated the biological significance of this interaction by determining whether Hxk2wca localized in the SUC2–Mig1 repressor complex in a glucose-dependent manner. To test this hypothesis, we used ChIP assays. Our results show that, in cells grown in high-glucose medium, both Hxk2 and Hxk2wca proteins were recruited to a DNA fragment of the SUC2 promoter containing the Mig1-binding site (Figure 7A, lanes 1 and 3). Conversely, in low-glucose medium, Hxk2 and Hxk2wca binding to the SUC2–Mig1 complex was abolished (Figure 7A, lanes 2 and 4).
Interestingly, binding of the Hxk2 protein to the Mig1 site of the SUC2 promoter was dependent on the presence of the decapeptide between Lys6 and Met15, since in W303.1Awrf cells, no binding of Hxk2wrf (lacks interaction with Mig1) to the SUC2 promoter was observed under any growth condition (Figure 7A, lanes 5 and 6). The lack of DNA amplification cannot be attributed to lack of expression as the Hxk2wrf protein because it was detected by Western blotting using an anti-Hxk2 antibody (Figure 7B). No DNA amplification was observed when we used cells without Hxk2 or oligonucleotides to amplify the ACT1 promoter by PCR.
Taking all of these results together, we suggest that Hxk2wca forms a complex with Mig1 and Snf1 similar to that of Hxk2, and that this complex is involved in the glucose-regulated expression of the SUC2 gene. Thus Hxk2wca maintains its regulatory function because it maintains the glucose signalling wild-type machinery, whereas Hxk2wrf mutant protein lacks the regulatory function, because it is unable to form the repressor complex with Mig1 protein.
The role of the Hxk2 protein in glucose repression has been a matter of controversy for many years. Our data challenge the view that its role in glucose-repression signalling is directly related to its catalytic activity. This view was based on the inverse correlation between Hxk2 and invertase activity in a collection of random Hxk2 mutants [1,12]. Although previous results have shown Hxk2 mutants with low catalytic activity that are still functional in glucose signalling , a complete separation of the two activities never has been achieved. We have used the accumulated knowledge about the structure of the catalytic and regulatory domains of the Hxk2 protein to design a set of new mutations. We probed the role of these Hxk2 mutations in the following Hxk2 functions: catalytic activity, regulatory activity and interactions with proteins essential for these functions. By screening for suppression of the catalytic activity while maintaining the regulatory function and suppression of the regulatory function and maintaining the catalytic activity in the Hxk2 protein, we have isolated novel alleles of the HXK2 gene that have been long sought after. The results confirm what has been suspected for some time: that Hxk2 mediates catalytic and regulatory activities through different domains in the protein. The two separation-of-function mutants isolated, Hxk2wca and Hxk2wrf, are remarkable since they convert the Hxk2 from a bifunctional protein into a single-function protein with activity as a mediating factor in transcription (Hxk2wca) or as an enzyme with hexose-phosphorylating activity (Hxk2wrf).
The Hxk2wca mutant protein encodes an Hxk2 protein without the last eight C-terminal amino acids and a replacement of Ser304, which is located in the active centre of the enzyme, with phenylalanine. The Hxk2wrf mutant is an Hxk2 protein with an internal deletion between amino acids Lys6 and Met15. Since there is no overlap between the two domains controlling the Hxk2 catalytic and regulatory functions respectively, in the present study, we demonstrated that a correlation between catalytic activity and regulatory function in this protein does not exist.
In the glucose-repression signalling pathway, two major mediators are the proteins Mig1 and Hxk2. Under high-glucose conditions, nuclear Hxk2 interacts both with Mig1 and the Snf1 subunit of the Snf1 protein kinase complex to establish the repression state of several genes. Hxk2 interacts strongly with Mig1 in high-glucose-grown cells and previous findings implicate Ser311 of Mig1 as a critical residue for this interaction . The interaction between Snf1 and Hxk2 was detected both under high- and low-glucose conditions and the data suggest that both proteins interact constitutively .
In low-glucose-grown cells, the Hxk2 interaction with Mig1 is abolished and a transient increase in interaction between Snf1 and Mig1 was detected . This interaction pattern potentially stimulates Mig1 phosphorylation by Snf1 kinase at Ser311. Phosphorylation of Ser311 results in most Mig1 being exported from the nucleus to the cytoplasm, and, ultimately, the derepression of genes regulated by the Hxk2–Mig1 glucose-repression signalling pathway.
To analyse the mechanism by which the loss-of-function mutants operate through the regulatory machinery, we have determined protein–protein interactions with Mig1 and Snf1 proteins, and protein–DNA interactions with the Mig1-binding site of the SUC2 promoter. Since the Hxk2–Mig1 complex plays an essential role in glucose-repression signalling of several genes [7,9], detailed knowledge of the interaction between Hxk2wca and Hxk2wrf mutant proteins with Mig1 is required to understand how these two mutants operate in the glucose-repression mechanism. One of the important observations described in the present study is that two-hybrid assays, co-immunoprecipitation and GST pull-down experiments with purified Mig1 protein demonstrated that Hxk2wrf does not interact with Mig1 under both high- and low-glucose conditions. The lack of interaction between Hxk2wrf and Mig1 implies a loss of regulatory function. However, two-hybrid assays, co-immunoprecipitation and GST pull-down experiments with purified Mig1 protein demonstrated that Hxk2wca interacted with Mig1 under high- but not low-glucose conditions, a similar behaviour to the wild-type Hxk2.
Although the interaction between Snf1 and Hxk2wrf or Hxk2wca was detected both under high- and low-glucose conditions and our data suggest that these proteins interact constitutively with Snf1, a loss of regulatory function was detected in the Hxk2wrf mutant protein. Lack of the regulatory function of the Hxk2wrf protein could be explained because the Hxk2 protein operates as a bridge between Snf1 and Mig1 and is required to inhibit Mig1 phosphorylation at Ser311 by Snf1 kinase under high-glucose conditions . Since Hxk2wrf has lost its interaction capacity with Mig1 under high-glucose conditions, the Mig1 protein could be phosphorylated by Snf1, and both Mig1 and Hxk2 are exported from the nucleus to the cytoplasm by the Msn5  and Xpo1 (Crm1) exporters  respectively. Thus, under high-glucose conditions, several genes controlled by the Mig1 repressor complex are expressed because the Hxk2wrf protein has lost its regulatory function.
This hypothesis is supported by the following facts: (i) although it has been reported that Snf1 is inactive under high-glucose conditions , it has also been described that the non-phosphorylated form of Snf1 may have a direct role in controlling the expression of the TRK1 and TRK2 genes of the Trk high-affinity potassium-uptake system ; (ii) in the absence of Hxk2, the Snf1 protein kinase complex assumes an active conformation even in the presence of glucose [9,33], because the Mig1 protein from both high- and low-glucose-grown cells has decreased electrophoretic mobility, indicating that the protein is phosphorylated ; (iii) in the absence of Snf1 or both Hxk2 and Snf1, Mig1 has an increased mobility pattern, indicating that the protein is dephosphorylated [9,33,37]; (iv) in Hxk2wrf mutants, lacking a domain required for Mig1–Hxk2 interaction, Mig1 is phosphorylated under both high- and low-glucose conditions (Figure 5A). Thus our data strongly suggest that the Hxk2 interaction with Mig1 under high-glucose conditions might prevent phosphorylation of Mig1 by Snf1.
In the present study, we have demonstrated that Hxk2wrf is unable to signal glucose repression in S. cerevisiae because it was not present in the Mig1 repressor complex of the SUC2 promoter. Furthermore, we have also demonstrated that Hxk2wca is able to signal glucose repression in S. cerevisiae because it is physically associated with the Mig1 protein repressor, and ChIP assays confirmed that both Hxk2 and Hxk2wca interacted with Mig1 in DNA fragments containing the MIG1 site of the SUC2 promoter. Therefore our results suggest that both Hxk2wca and Hxk2 use similar mechanisms to regulate glucose signalling in yeast.
Rafael Peláez performed experiments and analysed data. Pilar Herrero designed and performed experiments. Fernando Moreno designed the study, analysed data and wrote the paper.
We thank J.M. Gancedo for critical reading of the paper and fruitful discussions.
This work was supported by the Ministerio de Ciencia e Innovación (MICINN), Spain [grant number BFU2007-66063-C02-02]. R.P. was supported by a fellowship provided by the Fundación para el Fomento en Asturias de la Investigación Científica Aplicada y la Tecnología (Spain).