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Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in Saccharomyces cerevisiae: characterizing the effects of a phosphatase subunit on the yeast proteome

1999; Springer Nature; Volume: 18; Issue: 15 Linguagem: Inglês

10.1093/emboj/18.15.4157

1460-2075

Geoffrey R. Alms, Pascual Sanz, Marian Carlson, Timothy Haystead,

Metabolism, Diabetes, and Cancer

Article2 August 1999free access Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in Saccharomyces cerevisiae: characterizing the effects of a phosphatase subunit on the yeast proteome Geoffrey R. Alms Geoffrey R. Alms Department of Pharmacology, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA, 22908 USA Search for more papers by this author Pascual Sanz Pascual Sanz Instituto Biomedicina de Valencia (CSIC), Jaime Roig 11, 46010 Valencia, Spain Search for more papers by this author Marian Carlson Marian Carlson Department of Microbiology, Columbia University, New York, NY, USA Search for more papers by this author Timothy A.J. Haystead Corresponding Author Timothy A.J. Haystead Department of Pharmacology, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA, 22908 USA Search for more papers by this author Geoffrey R. Alms Geoffrey R. Alms Department of Pharmacology, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA, 22908 USA Search for more papers by this author Pascual Sanz Pascual Sanz Instituto Biomedicina de Valencia (CSIC), Jaime Roig 11, 46010 Valencia, Spain Search for more papers by this author Marian Carlson Marian Carlson Department of Microbiology, Columbia University, New York, NY, USA Search for more papers by this author Timothy A.J. Haystead Corresponding Author Timothy A.J. Haystead Department of Pharmacology, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA, 22908 USA Search for more papers by this author Author Information Geoffrey R. Alms1, Pascual Sanz2, Marian Carlson3 and Timothy A.J. Haystead 1 1Department of Pharmacology, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA, 22908 USA 2Instituto Biomedicina de Valencia (CSIC), Jaime Roig 11, 46010 Valencia, Spain 3Department of Microbiology, Columbia University, New York, NY, USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:4157-4168https://doi.org/10.1093/emboj/18.15.4157 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Protein phosphatase 1 (Glc7p) and its binding protein Reg1p are essential for the regulation of glucose repression pathways in Saccharomyces cerevisiae. In order to identify physiological substrates for the Glc7p–Reg1p complex, we examined the effects of deletion of the REG1 gene on the yeast phosphoproteome. Analysis by two-dimensional phosphoprotein mapping identified two distinct proteins that were greatly increased in phosphate content in reg1Δ mutants. Mixed peptide sequencing identified these proteins as hexokinase II (Hxk2p) and the E1α subunit of pyruvate dehydrogenase. Consistent with increased phosphorylation of Hxk2p in response to REG1 deletion, fractionation of yeast extracts by anion-exchange chromatography identified Hxk2p phosphatase activity in wild-type strains that was selectively lost in the reg1Δ mutant. The phosphorylation state of Hxk2p and Hxk2p phosphatase activity was restored to wild-type levels in the reg1Δ mutant by expression of a LexA–Reg1p fusion protein. In contrast, expression of LexA–Reg1p containing mutations at phenylalanine in the putative PP-1C-binding site motif (K/R)(X)(I/V)XF was unable to rescue Hxk2p dephosphorylation in intact yeast or restore Hxk2p phosphatase activity. These results demonstrate that Reg1p targets PP-1C to dephosphorylate Hxk2p in vivo and that the motif (K/R)(X) (I/V)XF is necessary for its PP-1 targeting function. Introduction The catalytic subunit of protein phosphatase 1 (PP-1C) is amongst the most conserved proteins in nature (Cohen and Cohen, 1989). The enzyme is ubiquitously expressed at high levels in all cell types with an intracellular protein concentration of ∼10 μM. In vitro, the free catalytic subunit displays indiscriminate substrate specificity that overlaps with other ubiquitously expressed family members, including PP-2A and PP-2B (Cohen and Cohen, 1989; Shenolickar and Nairn, 1991). However, in vivo, PP-1C is associated with a number of regulatory proteins that either inhibit its activity or specifically target the phosphatase to a selective set of substrates or intracellular location (Hubbard and Cohen, 1993; Campos et al., 1996; Egloff et al., 1997; Damer et al., 1998). To date, at least 20 PP-1 regulatory subunits have been identified in mammals and yeast; however, only three of these proteins, M110 (MYTP1), PTG (PPP1R5) and PP-1GM, have been demonstrated to act in a targeting capacity both in intact cells and in vitro. The M110 subunit binds PP-1C in vitro and increases its smooth muscle myosin phosphatase activity by a factor of 20 whilst suppressing non-specific activity towards other substrates (Alessi et al., 1994; Shirazi et al., 1994; Simuzu et al., 1994; Haystead et al., 1995). Addition of both native and recombinant M110 to β-escin-permeabilized smooth muscles enhances the ability of PP-1C to bring about relaxation following Ca2+-induced contraction (Alessi et al., 1994; Shirazi et al., 1994; Haystead et al., 1995; Gailly et al., 1996; Johnson et al., 1996). In vitro, PTG binds and targets PP-1C to glycogen, glycogen synthase and phosphorylase a (Doherty et al., 1996; Printen et al., 1997). Overexpression of PTG in hepatocytes results in hyperglycogen accumulation with a concomitant 4-fold activation of glycogen synthase and 40% inhibition of phosphorylase activity (Berman et al., 1998). In skeletal muscle, the GM subunit has been proposed to target PP-1C to regulate glycogen synthase dephosphorylation in response to adrenaline and insulin (for a review see Hubbard and Cohen, 1993). Phosphorylation of GM by protein kinase A (PKA) causes dissociation of PP-1C and thereby prevents activation of glycogen synthase. Conversely, phosphorylation of GM at a second distinct site by the insulin-stimulated protein kinase p90rsk (MAPKAP1) enhances association of the subunit with PP-1C to promote dephosphorylation and activation of glycogen synthase (Dent et al., 1988). The physiologically relevant targets of the other PP-1 regulatory subunits have yet to be defined. Indeed, because PP-1C has been implicated in so many cellular events, identifying the substrates for any of its putative targeting subunits is a seemingly intractable problem. In Saccharomyces cerevisiae, PP-1C is encoded by the essential gene GLC7 (DIS2S1) and genetic analysis has shown that the phosphatase participates in the regulation of many processes, including glucose repression pathways, glycogen accumulation, sporulation, cell cycle progression and protein translation (for a review see Stark, 1996). Like its mammalian counterpart, Glc7p is regulated by interaction with many distinct regulatory targeting subunits. In S.cerevisiae, genetic studies have implicated the Glc7p-binding protein Reg1p in the regulation of glucose repression pathways (Tu and Carlson, 1995; Huang et al., 1996). Like other yeast, S.cerevisiae thrive on a variety of carbon sources, but glucose and fructose are preferred. When one of these sugars is present, the enzymes required for gluconeogenic pathways and for the utilization of alternative carbon sources are synthesized at low rates or not at all (for a review see Gancedo, 1998). The precise mechanism by which glucose signals to the nucleus to repress genes controlling expression of enzymes involved in gluconeogenesis and alternative carbon source utilization is not known (Gancedo, 1998). Glucose itself or glucose 6-phosphate (G6P) may be important mediators. Genetic and biochemical evidence suggests that Reg1p physically associates and functions with Glc7p in intact cells to regulate the glucose repression pathway. In two-hybrid analysis, Reg1p binds Glc7p and both proteins can be co-immunoprecipitated from yeast extracts (Tu and Carlson, 1995). Furthermore, deletion of REG1 in a wild-type background results in inhibition of glucose repression, accumulation of intracellular glycogen, slow growth and increased cell size (Huang et al., 1996). Reg1p also appears to have selective functions in the regulation of glucose repression by specifically antagonizing the function of the Snf1 kinase, which is a negative regulator of the glucose repression pathways (Ludin et al., 1998). Reg1p shows a glucose-dependent association with the Snf1 kinase and may provide a means for dephosphorylation and inactivation of the enzyme by Glc7p. Here, we hypothesized that comparison of the wild-type yeast phosphoproteome with reg1Δ mutants and strains containing Reg1p with point mutations in its putative PP-1C-binding site would identify physiologically relevant substrates of the Reg1p–Glc7p complex. This is because substrate targets would become selectively hyperphosphorylated as a result of an imbalance of the normal kinase–phosphatase equilibrium that governs the steady-state phosphorylation of the protein(s) under basal conditions. To test this hypothesis, two-dimensional SDS–PAGE phosphoprotein maps were prepared from 32P-labeled wild-type and mutant strains grown in the presence and absence of glucose. A major increase in phosphate content was observed in the glycolytic enzyme hexokinase II (Hxk2p) and the E1α subunit of pyruvate dehydrogenase in wild-type strains grown in the absence of glucose, reg1Δ strains, and strains expressing LexA–Reg1p with point mutations in the putative PP-1C-binding site. Subsequent fractionation and assay with 32P-labeled Hxk2p identified hexokinase phosphatase activity in the wild-type strains that was absent in both reg1Δ and LexA–Reg1p point mutant strains. The effects of the mutations on the ability of Reg1p to repress glucose-induced expression of the SUC2 gene was also tested. Wild-type LexA–Reg1p was shown to complement the reg1Δ mutation effectively, reducing SUC2 expression 130-fold in glucose-grown cells, whereas the mutant forms of LexA–Reg1p reduced SUC2 expression only 3-fold. These results demonstrate that Reg1p targets PP-1C towards Hxk2p to promote its dephosphorylation in vivo and are consistent with previous work by Randez-Gil et al. (1998), suggesting an involvement of Reg1p–Glc7p in the dephosphorylation of Hxk2p. In addition, our findings show the utility of functional proteomic analysis for the identification of physiological substrates for protein phosphatases. Results The effects of REG1 deletion on the yeast phosphoproteome To examine the phenotypic effects of disruption of the REG1 gene on the yeast phosphoproteome, reg1Δ mutant (MCY3278) and wild-type (FY250) strains from the same genetic background were labeled to steady state with either [35S]methionine or [32P]orthophosphate and grown in the presence or absence of glucose. Following two-dimensional SDS–PAGE and autoradiography, the autoradiograms from each radiolabeled strain were made into digital images (Figure 1). To determine whether REG1 deletion affected the overall pattern of protein expression, autoradiograms from [35S]methionine-labeled yeast were compared using the BIORAD Melanie II 2D-analysis program. This analysis did not reveal any obvious differences in the expression of any one protein as a result of REG1 deletion (compare Figure 1A with B, and C with D). Analysis by Melanie of the autoradiograms prepared from 32P-labeled yeast identified ∼80 distinct phosphoproteins of varying abundance in each gel (Figure 1E–H). To identify phosphoproteins that were increased in phosphate content as a result of REG1 deletion, Melanie compared Figure 1E (wild type) with F (reg1Δ). Four groups of spots were identified to be increased in phosphate content in this analysis, labeled Hxk2p (a) and Hxk2p (b), E1, heat shock protein 60 (hsp60) and spot C (the nomenclature was derived following identification of the proteins by mixed peptide sequencing, see below). None of the other 80 or so phosphoproteins detected by Melanie in the two autoradiograms showed any significant differences in phosphate content, suggesting that spots Hxk2p (a), Hxk2p (b), hsp60, E1 and C may be target substrates for the Reg1p–Glc7p complex. To test the effects of glucose starvation on overall protein phosphorylation in the wild-type strain, Melanie compared Figure 1G with E. This analysis showed that three of the proteins that were increased in phosphate content following REG1 deletion (Figure 1F) were also hyperphosphorylated in the wild type as a result of glucose starvation (Figures 1G and 2A). Importantly, comparison of Figure 1H with F showed that the absence of glucose did not cause additional proteins to be phosphorylated in the reg1Δ strain (Figure 2A). Since REG1 deletion was found to mimic the effects of glucose starvation on the yeast phosphoproteome, this finding further supports the hypothesis that Reg1p plays a critical role in the regulation of glucose-sensing pathways. Figure 1.The effects of REG1 deletion and glucose starvation on the yeast phosphoproteome. Yeast strains MCY3278 and FY250 were labeled to steady state with [35S]methionine (A–D) or [32P]orthophosphate (E–H) and whole-cell extracts characterized by two-dimensional SDS–PAGE and autoradiography. Note, gels for protein detection were run separately from those detecting changes in phosphorylation. Spots of interest were identified by mixed peptide sequencing following transfer of the gels to PVM (Table I). Molecular weight (kDa) and isoelectric points (pI) are theoretical values calculated by the Expasy compute pI/MW tool (http://expasy.hcuge.ch/ch2d/pi_tool.htm.) and are derived from the primary amino acid sequence of the identified proteins. Spot D in (F) is an exposure artifact and was not reproduced in four other separate experiments. Download figure Download PowerPoint Figure 2.The effects of REG1 deletion and glucose starvation on protein phosphorylation and expression in yeast. Data shown in (A) were derived after densometric analysis by the Melanie program of the autoradiograms shown in Figure 1. Data in (B) show the pmol amounts of each protein that was subjected to mixed peptide sequencing. Open bars represent data from the wild-type strain. Filled bars represent data from the reg1Δ strain. Data shown are mean values (±SDM) from five separate experiments. Download figure Download PowerPoint To determine if deletion of the REG1 gene or the presence of raffinose result in significant alterations of intracellular ATP concentration, the nucleotide content of the wild-type and reg1Δ strain was measured. This measurement would indicate whether the observed alterations in phosphorylation state in the reg1Δ strain, or following glucose starvation in the wild-type case, were likely to be due to differences in either the cellular ATP pool or the specific activity of the γ-phosphate of the nucleotide. However, in three separate measurements by reverse-phase HPLC, the overall amounts of GTP, ATP, ADP and AMP recovered from both strains did not vary significantly (data not shown). Moreover, when grown in dextrose or raffinose, the specific activity of the γ-phosphate of ATP from wild-type or reg1Δ strains varied by <11.5 ± 3.5% (SDM, n = 3) with respect to one another. Identification of phosphoproteins regulated by Reg1p and glucose by mixed peptide sequencing To identify 32P-labeled proteins that were phosphorylated in response to REG1 deletion and glucose starvation, the two-dimensional gels were transferred to polyvinyl membrane (PVM) stained with amido black and the membranes autoradiographed. Eight phosphoproteins were selected for mixed peptide sequencing. Five were increased in phosphate content in response to REG1 deletion, namely Hxk2p spots a and b, hsp60, E1α and spot C. Three others, hsp71, elongation factor 1 (EF1) and initiation factor 5A (IF5A), were unaffected by the absence of Reg1p and were used as internal markers in mixed peptide sequencing to ensure that each gel was matched for protein loading (Figure 2B). Of the eight proteins that were selected for mixed peptide sequencing, all but one was identified unambiguously in the yeast database by the FASTF algorithm. The identified proteins included hsp71, hsp60, EF1, IF5A, the E1α subunit of pyruvate dehydrogenase (E1) and Hxk2p a and b (Table I). Spot C was not identified because it was below our sequencing sensitivity (<100 fmol). Expectation scores for the identified proteins ranged from 5.4e-75 for Hxk2p spot b to 7.6e-8 for the E1α subunit. In contrast, expectation scores for the next highest scoring non-related proteins ranged from 0.001 to 1.2. The identification of Hxk2p in this present study is consistent with previous evidence that glucose-induced dephosphorylation of Hxk2p in vivo is blocked in Glc7p point mutants (Glc7pT152K) and reg1Δ mutants (Randez-Gil, 1998). Table 1. Identification of phosphoproteins by mixed peptide sequencing The sequence is listed in order of PTH amino acids that were recovered after each Edman cycle. The most abundant is listed first. FASTF was used to search and match the mixed sequences to the yeast NR database. The scoring matrix was MD20, with expectation and score values set to 95% of the total hexokinase phosphatase activity in S.cerevisiae is present in the soluble fraction. This activity required up to 100 nM okadaic acid to inhibit it completely, indicating that it is a type 1 serine/threonine phosphatase (data not shown). To characterize yeast hexokinase phosphatase activity further, cell extracts were prepared from MCY3278 and FY250 strains, fractionated by anion-exchange chromatography and fractions assayed for phosphatase activity using 32P-labeled Hxk2p and phosphorylase a as the substrates (Figure 4A and B). Figure 4A shows that fractions from wild-type yeast contained a major and a minor peak of Hxk2p phosphatase activity. Significantly, when fractions were assayed from the reg1Δ strain, the major Hxk2p phosphatase peak was absent. This result demonstrates that Reg1p targets PP-1C (Glc7p) to dephosphorylate Hxk2p. When column fractions from both strains were assayed with phosphorylase a as the substrate, no significant difference in phosphatase activity was observed (Figure 4B). When assayed in the presence of 1 nM okadaic acid, all phosphorylase phosphatase activity was abolished in the column fractions, whereas hexokinase phosphatase activity was not affected (data not shown). This suggests that the majority of the phosphorylase phosphatase activity detected in the column fractions was due to PP-2A. The finding that both the wild-type and mutant strains contained phosphorylase phosphatase activity that was not affected by REG1 deletion suggests that Reg1p is selective in its actions towards Hxk2p. This finding is consistent with the general hypothesis of PP-1 regulation in which its regulatory subunits target PP-1C towards a selective set of substrates (Hubbard and Cohen, 1993). To determine the effects of REG1 deletion on PP-1C expression in the yeast cytosol, anion-exchange fractions were Western blotted with anti-PP-1C antibody (Figure 4C). In the wild-type column fractions, cross-reactivity with PP-1C was observed in two major areas, fraction 4 (data not shown) and between fractions 26 and 40. In the reg1Δ fractions, cross-reactivity was only observed in fraction 4, with no evidence of PP-1C between fractions 26 and 40. The presence of PP-1C in fractions 26–40 in the wild-type case is therefore due to binding of the phosphatase to Reg1p. These findings further support the hypothesis that Reg1p targets PP-1C to dephosphorylate Hxk2p in vivo. The finding that column fractions from the reg1Δ strain contained very little PP-1C by Western blot analysis is consistent with observations by others (Hubbard and Cohen, 1993) and our laboratory (Campos et al., 1996; Damer et al., 1998) that the majority of cellular PP-1 is localized in the particulate fraction. Our data suggest that in yeast, the Reg1p–Glc7p complex represents the major pool of PP-1 that is present in the cytosol. Figure 3.Preparation of yeast hexokinase II as a phosphoprotein substrate. (A) The result of a time course study in which 0.25 mg of recombinant Hxk2p was phosphorylated for 30 min with the indicated amounts of PKA. In (B), 0.25 mg of Hxk2p was phosphorylated with 400 ng of PKA for 30 min and then digested with CNBr. The digest was applied to a reverse-phase C18 column and developed with a linear gradient (1%/min) of acetonitrile in 0.1% TFA. Fractions were collected (1.0 ml) and the amount of radioactivity (c.p.m.) determined by Cerenkov counting. Fraction 27, which contained 90% of the total radioactivity, was sequenced in a 494 Applied Biosystems automated sequencer. To identify the precise amino acid that was phosphorylated in the protein, in insert (C), 100 pmol of Hxk2p was digested with endolyslpeptidase C and the major phosphopeptide purified by reverse-phase HPLC. Fifty picomoles of the purified peptide was sequenced, the remaining half was coupled to a solid support for identification of the 32P-labeled amino acid by collecting radioactivity after each Edman cycle (Fadden et al., 1998). Download figure Download PowerPoint Figure 4.Assay of hexokinase phosphatase activity in yeast extracts. Cell extracts were prepared from wild-type (●) and reg1Δ (○) yeast and fractionated by anion-exchange chromatography. Column fractions were assayed for Hxk2p (A) and phosphorylase (B) phosphatase activity. In (C), column fractions from wild-type and reg1Δ yeast were Western blotted with a rabbit anti-PP-1C antibody. Download figure Download PowerPoint The effects of disruption of the Reg1p PP-1C-binding site on the yeast phosphoproteome Recently, Barford and colleagues identified the motif (K/R)(V/I)XF, or (K/R)X(I/V)XF, in Reg1p and some other yeast PP-1 subunits, as a putative PP-1C-binding site that is present in all known mammalian and yeast PP-1 regulatory subunits (Egloff et al., 1997). To probe the functional significance of the (K/R)X(I/V)XF motif in Reg1p, point mutations were introduced into a LexA–Reg1p fusion protein at the phenylalanine residue (F468 in the Reg1p sequence). Two substitutions were made, F468D and F468R, to introduce a positive or negative charge into the binding site in place of the hydrophobic phenylalanine residue (note F468 is absolutely conserved in the primary sequences of all 20 of the mammalian and yeast PP-1 regulatory subunits reported to date). Plasmids containing LexA–Reg1p, LexA–Reg1pF468R and LexA–Reg1pF468D fusions were transformed into the reg1Δ strain. The transformed strains were incubated with 32P or [35S]methionine and extracts prepared for two-dimensional SDS–PAGE and autoradiography (Figure 5). Western analysis of LexA fusion protein immunoprecipitates prepared from each strain with LexA antibody confirmed that the fusion protein was expressed in all three cases (Figure 6A). Figure 5A–C shows that introduction of the LexA–Reg1p fusion proteins into the yeast proteome does not result in dramatic changes in the overall pattern of [35S]methionine-labeled proteins when the gels were compared with one another. Significantly, however, Figure 5D shows that introduction of LexA–Reg1p into the reg1Δ strain completely rescues the normal pattern of phosphoprotein labeling that was observed in the FY250 strain earlier. In particular, the phosphate content of both Hxkp2 spots was restored to basal levels observed in the wild-type state. In contrast, introduction of aspartic acid and arginine residues at F468 prevented reversion of the overall two-dimensional phosphoprotein pattern back to the wild-type phenotype (compare Figure 5E and F with D). In the case of the F468D mutation, the phosphorylation state of Hxk2p (a), Hxk2p (b) and E1α were increased 5.4 ± 1.2-, 6.5 ± 2.2- and 3.2 ± 0.52-fold (SDM, n = 2), respectively, compared with strains transformed with wild-type LexA–Reg1p. Similar effects were also observed with the F468R mutants. None of the other 80 or so phosphoproteins detected in Figure 5D–F were altered significantly in phosphorylation state as a result of the transformations. Figure 5.The effects of disruption of the Reg1p PP-1C-binding motif on the yeast phosphoproteome. MCY3278 (REG1 deleted) was transfected with plasmids expressing LexA–Reg1p, LexA–Reg1pF468D and LexA–Reg1pF468R fusion proteins. The transfected yeast were labeled to steady state with [35S]methionine (A–C) or [32P]orthophosphate (D–F) and whole-cell extracts characterized by two-dimensional SDS–PAGE and autoradiography. The indicated proteins were identified by mixed peptide sequencing following transfer of the gels to PVM. Download figure Download PowerPoint Figure 6.The effects of point mutations at F468 on the association of PP-1C with Reg1p. Cell extracts were prepared from MCY3278 strains transfected with plasmids expressing LexA–Reg1p (●), LexA–Reg1pF468D (▵) and LexA–Reg1pF468R (○) fusion proteins. In (A), extracts from each strain were immunoprecipitated with the LexA antibody then blotted for the expression of the LexA fusion protein. In (B), extracts from each strain were fractionated by anion-exchange chromatography and assayed for Hxk2p phosphatase activity. In (C), the LexA immunoprecipitates were blotted with an anti-rabbit PP-1C antibody. Download figure Download PowerPoint To test whether the two point mutations disrupted Hxk2p phosphatase activity of the Reg1p–Glc7p complex, cell extracts were fractionated by anion-exchange chromatography and assayed with 32P-labeled Hxk2p (Figure 6B). Figure 6B shows that transformation of LexA–Reg1p into the MCY3278 strain restores Hxk2p phosphatase activity. This finding further demonstrates that the LexA–Reg1p fusion protein is expressed as a fully functional Glc7p targeting subunit. However, when the column fractions from the F468D and F468R mutants were assayed with 32P-labeled Hxk2p, no evidence of phosphatase activity towards the substrate was detected. These observations are consistent with the increased phosphorylation of Hxk2p observed in these mutants, and suggest that mutation of F468 is sufficient to inhibit Reg1p function in vivo. To examine further whether introduction of aspartic acid or arginine at F468 disrupts Reg1p binding to Glc7p, the LexA–Reg1p fusion protein immunoprecipitates were blotted for PP-1C. Figure 6C shows that immunoprecipitates from LexA–Reg1p extracts contain Glc7p whereas those from the F468R and F468D mutants contained greatly reduced cross-reactivity at 37 kDa. Two-hybrid analysis further confirmed that the two point mutations also decreased interaction of Reg1p with Glc7