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Originally published In Press as doi:10.1074/jbc.M701272200 on June 1, 2007

J. Biol. Chem., Vol. 282, Issue 30, 21838-21847, July 27, 2007
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Expression of Human Protein Phosphatase-1 in Saccharomyces cerevisiae Highlights the Role of Phosphatase Isoforms in Regulating Eukaryotic Functions*Formula

Jennifer A. Gibbons{ddagger}1, Lukasz Kozubowski{ddagger}, Kelly Tatchell§, and Shirish Shenolikar{ddagger}2

From the {ddagger}Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and the §Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130

Received for publication, February 12, 2007 , and in revised form, May 31, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human (PP1) isoforms, PP1{alpha}, PP1beta, PP1{gamma}1, and PP1{gamma}2, differ in primary sequences at N and C termini that potentially bind cellular regulators and define their physiological functions. The GLC7 gene encodes the PP1 catalytic subunit with >80% sequence identity to human PP1 and is essential for viability of Saccharomyces cerevisiae. In yeast, Glc7p regulates glycogen and protein synthesis, actin cytoskeleton, gene expression, and cell division. We substituted human PP1 for Glc7p in yeast to investigate the ability of individual isoforms to catalyze Glc7p functions. S. cerevisiae expressing human PP1 isoforms were viable. PP1{alpha}-expressing yeast grew more rapidly than strains expressing other isoforms. On the other hand, PP1{alpha}-expressing yeast accumulated less glycogen than PP1beta-or PP1{gamma}1-expressing yeast. Yeast expressing human PP1 were indistinguishable from WT yeast in glucose derepression. However, unlike WT yeast, strains expressing human PP1 failed to sporulate. Analysis of chimeric PP1{alpha}/beta subunits highlighted a critical role for their unique N termini in defining PP1{alpha} and PP1beta functions in yeast. Biochemical studies established that the differing association of PP1 isoforms with the yeast glycogen-targeting subunit, Gac1p, accounted for their differences in glycogen synthesis. In contrast to human PP1 expressed in Escherichia coli, enzymes expressed in yeast displayed in vitro biochemical properties closely resembling PP1 from mammalian tissues. Thus, PP1 expression in yeast should facilitate future structure-function studies of this protein serine/threonine phosphatase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian protein phosphatase-1 (PP1)3 controls a wide range of physiological events, including learning and memory (1), glycogen metabolism (2), and protein translation (3). Three human PP1 genes encode four isoforms, termed {alpha}, beta (or {delta}), {gamma}1, and {gamma}2. In addition, more than 60 mammalian PP1 regulators have been identified (4). Thus, cellular functions of PP1 appear to be dictated by complexes containing a catalytic subunit and one or more regulators that define its subcellular localization and substrates. Several mammalian regulators show preferential recruitment of specific PP1 isoforms. Thus, neurabins, which target PP1 to the neuronal cytoskeleton, preferentially interact with PP1{gamma}1 (5, 6). GADD34 targets PP1{alpha} to the endoplasmic reticulum to regulate protein translation (7), and the glycogen-targeting subunit, GM, binds PP1beta to regulate glycogen metabolism in skeletal muscle (8). However, the structural determinants that promote selective binding of PP1 isoforms by regulators remain poorly understood.

Saccharomyces cerevisiae is unique in that it is the only known eukaryote with a single PP1 gene, GLC7, which is essential for viability (9). This enabled mutagenesis studies of GLC7 that yielded an array of active PP1 catalytic subunits that helped to define the mechanism of PP1 regulation by mammalian inhibitor-1 (10), inhibitor-2 (11), and NIPP-1 (12). Expression of human inhibitor-1 in yeast also provided insights into the structure-function relationship for this PP1 regulator (13) and identified novel Glc7p-regulated events in yeast (10, 14). These studies emphasized the conservation of PP1 structure and regulation in eukaryotes.

As in higher eukaryotes, Glc7p regulates multiple physiological events in yeast, including glycogen metabolism (9), sporulation (15), and transcriptional responses (16). Thus, analysis of GLC7 phenotypes provides a broad readout of PP1 functions. We utilized a gene replacement strategy to substitute Glc7p in S. cerevisiae with human PP1 isoforms. These studies established that human PP1 supported growth and viability of yeast in the absence of GLC7. Additional studies highlighted differences in the assembly of human PP1 isoforms into functional complexes containing yeast PP1 regulators, thereby accounting for their differing ability to control aspects of yeast physiology. These studies provided direct experimental evidence for distinct physiological roles of PP1 isoforms and highlighted structural elements at their N termini that may direct PP1 functions in eukaryotic cells.

Most structural and biochemical studies of human PP1 have focused on enzymes expressed in Escherichia coli (17). These Mn2+-requiring enzymes differ from native PP1 purified from mammalian tissues, which contains Zn2+ and Fe2+ in the active site, and show enhanced dephosphorylation of p-nitrophenyl phosphate (pNPP) (18) and impaired regulation by proteins such as inhibitor-1 (19) and neurabins (20). Although expression in insect cells (20, 21) and Pichia pastoris (18) yielded PP1 with improved biochemical characteristics, low yield (20) and insolubility (21) of these overexpressed proteins have severely limited the widespread use of these eukaryotic expression systems. Human PP1 expressed in S. cerevisiae demonstrated biochemical properties more closely resembling PP1 from mammalian tissues than the enzyme expressed in E. coli, and provided a novel expression system that should greatly facilitate future mechanistic studies of this protein serine/threonine phosphatase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—DNA was purified using QIAquick extraction, PCR purification kits, and QIAprep spin miniprep kits (Qiagen). Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs. Pfu Turbo DNA polymerase (Stratagene), Platinum Taq DNA polymerase, and dNTPs (Invitrogen) were obtained from indicated sources. [{gamma}-32P]ATP was from PerkinElmer Life Sciences. Glutathione-Sepharose was from GE Healthcare, and isopropyl beta-D-thiogalactopyranoside was from Gold Biotechnologies. Phosphorylase kinase was obtained from Sigma, and phosphorylase b was from Calzyme Laboratories, Inc. Microcystin-LR was from Alexis Biochemicals. Protein concentration was estimated using Bio-Rad protein assay with bovine serum albumin as standard.

Yeast Strains—Yeast strains (supplemental Table 1) were derived from the parent strain, KT1900, and were congenic with KT1112 (MATa ura3-52 leu2 his3) (22). Yeast were grown on YPD (2% tryptone, 1% yeast extract, and 2% glucose) medium (BD Biosciences) at 30 °C except when noted. Complete synthetic medium (CSM) and media lacking specific amino acids were prepared with yeast nitrogen base (BD Biosciences), 2% glucose, amino acid supplement mixtures (QBiogene), and copper sulfate added when needed. Yeast strains were sporulated at 24 °C on solid medium containing 1% potassium acetate, 0.1% yeast extract, and 0.05% dextrose. Strains transformed with plasmid pNC160 (JG1-15) lost GFP-Glc7p-expressing plasmid when grown on SC-Trp plates (which retained pNC160) and 1 mg/ml 5-fluoroorotic acid (5-FOA) (Toronto Research Chemicals) for counterselection.

PP1 genotypes were confirmed by colony PCR and expression of Glc7p and human PP1 confirmed by immunoblotting yeast lysates. Strains with GLC7 or human PP1 integrated at the HIS3 locus were created by transforming JG24 with integrating plasmid pRS303 (pJG24-29) cut with BstXI within the HIS3 locus. Transformants sporulated and tetrads were dissected to select JG16-23 strains. Diploid strains, JG25-28, were obtained by mating isogenic haploid mutants. Haploid strains, JG40-49, were generated by integrating the PP1-expressing plasmid into the HIS3 locus of KT1900 and selecting strains for loss of the GFP-Glc7p-expressing maintenance plasmid by growth on CSM containing 5-FOA. JG50 was created by integrating pRS303 plasmid into JG51, and pRS316 plasmid was eliminated by growth on CSM containing 5-FOA.

Expression Plasmids—Primers (supplemental Table 2) were utilized to generate mutant PP1 cDNAs that were inserted into expression plasmids (supplemental Table 3). The CUP1 promoter was used to express Glc7p and human PP1 catalytic subunits in yeast with the exception of plasmids derived from pJG24-29, which used the native GLC7 promoter and terminator (derived from p1855). Hemagglutinin (HA) tag was inserted into selected plasmids (pJG9, -11, -13, -15, and -17). Chimeras of PP1{alpha} and PP1beta used two-step PCR with PP1{alpha} N and C terminus with overlapping PP1beta catalytic core sequences (JAG143-146) and PP1{alpha} catalytic core with overlapping PP1beta N or C terminus (JAG143 and -148 or JAG147 and -146). All PCR products were purified, cut with EcoRI, and ligated into pNC160.

Fluorescence Microscopy—Indirect immunofluorescence of yeast was undertaken as described (23). Log phase yeast, fixed in 4% formaldehyde and permeabilized in solution B (100 mM K2PO4, pH 7.5, and 1.2 mM sorbitol), containing ~450 units of lyticase (Sigma) and 0.5% 2-mercaptoethanol, were stained with rabbit anti-HA antibody (Roche Applied Science) at 1:200 dilution and Cy3-conjugated goat anti-rabbit secondary antibody at 1:200 dilution (Jackson ImmunoResearch) and costained with 4,6-diamidino-2-phenylindole (Sigma) or rabbit anti-histone H2A antibody (1:200) to visualize nuclei. Zeiss Imager A1 Axioscope was used with the images captured on Hamamatsu ORCA ER digital camera interfaced with Meta-Morph software (Universal Imaging, Silver Spring, MD).

Yeast Phenotypes—Glycogen accumulation was estimated by patching yeast on CSM plates at 30 °C for 24 or 48 h. Plates were inverted over iodine crystals to stain glycogen and photographed. DH3 (deletion of glycogen synthase), EG328-1A (the parent strain), and WW10 (deletion of both PCL8 and PCL10) were used as reference for low, normal, and high levels of glycogen. Glucose derepression was performed as described (24). Plates were incubated at 30 °C under anaerobic conditions (chambers provided by Annette Golonka, Duke University). All yeast phenotypic studies were undertaken using at least six independent colonies.

Yeast Glycogen Content—Quantitative glycogen assays were performed as described (25) with the following changes. Culture growth was supplemented with 0.1 mM CuSO4 as indicated. Total glucose was analyzed using Amplex Red glucose assay kit (Molecular Probes).

PP1 Catalytic Subunits—Recombinant PP1 catalytic subunits were expressed in E. coli as described (26). Native PP1 catalytic subunits were purified from rabbit skeletal muscle (27). PP1 catalytic subunits were partially purified from yeast strains. Yeast were grown at 30 °C to A600 of 0.6–0.8, and PP1 expression was induced with 1 mM CuSO4 for 3–4 h prior to sedimenting yeast by centrifugation. The yeast pellet was resuspended in YeastBuster Protein Extraction Reagent and THP solution (Novagen), which included protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A), and lysed as described by the manufacturer. Lysed yeast were subjected to centrifugation at 30,000 x g for 30 min at 4 °C and purified via heparin-agarose essentially as described previously (26). Most studies utilized batch elution with 0.5 M NaCl following extensive washing with 0.1 M NaCl and yielded PP1 catalytic subunits with 50–55% purity. Elution using a linear gradient from 0.1 to 0.5 M NaCl (26) or affinity purification on Microcystin-Sepharose (supplemental Fig. 3) increased the enzyme purity to greater than 70–80%.


Figure 1
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  		FIGURE 1.
Structure of human and yeast PP1. A shows schematic structures of the four human PP1 isoforms and the budding yeast PP1 catalytic subunit Glc7p. The central catalytic core (filled bars) shares greater than 96% sequence identity among human enzymes and 90% identity with Glc7p. The variable N and C termini were aligned using ClustalW (52). The amino acid numbers correspond to primary sequence of human PP1{alpha}. B shows schematic of chimeras of human PP1{alpha} and PP1beta analyzed in this study. PP1{alpha}306 represents amino acids 1–306 of human PP1{alpha}.

 
Phosphorylase a Phosphatase—Enzyme assays were undertaken as described previously (26).

Hydrolysis of p-Nitrophenyl Phosphate—Dephosphorylation of pNPP required 10-fold more PP1 catalytic subunit than used in the above phosphorylase a phosphatase assays. PP1 catalytic subunits diluted into 20 mM 4-nitrophenyl phosphate (Serva Electrophoresis), 1 mg/ml bovine serum albumin, 20 mM Tris-HCl, pH 8, 5 mM MgCl2, and 0.1% (v/v) 2-mercaptoethanol were incubated at 37 °C for 10 min. Assays with PP1 catalytic subunits expressed in E. coli included 1 mM MnCl2. Assays were terminated with addition of an equal volume of 250 mM NaOH and analyzed at A410.

Recombinant PP1 Regulators—GST-Gac1p, GST-Gip2p, GST-Pig2p, and GST-neurabin (NrbI) (residues 374–516) fusion proteins were expressed in E. coli and purified as described previously (5).

PP1 Binding by Regulators—Sedimentation of human PP1 isoforms from yeast lysates using GST fusions of targeting subunits was undertaken as described (26). Bound PP1 was detected by immunoblotting with anti-HA monoclonal antibody (Covance, Inc., or Roche Diagnostics). Immunoblotting with rabbit anti-3-phosphoglycerate kinase (PGK) antibody (Molecular Probes) established protein loading. Immunoreactivity was quantified using anti-mouse AlexaFluor 680 (Molecular Probes) and anti-rabbit IR Dye 800 (Rockland) secondary antibodies, respectively, using Odyssey infrared imaging system (Li-COR Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Yeast Strains Expressing Human PP1—Primary structures of human and S. cerevisiae PP1 (Fig. 1A) highlight a highly conserved catalytic core (filled bars) flanked by diverse N and C termini (open bars). To compare functional properties of the human and yeast PP1 catalytic subunits, we transformed GLC7: LEU2 (KT1900 and KT1901) strains with plasmids expressing HA-PP1 catalytic subunits under the inducible CUP1 promoter and selecting on CSM containing 5-FOA to eject the GFP-Glc7p maintenance plasmid. This generated strains that expressed individual WT human PP1 isoforms, a C-terminally truncated PP1{alpha}306, and chimeric PP1{alpha}/beta catalytic subunits that substituted either N or C termini from PP1{alpha} into PP1beta and vice versa (schematically shown in Fig. 1B).

Immunoblotting yeast lysates with anti-HA antibody allowed for direct comparison of expression levels of all PP1 catalytic subunits (Fig. 2A). Quantitation using Odyssey showed that basal expression of HA-Glc7p was 4–5-fold higher than any human PP1 (Fig. 2B). Addition of 0.1 mM CuSO4 to media increased expression of all PP1s by 2–3-fold but not the control protein, PGK. Growth on media containing 0.3 mM CuSO4 resulted in a further 0.2–2-fold increase in human PP1, but the expression of HA-Glc7p was either unchanged on modestly reduced when compared with growth in 0.1 mM CuSO4. This suggested that there was an upper limit for PP1 expression beyond which viability may be compromised (28).

Prior studies that expressed GFP-Glc7p in yeast (29) showed localization at bud neck and spindle poles. By contrast, HA-Glc7p was highly concentrated at nuclei (supplemental Fig. 1). Although cytoplasmic distribution was also noted, no immunofluorescence for HA-Glc7p was seen at bud neck or spindle poles. This may reflect the differing experimental strategies with the overexpression of GFP-Glc7p in the earlier studies compared with gene replacement used in this study. Compared with Glc7p, fewer yeast showed nuclear staining for human PP1, with PP1{alpha} most consistently localized to nuclei and PP1{gamma}1 and PP1{gamma}2 being excluded from nuclei in most cells. Although nuclear staining for human PP1 was variable, all yeast showed immunostaining for histone H2A. This suggested that as in mammalian cells, PP1 localization in yeast may be highly dynamic (29). Differences in PP1 isoforms was also suggested by frequent nucleolar localization of PP1beta and PP1{alpha}, PP1beta and PP1{gamma}1 at mitotic spindles and PP1beta and PP1{gamma}2 at bud scars.

Growth of Human PP1-expressing Yeast—Analysis of growth of multiple independent colonies showed that yeast expressing HA-Glc7p grew essentially like control WT yeast (Fig. 3). By comparison, all yeast expressing human PP1 catalytic subunits grew more slowly. Among these, yeast expressing PP1{alpha} or PP1{alpha}306 grew most rapidly, and PP1beta-expressing yeast grew the slowest. Similar results were obtained on liquid or solid media and rich or synthetic media (data not shown). Yeast expressing chimeric PP1 subunits, specifically PP1{alpha}/betaC with C terminus from PP1beta fused to the remainder of PP1{alpha} and PP1beta/{alpha}N, containing the N terminus of PP1{alpha}, grew almost 2-fold faster than yeast expressing PP1beta, PP1beta/{alpha}C or PP1{alpha}/betaN (Fig. 3B). These data suggested that deleting the C terminus (PP1{alpha}306) or substituting the C terminus from PP1beta had little effect on the ability of PP1{alpha} to support yeast growth. Hence, PP1{alpha}-, PP1{alpha}306-, PP1beta/{alpha}N,- and PP1{alpha}/betaC-expressing yeast grew at essentially similar rates. By contrast, PP1{alpha}/betaN, containing the N terminus from PP1beta, grew more slowly and resembled yeast expressing either PP1beta or PP1beta/{alpha}C. These data highlighted a unique contribution of N-terminal sequences in PP1{alpha} and PP1beta in directing yeast growth.


Figure 2
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  		FIGURE 2.
Expression of human and yeast PP1 in S. cerevisiae. A, yeast strains with HA-tagged PP1 catalytic subunits were grown to log phase and lysed as described under "Experimental Procedures." Cell lysates were subjected to SDS-PAGE, and PP1 expression was analyzed by immunoblotting (IB) with anti-HA antibody. Immunoblotting for PGK provided a control for protein loading. B shows quantitation of HA-PP1 expression in yeast grown under basal conditions and in media with and without CuSO4, respectively, from several representative Western blots analyzed using LiCOR Odyssey software.

 


Figure 3
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  		FIGURE 3.
Growth of yeast expressing human PP1. Yeast were grown to stationary phase, diluted to A600 = 0.3 in CSM, and grown at 30 °C. A600 was monitored at selected time intervals. A shows growth curves for yeast expressing human PP1. Yeast expressing individual PP1 catalytic subunits are shown as follows: PP1{alpha} (filled squares), PP1beta (open squares), PP1{gamma}1(filled triangles), PP1{gamma}2(open triangles), and PP1{alpha}306 (filled diamonds). B shows the growth of yeast expressing the human PP1{alpha}/beta chimeras, PP1{alpha}/betaN(filled triangles), PP1{alpha}/betaC(filled diamonds), PP1beta/{alpha}N(open triangles), and PP1beta/{alpha}C(shaded triangles). The growth of yeast expressing HA-Glc7p (open diamonds) and untransformed yeast (filled squares with dashed lines) was analyzed in parallel.

 
Rank order of growth of yeast expressing Glc7p and human PP1 isoforms in media containing 0.1 mM CuSO4 was identical to that in media lacking CuSO4 (Fig. 3A). However, discernible reduction in growth of yeast containing HA-Glc7p was observed at 0.1 mM CuSO4 (~15%) and 0.3 mM CuSO4 (30%; data not shown). As growth of untransformed or WT yeast was unaffected by either 0.1 or 0.3 mM CuSO4, this hinted at a growth inhibitory effect of high Glc7p levels (9). Hence, growth on 0.3 mM CuSO4 no longer differentiated yeast expressing Glc7p from those with human PP1.

We also generated yeast in which HA-PP1 cDNAs (behind CUP1 promoter) were inserted into an identical HIS3 locus. HA-Glc7p levels in the integrated strain were 3–4-fold higher than those expressing human PP1, similar to that noted above for plasmid-borne strains (data not shown). This suggested either enhanced translation or stability of Glc7p rather than gene dosage accounting for its higher levels in yeast. Most importantly, yeast containing integrated HA-PP1, either in presence or absence of CuSO4, grew identically to strains containing PP1-expressing plasmids (data not shown).

When compared at differing temperatures, WT S. cerevisiae, like yeast expressing HA-Glc7p, grew effectively on solid media at 16 °C (Fig. 4A), 30 °C (Fig. 4B), and 37 °C (Fig. 4C). By comparison, yeast expressing human PP1, both WT and chimeras, grew slower with fewer colonies seen at higher dilutions at all temperatures. Yeast expressing PP1beta, PP1{alpha}/betaN, and PP1beta/{alpha}C were notably impaired in growth at 37 °C, which was readily visible at lower dilutions. This highlighted the unique temperature sensitivity of yeast expressing PP1beta and PP1{alpha}/betaN {alpha} and suggested a key contribution of the unique PP1beta N terminus to this phenotype.

Ion homeostasis in yeast is known to regulated by Glc7p (30, 31). In contrast to WT and HA-Glc7p-expressing yeast, yeast expressing human PP1 catalytic subunits either failed to grow or grew slowly in media containing 0.1 M CsCl (Fig. 5A) or 0.9 M NaCl (Fig. 5B). Interestingly, strains expressing the chimeric PP1beta/{alpha}N catalytic subunit showed significant growth on media containing 0.1 mM CsCl. Thus, the fusion of PP1beta N terminus to the remainder of PP1{alpha} generated a unique human PP1 catalytic subunit that showed some capacity to regulate yeast ion homeostasis.


Figure 4
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  		FIGURE 4.
Temperature sensitivity of yeast expressing human PP1. Yeast were grown to stationary phase and serially diluted to an A600 of 1.0, 0.1, 0.01, and 0.001. These dilutions were spotted on YPD plates and grown at stated temperatures. Representative plates from at least three independent experiments are shown. A, 16 °C for 5 days; B, 30 °C for 3 days; C, 37 °C for 3 days. Arrows point to yeast strains showing significant thermal sensitivity.

 
Glycogen Accumulation in Yeast Expressing Human PP1—Glc7p is essential for glycogen synthesis (22) and promotes the dephosphorylation and activation of yeast glycogen synthase. Thus, glycogen accumulation, visualized in yeast by iodine staining (Fig. 6A), showed that less glycogen accumulated in yeast expressing human PP1 than either control yeast or yeast expressing HA-Glc7p. Despite similar PP1 levels, yeast expressing human isoforms also demonstrated differing glycogen content. Specifically, PP1beta-, PP1{gamma}1-, and PP1{gamma}2-expressing yeast accumulated more glycogen than yeast containing either PP1{alpha} or PP1{alpha}306. PP1{alpha}306-expressing yeast consistently showed a small increase in glycogen content compared with PP1{alpha}-expressing yeast, suggesting a potential negative role for PP1{alpha} C terminus in generating an active glycogen synthase phosphatase.


Figure 5
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  		FIGURE 5.
Salt sensitivity of yeast strains expressing human PP1. Yeast strains were grown to stationary phase and serially diluted to an A600 of 1.0, 0.1, 0.01, and 0.001. These dilutions were spotted on YPD plates containing the indicated concentrations of salt and grown for 5 days at 30 °C. Data shown are representative of at least three independent experiments. A shows growth on YPD with 0.1 M CsCl. B shows growth on YPD with 0.9 M NaCl.

 
Yeast expressing a chimeric phosphatase, PP1{alpha}/betaN, showed increased glycogen content compared with PP1{alpha}-expressing yeast, demonstrating the ability of the PP1beta N terminus to enhance the capacity of PP1{alpha} to promote glycogen synthesis. Conversely, incorporation of the PP1{alpha} N terminus generated PP1beta/{alpha}N and reduced the activity of PP1beta in promoting yeast glycogen accumulation. By comparison, PP1beta/{alpha}C accumulated glycogen to similar levels as yeast expressing PP1beta. These data emphasized the positive role for the N terminus in the ability of PP1beta to regulate yeast glycogen. When yeast were grown on media containing CuSO4 to increase PP1 levels, the overall glycogen content in all strains was elevated, but this did not alter the overall conclusion, namely the N terminus accounted for the unique ability of PP1beta to promote yeast glycogen synthesis.


Figure 6
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  		FIGURE 6.
Glycogen accumulation in yeast expressing human PP1. A, yeast were patched on plates containing either CSM (left panel) or CSM with 0.1 mM CuSO4 (right panel) and grown for 48 h at 30 °C. Plates were inverted over iodine crystals (described under "Experimental Procedures") and photographed. A representative photograph from several independent experiments is shown. To compare glycogen levels, we utilized the following reference yeast strains: DH3 (low glycogen), EG328-1A (normal glycogen), and WW10 (high glycogen). B, yeast grown for 24 h in CSM (left panel) or CSM with 0.1 mM CuSO4 (right panel) were sedimented and lysed, and their glycogen content was analyzed as total glucose as described under "Experimental Procedures." The average ± S.E. of three independent experiments is shown.

 
A quantitative assay that analyzed total glucose following digestion of yeast glycogen with amyloglucosidase (Fig. 6B) confirmed all the major findings discussed above. Most importantly, yeast expressing human PP1, in the presence or absence of CuSO4, accumulated 6–7-fold less glycogen than WT untransformed yeast or yeast expressing HA-Glc7p, highlighting the remarkable effectiveness of Glc7p to promote yeast glycogen synthesis. In the absence of CuSO4, yeast expressing PP1{alpha} or PP1{alpha}306 contained 5–10-fold less glycogen than yeast expressing PP1beta, PP1{gamma}1, or PP1{gamma}2. With the exception that glycogen content in PP1{alpha} (and PP1{alpha}306)-expressing yeast was elevated in the presence of CuSO4 to levels comparable with yeast expressing PP1beta and PP1{gamma}2, increasing expression of other human PP1 isoforms had only modest effects on glycogen content. WT and HA-Glc7p-expressing yeast also contained similar glycogen levels in presence or absence of CuSO4. When grown in the presence of CuSO4, the glycogen content of yeast expressing PP1{alpha}/betaN, PP1beta/{alpha}N and PP1beta/{alpha}C was identical, whereas PP1{alpha}/betaC-expressing yeast accumulated 50% less glycogen. This also demonstrated that the unique deficiency of PP1{alpha} in promoting glycogen synthesis could be overcome by elevating PP1{alpha} (or PP1{alpha}306) expression in yeast.

Binding of Human PP1 to Yeast PP1 Regulators—Gac1p is the primary PP1 targeting subunit controlling yeast glycogen synthesis (32). We utilized purified recombinant GST-Gac1p in pulldowns from lysates of yeast that were matched for their content of human PP1. To our surprise, Gac1p bound human PP1beta, PP1{gamma}1, and PP1{gamma}2 with near equal efficiency to Glc7p, when normalized for input PP1 (Fig. 7, A and B). By contrast, GST-Gac1p sedimented 5-fold less PP1{alpha} (and PP1{alpha}306). With the exception of Glc7p, the relative binding of GST-Gac1p to human PP1 isoforms paralleled the glycogen accumulation seen in yeast expressing these proteins.

Binding of chimeric PP1{alpha}/beta catalytic subunits by varying concentrations of GST-Gac1p (Fig. 7B) confirmed that PP1beta bound GST-Gac1p with near equal affinity to Glc7p. By contrast, GST-Gac1p demonstrated ~5-fold reduced affinity for PP1{alpha}. PP1beta/{alpha}C showed Gac1p binding equal to or better than PP1beta. By comparison, PP1beta/{alpha}N showed reduced Gac1p binding, which was indistinguishable from PP1{alpha}. Similarly, PP1{alpha}/betaC bound Gac1p similar to PP1{alpha}, whereas PP1{alpha}/betaN showed enhanced Gac1p binding, equivalent to PP1beta. These data also highlighted the unique contribution of PP1beta N terminus in enhancing Gac1p binding. By comparison, the presence of PP1{alpha} N terminus diminished Gac1p binding. The C termini of the human PP1 catalytic subunits appeared to be largely dispensable for Gac1p binding.

Two other yeast PP1 regulators, Gip2p and Pig2p, have also been implicated in regulating glycogen metabolism (30, 32). We analyzed the binding of human PP1 to purified GST-Gip2p and GST-Pig2p (Fig. 7A). Remarkably, PP1{alpha} and, even more striking, PP1beta bound GST-Gip2p more effectively than Glc7p. By comparison, GST-Gip2p bound PP1{gamma}1, PP1{gamma}2, and PP1{alpha}306 similar to or slightly weaker than Glc7p (Fig. 7C). All human PP1s bound GST-Pig2p with equivalent efficacy but 2-fold weaker than Glc7p (Fig. 7C). Lack of correlation between PP1 binding and glycogen accumulation suggested that yeast complexes containing Pig2p and Gip2p do not account for the differences in glycogen content noted in yeast expressing human PP1 isoforms.

Human PP1 Supports Selected Glc7p Functions—Glc7p binds Gip1p to regulate yeast sporulation (33). Analysis of diploid strains with human PP1beta, PP1{gamma}1, or PP1{gamma}2 integrated into the HIS3 locus established that these yeast did not sporulate, even when PP1 expression was elevated by yeast growth on media containing CuSO4. By comparison, control untransformed yeast and yeast expressing HA-Glc7p sporulated normally (data not shown). Transforming yeast expressing PP1{gamma}1 or PP1{gamma}2 with a Glc7p-expressing plasmid rescued the sporulation defect. This suggested that human PP1 either failed to bind Gip1p or transduce Glc7p signals required for sporulation.

Glc7p binds Reg1p to regulate glucose derepression. Under conditions of abundant glucose, this PP1 complex inhibits gene transcription required for metabolism of other carbon sources (34). Growth on 2-deoxyglucose activates this mechanism, but unable to metabolize this glucose analog, yeast fail to grow in media containing sucrose as a carbon source and 2-deoxyglucose. Like WT untransformed and HA-Glc7p-expressing yeast, all strains expressing human PP1 failed to grow under anaerobic conditions (supplemental Fig. 2). By comparison, the yeast strain KT1512, which lacked Reg1p, grew well (35). These data suggested that human PP1 isoforms were indistinguishable from Glc7p in regulating glucose derepression.


Figure 7
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  		FIGURE 7.
Binding of human PP1 by yeast PP1 regulators. A, representative immunoblot (IB) from several independent pulldowns of human PP1 catalytic subunits, and Glc7p in yeast lysates by 5 µg of GST, GST-Gac1p, GST-Gip2p, and GST-Pig2p is shown. Expression of Glc7p and human PP1 isoforms was equalized by varying CuSO4 in culture medium and is shown as load (L). PP1 sedimented (P) and that remaining soluble (S) are also shown in each case. B, quantitation of PP1 pulldowns from yeast lysates using 5 µg of GST-Gac1p is shown. PP1 eluted from glutathione-Sepharose beads as described under "Experimental Procedures" was subjected to immunoblotting and quantified using LiCOR Odyssey software. GST alone did not bind to any PP1 catalytic subunit (data not shown). Percent of PP1 in yeast lysates that was sedimented by GST-Gac1p (5 µg) was normalized to that seen with Glc7p (left panel). Average ± S.E. of four independent experiments is shown. Right panel shows representative data of total PP1 bound (in arbitrary units) in three independent pulldowns by increasing concentrations of GST-Gac1p for PP1{alpha} (filled squares), PP1beta (open squares), PP1{alpha}/betaN(filled triangles), PP1{alpha}/betaC(filled diamonds), PP1beta/{alpha}N(open triangles), PP1beta/{alpha}C(open diamonds), and Glc7p (filled squares with dashed line). C, PP1 catalytic subunits sedimented using 5 µg of GST fusion proteins containing two other yeast glycogen regulatory subunits were quantified as above. The results are shown as percent of Glc7p sedimented in the same assay. Average ± S.E. of three independent experiments is shown. Left panel shows results with GST-Gip2p, and right panel shows GST-Pig2p.

 
Biochemical Analysis of Human PP1 Expressed in Yeast—As current studies represented the first expression of human PP1 in budding yeast, we analyzed the biochemical properties of PP1 partially purified from yeast as described previously (11–13). Phosphorylase phosphatase activity of human PP1 preparations was potently inhibited by 2 nM microcystin-LR (MCLR), a known PP1 inhibitor (36) (Table 1). Moreover, unlike human PP1{alpha}306 expressed in E. coli, PP1 catalytic subunits from yeast did not require MnCl2 for activity and were not inhibited by 10 mM EDTA (17), resembling PP1 purified from rabbit skeletal muscle (data not shown). Unlike bacterially expressed PP1{alpha}306, whose activity against pNPP was inhibited by 2 nM MCLR, PP1 from rabbit skeletal muscle showed no detectable hydrolysis of pNPP. Although partially purified human PP1 from yeast displayed pNPP phosphatase activity, this was not inhibited by MCLR and thus represented contaminating yeast phosphatase(s).


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  		TABLE 1
Characterization of PP1 enzymatic activity PP1 catalytic subunits (equal amounts of protein) partially purified from yeast lysates (as described under "Experimental Procedures") were analyzed for phosphatase activity using phosphorylase a and pNPP as substrates. Hydrolysis of each substrate during 10 min of incubation in the absence and presence of 2 nM MCLR was compared. Assays were undertaken in duplicate, and the average of two experiments is shown.

 
Dose-response for inhibition of human PP1 expressed in yeast by okadaic acid was essentially identical with an IC50 of ~80 nM and significantly higher than PP2Ac purified from skeletal muscle (IC50 1 nM; Fig. 8A). Human and yeast PP1 catalytic subunits were potently inhibited by human inhibitor-2, a known PP1-specific inhibitor, and as anticipated, PP2Ac was not inhibited by inhibitor-2 (Fig. 8B). Human PP1{alpha} expressed in yeast showed 20-fold increased sensitivity to inhibition by GST-NrbI-(374–516), another PP1-specific regulator, when compared with the same enzyme expressed in bacteria (Fig. 8C). Finally, pulldowns with GST-NrbI-(374–516) showed that this PP1-binding peptide (5) bound human PP1{gamma}1 from yeast lysates with nearly 50-fold higher affinity than PP1beta, which was barely detectable using 10 µg of the bait (Fig. 8D). These data demonstrated that relative recognition of human PP1 isoforms expressed in yeast by neurabin displayed the isoform selectivity noted in mammalian neurons (5). Together, these studies demonstrated that human PP1 expressed in S. cerevisiae displayed biochemical properties that closely resembled those of PP1 from mammalian tissues.


Figure 8
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  		FIGURE 8.
Biochemical characterization of PP1 expressed in S. cerevisiae. A, phosphorylase phosphatase activity of Glc7p and human PP1 extensively purified from yeast lysates was analyzed in the presence of increasing concentrations of okadaic acid. The inhibition of PP1 (filled squares) and PP2A (filled triangles connected by dashed line) catalytic subunits purified to near homogeneity from rabbit skeletal muscle was compared with Glc7p (open squares joined by dashed line), human PP1{alpha} (filled diamonds), PP1{alpha}306 (filled circles), PP1beta (filled triangles connected by solid line), and PP1{gamma}1(open circles joined by dotted line). B, dose-dependent inhibition of PP1 and PP2A catalytic subunits purified from rabbit skeletal muscle by recombinant human Inhibitor-2 was compared with selected PP1 catalytic subunits (symbols shown in A) purified from yeast. C, dose-dependent inhibition of PP1{alpha} expressed in S. cerevisiae (filled diamonds) by a recombinant GST-neurabin peptide, GST-NrbI-(374–516), was compared with PP1{alpha} expressed in E. coli (open diamonds). PP2A catalytic subunit purified from rabbit skeletal muscle (filled triangles joined by dashed line) was included as a control. D, pulldowns of HA-PP1{gamma}1 and HA-PP1beta from yeast lysates, matched for equivalent protein expression by growth of yeast in CuSO4, were undertaken using GST (10 µg) and GST-NrbI-(374–516) (1.0 and 10 µg of protein) as bait. Immunoblotting of load or input (L) and sedimented (P) and soluble (S) fractions was undertaken using anti-HA antibody. Immunoblotting with anti-PGK was used as control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Most eukaryotes contain multiple PP1 genes. Thus, PP1 is encoded by 4 genes in Drosophila melanogaster, 3 mammalian genes, 7–11 plant genes, and up to 38 Caenorhabditis elegans genes. Multiple PP1 isoforms, PP1{alpha}, PP1beta, PP1{gamma}1, and PP1{gamma}2, are expressed in mammalian tissues, although PP1{gamma}2 is most highly expressed in testes, and PP1beta is the major isoform in skeletal muscle (37). Eukaryotic cells also contain more than 60 PP1 regulators. Thus, investigating the physiological role of PP1 isoforms and/or the molecular basis by which they are recruited by cellular regulators is challenging. Thus, it is currently unclear whether high levels of GM (PPP13A) and PP1beta or specific structural determinants in these two proteins promote their preferential association and define PP1beta/GM as a glycogen synthase phosphatase (8).

Expression of cellular PP1 regulators is closely linked to the content of PP1 catalytic subunits in mammalian tissues. Thus, deleting mouse GM (PPP13A) gene resulted in 60% reduction of overall muscle PP1 activity (38). By comparison, overexpression of GM in transgenic mice elevated muscle PP1 (39). Overexpression of PP1 catalytic subunits in mammalian cells has been confounded by compensatory reductions in endogenous PP1 levels (40). Thus, deletion of the mouse PP1{gamma} gene resulted in increased expression (41) and incorporation of PP1{alpha} in neuronal complexes containing neurabin (5). This highlighted the difficulties in studying PP1 functions in mammalian cells and suggested that novel approaches are needed to investigate the physiological functions of PP1.

To date, structural and functional studies of human PP1 have utilized enzymes expressed in E. coli (42). Three-dimensional structures of bacterially expressed PP1{alpha} (43), PP1beta (44), and PP1{gamma}1 (45) highlighted a highly conserved central domain, with a catalytic center containing two Mn2+ ions, but N and C termini unique to each PP1 isoform were not visualized. Cocrystallization of PP1 with a fragment of the myosin-targeting subunit (MYPT1) (44) and mutagenesis studies (20) hinted at a role for the PP1 C terminus in binding cellular regulators. By contrast, the inability to modify N-terminal sequences without compromising bacterial PP1 expression or inactivating the phosphatase4 has hampered efforts to investigate the role of the N-terminal sequences in PP1 function and regulation.

The presence of an essential gene, GLC7, encoding a single PP1 catalytic subunit in S. cerevisiae facilitated genetic studies that have helped to define the mode of action of several human PP1 regulators (11–13). This also suggested conserved PP1 function and regulation in this model eukaryote. Thus, current studies undertook a gene replacement strategy to generate yeast strains expressing individual human PP1 isoforms. Lower levels of human PP1, even in yeast grown on media containing CuSO4 which activated the CUP1 promoter to induce PP1, suggested a more efficient translation or increased stability of Glc7p in yeast. Consistent with their reduced PP1 content, yeast expressing human PP1 isoforms grew 3–5-fold slower than untransformed yeast or yeast expressing HA-Glc7p. The 4-fold faster growth for PP1{alpha}-expressing compared with PP1beta-expressing yeast enabled structure-function studies that analyzed PP1{alpha}/beta chimeras expressed in yeast. Substituting the PP1beta N terminus in PP1{alpha}/betaN reduced yeast growth, whereas substituting the PP1{alpha} N terminus enhanced growth in PP1beta/{alpha}N-expressing yeast. By contrast, switching C-terminal sequences in these two PP1 isoforms had little or no effect on growth. These studies not only noted the differing abilities of human PP1 isoforms to support yeast growth but also, for the first time, highlighted a critical role for their unique N-terminal sequences in dictating the cellular function of PP1 catalytic subunits.

Immunofluorescence showed that the highest concentration of human PP1 was in the yeast nuclei. Human PP1{gamma}1 was uniquely associated with yeast mitotic spindles, as also reported in HeLa cells (46). Nucleolar localization of human PP1beta in yeast was also consistent with prior observations in mammalian cells (47). Interestingly, the latter studies focused on an N-terminal arginine present in PP1beta and PP1{gamma}1 as critical for their nucleolar localization. Although PP1{alpha} was excluded from this compartment, the substitution, Q20R, successfully targeted this enzyme to nucleoli. Thus, specific N-terminal residues may play a key role in directing the subcellular localization of PP1 isoforms in both human and possibly yeast cells.

Yeast expressing human PP1 accumulated less glycogen than strains expressing Glc7p. PP1{alpha} strains were particularly notable in that they accumulated less glycogen than strains expressing other human isoforms. However, increasing PP1{alpha} expression by yeast growth on media containing CuSO4 appeared to overcome this deficit and may be explained by the reduced affinity of PP1{alpha} for the yeast glycogen-targeting subunit, Gac1p. Analysis of PP1{alpha}/beta chimeras showed that the enhanced ability of PP1beta to bind Gac1p and promote glycogen accumulation resulted from its unique N terminus. Thus, PP1{alpha}/betaN bound Gac1p more avidly than PP1{alpha}, whereas PP1beta/{alpha}N showed reduced Gac1p binding compared with PP1beta. Interestingly, studies in yeast that expressed a mutant Glc7p, R19A/K22A, showed that this strain failed to accumulate glycogen (48) and speculated that N-terminal residues may mediate Gac1p binding. It is noteworthy that Arg-19 is present Glc7p and human PP1beta, PP1{gamma}1 and PP1{gamma}2, which bound Gac1p with avidity similar to Glc7p. By comparison, as noted above, PP1{alpha} possessed glutamine and bound Gac1p weakly. Thus, specific N-terminal residues that mediate PP1 binding to cellular regulators may also dictate its localization in cells.

The ability of Glc7p to bind Gac1p with similar avidity to human PP1beta, PP1{gamma}1, and PP1{gamma}2 was somewhat surprising as Glc7p-expressing strains accumulated 5-fold higher glycogen. This could reflect the unique activity of the Glc7p-Gac1p complex as a yeast glycogen synthase phosphatase. Thus, there appears to be some discordance between binding of regulators and generation of a functional PP1 complex. This was also suggested in prior studies that identified PP1 mutations that retained effective neurabin binding, but compromised neurabin's ability to regulate phosphatase activity (26). In contrast to Gac1p, the pattern of human PP1 binding to Gip2p and Pig2p, also implicated in yeast glycogen metabolism (30, 32), did not correlate with the glycogen content in yeast and discounted these interactions as primary drivers of yeast glycogen accumulation.

Earlier in vitro studies had suggested that PP1 C termini may play a key role in its regulation by cellular proteins (49). However, current work failed to note significant functional consequences of deleting or substituting C-terminal sequences in human PP1. Similarly, a C-terminal deletion in Glc7p (glc7–305) appeared fully functional in promoting yeast glycogen synthesis, although a further truncation, glc7–299, did confer partial glycogen deficiency (48). This may reflect the fact that the PP1 C terminus may play a paradoxical role in regulating PP1, potentially inhibiting phosphatase activity of the isolated catalytic subunits (50), while contributing positively to allosteric modulation of PP1 by cellular regulators (20, 49).

It is noteworthy that yeast expressing human PP1 did not sporulate, a known Glc7p-regulated event (51). These strains also showed increased salt and temperature sensitivity compared with WT yeast, possibly indicating the reduced ability of human PP1 to bind or be regulated by selected yeast PP1-binding proteins. On the other hand, despite their lower expression in yeast, all human PP1 isoforms effectively catalyzed the events required for glucose derepression. This suggested that human PP1 may recruit some yeast regulators, such as Reg1p (35), as effectively or better than Glc7p.

As current studies represented the first expression of human PP1 in budding yeast, we also analyzed the biochemical properties PP1 partially purified from yeast lysates. Unlike human PP1 expressed in E. coli, these enzymes did not require added divalent cations, specifically Mn2+, were not inhibited by EDTA, and did not display detectable microcytin-LR-inhibited pNPP phosphatase activity. Sensitivity of PP1 enzymes purified from yeast to both okadaic acid and inhibitor-2 were entirely characteristic of PP1 isolated rabbit skeletal muscle. Finally, human PP1 expressed in yeast showed 50-fold higher sensitivity to the neuronal regulator, neurabin, which also selectively recruited PP1{gamma}1 over PP1beta from yeast lysates as previously seen in mammalian neurons. This raises the intriguing possibility that the generation of yeast strains expressing human PP1 provides a novel expression system that may provide both quantity and quality of human PP1 necessary to facilitate future studies directed at a better understanding of the structure and regulation of this key signaling molecule.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants DK52054 (to S. S.) and GM47789 (to K. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1–3 and Figs. 1–3. Back

1 Supported in part by National Institutes of Health Toxicology Training Grant T32ES07031. Back

2 To whom correspondence should be addressed: Molecular Pharmacology, Pfizer Global Research and Development, 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 734-622-7302; Fax: 734-622-1565; E-mail: Shirish.Shenolikar{at}Pfizer.com.

3 The abbreviations used are: PP1, protein phosphatase-1; 5-FOA, 5-fluoroorotic acid; CSM, complete synthetic medium; GST, glutathione S-transferase; HA, hemagglutinin; pNPP, p-nitrophenyl phosphate; PGK, anti-3-phosphoglycerate kinase; WT, wild type; MCLR, microcystin-LR. Back

4 J. H. Connor and S. Shenolikar, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Danny Lew, John Connor, Dennis Thiele, and members of his laboratory at Duke University for their advice and support during the course of this work.



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