Molecular Characterization of Ypi1, a Novel Saccharomyces cerevisiae Type 1 Protein Phosphatase Inhibitor
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m306157200
ISSN1083-351X
AutoresMaría Adelaida García-Gimeno, Iván Muñoz, Joaquı́n Ariño, Pascual Sanz,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoThe Saccharomyces cerevisiae open reading frame YFR003c encodes a small (155-amino acid) hydrophilic protein that we identified as a novel, heat-stable inhibitor of type 1 protein phosphatase (Ypi1). Ypi1 interacts physically in vitro with both Glc7 and Ppz1 phosphatase catalytic subunits, as shown by pull-down assays. Ypi1 inhibits Glc7 but appears to be less effective toward Ppz1 phosphatase activity under the conditions tested. Ypi1 contains a 48RHNVRW53 sequence, which resembles the characteristic consensus PP1 phosphatase binding motif. A W53A mutation within this motif abolishes both binding to and inhibition of Glc7 and Ppz1 phosphatases. Deletion of YPI1 is lethal, suggesting a relevant role of the inhibitor in yeast physiology. Cells overexpressing Ypi1 display a number of phenotypes consistent with an inhibitory role of this protein on Glc7, such as decreased glycogen content and an increased growth defect in a slt2/mpk1 mitogen-activated protein kinase-deficient background. Taking together, these results define Ypi1 as the first inhibitory subunit of Glc7 identified in budding yeast. The Saccharomyces cerevisiae open reading frame YFR003c encodes a small (155-amino acid) hydrophilic protein that we identified as a novel, heat-stable inhibitor of type 1 protein phosphatase (Ypi1). Ypi1 interacts physically in vitro with both Glc7 and Ppz1 phosphatase catalytic subunits, as shown by pull-down assays. Ypi1 inhibits Glc7 but appears to be less effective toward Ppz1 phosphatase activity under the conditions tested. Ypi1 contains a 48RHNVRW53 sequence, which resembles the characteristic consensus PP1 phosphatase binding motif. A W53A mutation within this motif abolishes both binding to and inhibition of Glc7 and Ppz1 phosphatases. Deletion of YPI1 is lethal, suggesting a relevant role of the inhibitor in yeast physiology. Cells overexpressing Ypi1 display a number of phenotypes consistent with an inhibitory role of this protein on Glc7, such as decreased glycogen content and an increased growth defect in a slt2/mpk1 mitogen-activated protein kinase-deficient background. Taking together, these results define Ypi1 as the first inhibitory subunit of Glc7 identified in budding yeast. In eukaryotic organisms, protein phosphatases play a key role in the control and integration of cellular physiology. Among them, type 1 protein phosphatases (PP1) 1The abbreviations used are: PP1type 1 protein phosphatasePP2Atype 2A protein phosphataseGSTglutathione S-transferaseHAhemagglutinin epitopeSCsynthetic complete.1The abbreviations used are: PP1type 1 protein phosphatasePP2Atype 2A protein phosphataseGSTglutathione S-transferaseHAhemagglutinin epitopeSCsynthetic complete. regulate a great variety of physiological processes in the cell such as carbohydrate and lipid metabolism, protein synthesis, and cell cycle progression (1Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (595) Google Scholar, 2Stark M.J. Yeast. 1996; 12: 1647-1675Crossref PubMed Scopus (170) Google Scholar, 3Zolnierowicz S. Bollen M. EMBO J. 2000; 19: 483-488Crossref PubMed Scopus (153) Google Scholar). type 1 protein phosphatase type 2A protein phosphatase glutathione S-transferase hemagglutinin epitope synthetic complete. type 1 protein phosphatase type 2A protein phosphatase glutathione S-transferase hemagglutinin epitope synthetic complete. The PP1 catalytic subunit (PP1c) is highly conserved throughout evolution. In most eukaryotes, several isoforms have been described (i.e. four in mammals), although in the yeast Saccharomyces cerevisiae only one PP1c is present, named Glc7, which is essential for cell viability (4Feng Z.H. Wilson S.E. Peng Z.Y. Schlender K.K. Reimann E.M. Trumbly R.J. J. Biol. Chem. 1991; 266: 23796-23801Abstract Full Text PDF PubMed Google Scholar, 5Clotet J. Posas F. Casamayor A. Schaaff-Gerstenschlager I. Arino J. Curr. Genet. 1991; 19: 339-342Crossref PubMed Scopus (28) Google Scholar). Similar to its mammalian counterpart, Glc7 participates in the regulation of many different cellular processes such as glycogen metabolism, glucose repression, ion homeostasis, mitosis, meiosis, sporulation, vacuole fusion, endocytosis, polyadenylation termination, and cell wall integrity (6Cannon J. Pringle J.R. Fiechter A. Khalil M. Genetics. 1994; 136: 485-503Crossref PubMed Google Scholar, 7Francisco L. Wang W. Chan C.S.M. Mol. Cell. Biol. 1994; 14: 4731-4740Crossref PubMed Scopus (239) Google Scholar, 8Hisamoto N. Sugimoto K. Matsumoto K. Mol. Cell. Biol. 1994; 14: 3158-3165Crossref PubMed Scopus (92) Google Scholar, 9Tu J. Carlson M. Mol. Cell. Biol. 1994; 14: 6789-6796Crossref PubMed Scopus (81) Google Scholar, 10Wek R.C. Cannon J.F. Dever T.E. Hinnebusch A.G. Mol. Cell. Biol. 1992; 12: 5700-5710Crossref PubMed Google Scholar, 11Tachikawa H. Bloecher A. Tatchell K. Neiman A.M. J. Cell Biol. 2001; 155: 797-808Crossref PubMed Scopus (79) Google Scholar, 12Chang J.S. Henry K. 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Biochemistry. 1999; 38: 16952-16957Crossref PubMed Scopus (89) Google Scholar). Among them, inhibitor-1 and inhibitor-2 are of special interest because they represent two different ways of inhibiting PP1c phosphatase activity. Inhibitor-1 and its structural homologue DARPP-32 require phosphorylation by the cAMP-dependent protein kinase A to gain PP1c-inhibitory capacity. In contrast, inhibitor-2 inhibits PP1c only in its dephosphorylated form (16Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar, 21Eto M. Karginov A. Brautigan D.L. Biochemistry. 1999; 38: 16952-16957Crossref PubMed Scopus (89) Google Scholar, 22Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: D961-D972Crossref PubMed Google Scholar, 23Connor J.H. Frederick D. Huang H.B. Yang J. Helps N.R. Cohen P.T.W. Nairn A.C. DePaoli-Roach A. Tatchell K. Shenolikar S. J. Biol. Chem. 2000; 275: 18670-18675Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Most of the PP1c inhibitors present the consensus PP1c binding motif described above, but several reports have shown that the association of inhibitory proteins to PP1c may involve additional contacts (23Connor J.H. Frederick D. Huang H.B. Yang J. Helps N.R. Cohen P.T.W. Nairn A.C. DePaoli-Roach A. Tatchell K. Shenolikar S. J. Biol. Chem. 2000; 275: 18670-18675Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 24Kwon Y.G. Huang H.B. Desdouits F. Girault J.A. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3536-3541Crossref PubMed Scopus (106) Google Scholar, 25Connor J.H. Quan H.N. Ramaswamy N.T. Zhang L. Barik S. Zheng J. Cannon J.F. Lee E.Y. Shenolikar S. J. Biol. Chem. 1998; 273: 27716-27724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 26Huang H.B. Horiuchi A. Watanabe T. Shih S.R. Tsay H.J. Li H.C. Greengard P. Nairn A.C. J. Biol. Chem. 1999; 274: 7870-7878Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Mammalian inhibitor-1 and inhibitor-2 can also inhibit the yeast PP1 phosphatase Glc7 (25Connor J.H. Quan H.N. Ramaswamy N.T. Zhang L. Barik S. Zheng J. Cannon J.F. Lee E.Y. Shenolikar S. J. Biol. Chem. 1998; 273: 27716-27724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 27Dignam S.S. Koushik J.S. Wang J. Trumbly R.J. Schlender K.K. Lee E.Y. Reimann E.M. Arch. Biochem. Biophys. 1998; 357: 58-66Crossref PubMed Scopus (5) Google Scholar, 28Zheng J. Khalil M. Cannon J.F. J. Biol. Chem. 2000; 275: 18070-18078Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). However, no yeast homologue of inhibitor-1 has been described yet, and the yeast homologue of mammalian inhibitor-2, Glc8 (29Tung H.Y. Wang W. Chan C.S. Mol. Cell. Biol. 1995; 15: 6064-6074Crossref PubMed Scopus (86) Google Scholar), functions as an activator rather than as an inhibitor of Glc7 (30Nigavekar S.S. Tan Y.S. Cannon J.F. Arch. Biochem. Biophys. 2002; 404: 71-79Crossref PubMed Scopus (32) Google Scholar). Recently, in a two-hybrid screening of a human brain cDNA library searching for potential mammalian PP1c regulatory proteins, a novel PP1 inhibitor, namely inhibitor-3, was identified. This protein shared 21% identity with a protein of unknown function encoded by the yeast YFR003c open reading frame (31Zhang J. Zhang L. Zhao S. Lee E.Y. Biochemistry. 1998; 37: 16728-16734Crossref PubMed Scopus (51) Google Scholar). It was also demonstrated by two-hybrid analysis that the Yfr003c protein could interact with Glc7 (32Tu J. Song W. Carlson M. Mol. Cell. Biol. 1996; 16: 4199-4206Crossref PubMed Scopus (89) Google Scholar, 33Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. Nature. 2000; 403: 623-627Crossref PubMed Scopus (3882) Google Scholar, 34Ito T. Chiba T. Ozawa R. Yoshida M. Hattori M. Sakaki Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4569-4574Crossref PubMed Scopus (2912) Google Scholar). Therefore, this protein could be a good candidate for an endogenous inhibitor of Glc7 phosphatase activity. Ppz1 and Ppz2 are PP1-related phosphatases involved in saline tolerance, cell wall integrity, cell cycle progression, and protein translation regulation, and, very recently, they have also been related to regulation of K+ and pH homeostasis (35Arino J. Eur. J. Biochem. 2002; 269: 1072-1077Crossref PubMed Scopus (35) Google Scholar, 36Yenush L. Mulet J.M. Arino J. Serrano R. EMBO J. 2002; 21: 920-929Crossref PubMed Scopus (123) Google Scholar). Among them, Ppz1 appears to be more relevant than Ppz2 in regulating the functions mentioned above (35Arino J. Eur. J. Biochem. 2002; 269: 1072-1077Crossref PubMed Scopus (35) Google Scholar). Recent results indicate that Ppz phosphatases and Glc7 might have overlapping functions to some extent and that Ppz1 shares a subset of Glc7 regulatory subunits to fulfill its function (37Venturi G.M. Bloecher A. Williams-Hart T. Tatchell K. Genetics. 2000; 155: 69-83PubMed Google Scholar). Interestingly, the Yfr003c protein has also been reported to interact with Ppz1 in a two-hybrid analysis (37Venturi G.M. Bloecher A. Williams-Hart T. Tatchell K. Genetics. 2000; 155: 69-83PubMed Google Scholar). In this sense, Yfr003c could also be a good candidate for an inhibitor of Ppz1 phosphatase activity in the same way as Hal3, a specific inhibitor of this type of phosphatases (38de Nadal E. Clotet J. Posas F. Serrano R. Gomez N. Arino J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7357-7362Crossref PubMed Scopus (91) Google Scholar), which appears to regulate all known functions of Ppz1 (35Arino J. Eur. J. Biochem. 2002; 269: 1072-1077Crossref PubMed Scopus (35) Google Scholar). In this report, we provide both in vitro and in vivo evidence demonstrating that the protein encoded by YFR003c is an inhibitor of the type 1 protein phosphatase Glc7 and, to some extent, perhaps of Ppz1. Hence, we propose the name Ypi1 (for yeast phosphatase inhibitor 1) for this protein. Strains and Culture Conditions—Escherichia coli DH5α was used as the recipient cell for all plasmids and constructions. Yeast strains used in this work are listed in Table I. The ypi1Δ heterozygous null mutant was constructed using a diploid strain, M5 (see Table I), by a one-step short flanking kanamycin disruption method (39Wach A. Brachat A. Pöhlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2218) Google Scholar). The disruption cassette was generated by PCR using as template plasmid pFA6a-kanMX4 and primers YFRdel-1 and YFRdel-2 (see below). In this way, we disrupted by homologous recombination the complete YPI1 (YFR003c) open reading frame (from +1 ATG to the stop codon) in one of the two wild type alleles of the diploid. Mutants were confirmed by genomic PCR analyses using specific nucleotides for the wild type allele (oligonucleotides YFR-1 and YFR-2) and for the disrupted allele (oligonucleotides YFRPR-1, outside the disruption cassette, and YFRdel-2, inside the KanMX4 selection marker). Tetrad analysis was performed by standard methods, and the presence of the disruption cassette in the viable spore progeny was scored by its associated phenotype (growth in YPD containing 200 μg/ml Geneticin plates).Table IStrains used in this workName of strainGenotypeReferenceFY250MATα his3 leu2 trp1 ura345Sanz P. Alms G.R. Haystead T.A.J. Carlson M. Mol. Cell. Biol. 2000; 20: 1321-1328Crossref PubMed Scopus (177) Google ScholarMCY3000FY250 glc7-T152K45Sanz P. Alms G.R. Haystead T.A.J. Carlson M. Mol. Cell. Biol. 2000; 20: 1321-1328Crossref PubMed Scopus (177) Google ScholarM5MATa/MATα his4/+ leu2/leu2 trp1/trp1 ura3/ura363van Heusden G.P. Griffiths D.J. Ford J.C. Chin A.W.T.F. Schrader P.A. Carr A.M. Steensma H.Y. Eur. J. Biochem. 1995; 229: 45-53Crossref PubMed Scopus (138) Google ScholarJA100MATa his4 leu2 trp1 ura3 can-1r49Clotet J. Gari E. Aldea M. Arino J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (67) Google ScholarJA110JA100 sit4::TRP149Clotet J. Gari E. Aldea M. Arino J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (67) Google ScholarJC002JA100 sit4::TRP1 tetO:HAL355Simon E. Clotet J. Calero F. Ramos J. Arino J. J. Biol. Chem. 2001; 276: 29740-29747Abstract Full Text Full Text PDF PubMed Scopus (45) Google ScholarJC10JA100 mpk1::LEU264Vissi E. Clotet J. de Nadal E. Barcelo A. Bako E. Gergely P. Dombradi V. Arino J. Yeast. 2001; 18: 115-124Crossref PubMed Scopus (21) Google Scholar Open table in a new tab Standard methods for genetic analysis and transformation were used. Yeast cultures were grown in rich medium (YPD) or synthetic complete (SC) medium lacking appropriate supplements to maintain selection for plasmids (40Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990: 164-165Google Scholar), containing the indicated carbon sources. slt2/mpk1Δ mutants were selected on plates containing 1 m sorbitol. Oligonucleotides—The following oligonucleotides were used in the present study: YFR-1, GTCTGAATTCATGAGTGGAAATCAAATGG; YFR-2, TTTCGTCGACCAAAGCCTCAGTCCTTC; YFRPR-1, CCGGAATTCCTCCGGTACCCGATTGAGGCATC; YFRdel-1, TGCCAGGAGTTGCGAGCTAAGTCTTCAATTAAGTCTATAAGGATGCGTACGCTGCAGGTCGAC; YFRdel-2, TTGCTGCTTCATCGAATATTTTGGCTTTCGTTGTACAAAGCCTCAATCGATGAATTCGAGCTCG; YFRW53A-1, CTACAAGGCACAAATGTAAGAgctGAAGAAAATGTGATTGACAATG; YFRW53A-2, CATTGTCAATCACATTTTCTTCagcTCTTACATTGTGCCTTGTAG. New restrictions sites are underlined. The ATG initiating codon is denoted in boldface type. Mutated codons are in lowercase letters. Plasmids—All plasmids containing the YPI1 (YFR003c) open reading frame were constructed by inserting the PCR-derived open reading frame (from +1 ATG to the stop codon) obtained using primers YFR-1 and YFR-2 and genomic DNA from strain FY250 as template. The PCR product was sequenced to confirm that no modifications were introduced by the Taq polymerase. The PCR product was subcloned into the EcoRI and SalI sites of the plasmids used in this work: pWS93 (41Song W. Carlson M. EMBO J. 1998; 17: 5757-5765Crossref PubMed Scopus (102) Google Scholar), to tag Ypi1 protein with 3× HA epitopes (plasmid pWS-Ypi1); pGEX-6P-1 (Amersham Biosciences) to express a GST-Ypi1 fusion protein in E. coli (plasmid pGEX-Ypi1); and pUC18 (plasmid pUC-Ypi1). The YPI1(W53A) mutant form was obtained using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). Plasmid pUC-Ypi1 was used as template in the PCRs using oligonucleotides YFRW53A-1 and YFRW53A-2 described above. The appearance of a new restriction site, AluI, was used to select the putative mutant, which was fully sequenced to check for the correct introduction of the mutation and the absence of unwanted changes. The plasmid obtained was called pUC-Ypi1W53A. An EcoRI-SalI fragment from pUC-Ypi1W53A was subcloned into pWS93 and pGEX-6P-1 to express the mutated protein in yeast (plasmid pWS-Ypi1W53A) and E. coli (plasmid pGEX-Ypi1W53A), respectively. The construction of an N-terminally deleted (Δ1–344) form of Ppz1, containing only the catalytic domain, as a GST fusion in plasmid pGEX-KT has been described previously (42Clotet J. Posas F. de Nadal E. Arino J. J. Biol. Chem. 1996; 271: 26349-26355Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). GST-Glc7 was obtained from Dr. C. S. Chan (29Tung H.Y. Wang W. Chan C.S. Mol. Cell. Biol. 1995; 15: 6064-6074Crossref PubMed Scopus (86) Google Scholar). GST-Hal3 was obtained by subcloning an EcoRI-XhoI fragment from plasmid YPGE15 (36Yenush L. Mulet J.M. Arino J. Serrano R. EMBO J. 2002; 21: 920-929Crossref PubMed Scopus (123) Google Scholar) into pGEX6P-1. High copy expression of HAL3 in yeast (plasmid pHAL3) was accomplished by cloning a 3.2-kbp EcoRI-HindIII genomic fragment containing the entire gene into the same sites of plasmid YEplac195 (43Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2500) Google Scholar). Expression of Recombinant Proteins in E. coli—Purification of the fusion proteins GST-Glc7, GST-Ppz1Δ1–344, GST-Ypi1, GST-Ypi1W53A, and GST-Hal3 was carried out as described in Ref. 44Zhang A.J. Bai G. Deans-Zirattu S. Browner M.F. Lee E.Y. J. Biol. Chem. 1992; 267: 1484-1490Abstract Full Text PDF PubMed Google Scholar, with some modifications. E. coli transformants harboring the different GST fusions were grown in 500 ml of LB/ampicillin, supplemented with 0.5 mm MnCl2 only for purification of GST-Glc7 and GST-Ppz1Δ1–344. Transformants were grown at 37 °C until the absorbance at 600 nm reached a value of about 0.3. Isopropyl-1-thio-β-d-galactopyranoside was then added to a concentration of 0.1 mm, and cultures were grown overnight at 25 °C. Cells were harvested and resuspended in 20 ml of sonication buffer (50 mm Tris-HCl, pH 7.6, 0.2 mm EGTA, 150 mm NaCl, 10% glycerol, 0.1% Triton X-100, 2 mm dithiothreitol, 2 mm phenylmethylsulfonyl fluoride, and complete protease inhibitor mixture (Roche Applied Science)). This buffer was made 2 mm MnCl2 when purifying GST-Glc7 and GST-Ppz1Δ1–344 fusion proteins. Cells were disrupted by sonication, and the fusion proteins were purified by passing the extracts through a 1-ml bed volume of glutathione-Sepharose columns (Amersham Biosciences). To remove the GST moiety from GST fusions to Ypi1 and Ypi1W53A, the fusion proteins bound to the glutathione-Sepharose beads were treated with PreScission Protease (Amersham Biosciences) during 4–5 h at 4 °C following the manufacturer's instructions. GST-Glc7, GST-Ppz1Δ1–344, GST-Hal3, and GST proteins were eluted from the column with 10 mm glutathione. Samples were stored at –80 °C. Pull-down Assays and Immunoblot Analysis—Preparation of yeast protein extracts for pull-down assays was essentially as described previously (45Sanz P. Alms G.R. Haystead T.A.J. Carlson M. Mol. Cell. Biol. 2000; 20: 1321-1328Crossref PubMed Scopus (177) Google Scholar). Extraction buffer was 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 0.1% Triton X-100, 1 mm dithiothreitol, and 10% glycerol and contained 2 mm phenylmethylsulfonyl fluoride and complete protease inhibitor mixture (Roche Applied Science). The E. coli protein extracts were prepared as described above. Pull-down assays were carried out as follows. Fusion proteins (GST-Glc7 and GST-Ppz1Δ1–344) made in E. coli were allowed to bind to glutathione-Sepharose (Amersham Biosciences) affinity matrix for 1 h at 4 °C with gentle shaking. Then the beads were washed four times with extraction buffer (see above). Yeast extracts (500 μg) were then incubated with the beads for an additional 1 h at 4 °C and again washed four times with extraction buffer. Proteins retained by the affinity system were detected by SDS-PAGE followed by immunoblot using anti-GST polyclonal (Amersham Biosciences) or anti-HA monoclonal (Roche Applied Science) antibodies and chemiluminiscence reagents (ECL; Amersham Biosciences). Protein Phosphatase Assays—Protein phosphatase activity using p-nitrophenylphosphate as substrate was determined essentially as described in Ref. 46Silberman S.R. Speth M. Nemani R. Ganapathi M.K. Dombradi V. Paris H. Lee E.Y. J. Biol. Chem. 1984; 259: 2913-2922Abstract Full Text PDF PubMed Google Scholar. The reaction buffer was 50 mm Tris-HCl, pH 7.5, 0.1 mm EGTA, 2 mm MnCl2, and 1 mm dithiothreitol. Samples were incubated for 10 min at 30 °C, and then the reaction was stopped by adding 1% Tris (final concentration). For phosphatase inhibition assays, different amounts of the purified inhibitors were incubated with the purified phosphatases during 5 min at 30 °C, prior to the addition of p-nitrophenylphosphate. Alternatively, we used the N-terminal domain of the Reg1 protein tagged with HA (HA-Reg11–443) as endogenous protein substrate. This protein showed a clear change in electrophoretic mobility after shifting cells from medium containing high (4%) glucose to low (0.05%) glucose, due to phosphorylation of the protein (45Sanz P. Alms G.R. Haystead T.A.J. Carlson M. Mol. Cell. Biol. 2000; 20: 1321-1328Crossref PubMed Scopus (177) Google Scholar). Since Reg1 is dephosphorylated by Glc7 in response to glucose, for these assays we used the mutant allele glc7-T152K (strain MCY3000), which is defective in dephosphorylating Reg1 (45Sanz P. Alms G.R. Haystead T.A.J. Carlson M. Mol. Cell. Biol. 2000; 20: 1321-1328Crossref PubMed Scopus (177) Google Scholar). MCY3000 transformants expressing HA-Reg11–443 were grown until exponential phase (A600 around 0.4–0.7) in SC medium containing 4% glucose as carbon source, and shifted to a medium containing 0.05% glucose during 20 min. Cells were then harvested, and yeast crude extract was obtained as described above. One μg of this extract was incubated for 20 min at 30 °C with 1.8 μg of GST-Glc7 or GST-Ppz1Δ1–344 in a buffer containing 50 mm Tris-HCl, pH 7.5, 0.1 mm EGTA, 2 mm MnCl2 and 1 mm dithiothreitol. The reaction was stopped by boiling the samples for 3 min in electrophoresis sample buffer. Then the phosphorylation status of HA-Reg11–443 was analyzed by SDS-PAGE and immunoblot. When potential phosphatase inhibitors were assayed, different amounts of the purified inhibitors were incubated with the purified phosphatases during 5 min at 30 °C, prior to the addition of the yeast crude extract. Measurement of Glycogen Content—Wild type strain JA100 containing plasmids pWS93 or pWS-Ypi1 were grown on YPD until the indicated absorbance at 660 nm and then ∼200 mg (wet weight) of fresh cells were collected by filtration. Cells were disrupted, and glycogen was measured essentially as in Ref. 47Arino J. Posas F. Clotet J. Methods Mol. Biol. 1998; 93: 305-313PubMed Google Scholar. Glucose released by glycogen hydrolysis was measured using a glucose-oxidase-based commercial kit. Phenotypic Analyses and Other Techniques—The effect of the overexpression of Ypi1 was monitored on plates by "drop tests" as previously described (48Posas F. Camps M. Arino J. J. Biol. Chem. 1995; 270: 13036-13041Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Briefly, cells were grown on SC medium lacking uracil for ∼48 h, and absorbance at 660 was measured and adjusted to 0.05. Serial dilutions (1:5) were made, and 3 μl of each dilution was deposited on the indicated culture media. To monitor recovery from α-factor arrest, strain JA110 (sit4) was transformed with plasmid pWS93 or pWS-Ypi1, and cells were grown until an absorbance at 660 nm of 0.6 was reached. Recovery from α factor arrest was performed as in Ref. 49Clotet J. Gari E. Aldea M. Arino J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (67) Google Scholar. Budding index was monitored as in Ref. 49Clotet J. Gari E. Aldea M. Arino J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (67) Google Scholar, and DNA content was determined by flow cytometry as in Ref. 50Gallego C. Gari E. Colomina N. Herrero E. Aldea M. EMBO J. 1997; 16: 7196-7206Crossref PubMed Scopus (136) Google Scholar. Yfr003c Belongs to a Highly Conserved Family of Proteins Including a PP1 Protein Phosphatase Inhibitor—YFR003c encodes a small protein (155 residues; 18 kDa, estimated molecular mass) very rich in hydrophilic residues (Asp + Glu content 19.4%; Ser + Thr content 14.8%; Lys + Arg content 16.8%) that shows an aberrant mobility in SDS-PAGE (it runs as a protein of around 30 kDa) and that is heat-stable (see below). All of these properties make this protein very similar to PP1 phosphatase inhibitors described in mammalian cells (16Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar, 17Aggen J.B. Nairn A.C. Chamberlin R. Chem. Biol. 2000; 7: R13-R23Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). A protein BLAST analysis revealed that Yfr003c was highly homologous to a family of small proteins, one of which has been described as PP1 phosphatase inhibitor (Fig. 1). In fact, Yfr003c was previously postulated as the putative yeast homologue of mammalian PP1 inhibitor-3 (31Zhang J. Zhang L. Zhao S. Lee E.Y. Biochemistry. 1998; 37: 16728-16734Crossref PubMed Scopus (51) Google Scholar). It is also important to notice that Yfr003c was strongly conserved throughout all eukaryotes, with homologues in yeast, insects, plants, worms, and mammals. Fig. 1A shows a phylogenetic tree of all of the Yfr003c homologues using the Genebee service (available on the World Wide Web at www.genebee.msu.su). The Yfr003c Gene Product Interacts Physically with Glc7 and the PP1-related Phosphatase Ppz1—The yeast protein encoded by YFR003c (hereafter referred to YPI1, for yeast phosphatase inhibitor 1) was initially identified in a two-hybrid screening searching for Glc7-interacting proteins using LexA-Glc7 as bait (32Tu J. Song W. Carlson M. Mol. Cell. Biol. 1996; 16: 4199-4206Crossref PubMed Scopus (89) Google Scholar). Yeast two-hybrid global analyses have also shown protein-protein interaction between Ypi1 and Glc7 (33Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. Nature. 2000; 403: 623-627Crossref PubMed Scopus (3882) Google Scholar, 34Ito T. Chiba T. Ozawa R. Yoshida M. Hattori M. Sakaki Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4569-4574Crossref PubMed Scopus (2912) Google Scholar). To verify this interaction by an alternative experimental approach, we used an affinity pull-down assay system based on the expression of a GST-Glc7 fusion protein in bacteria. Purified GST-Glc7 was then used to bind HA-tagged-Ypi1 expressed in yeast cells. As shown in Fig. 2A (lane 2), HA-Ypi1 was detected in the fraction retained by GST-Glc7, corroborating the specific interaction between Glc7 and Ypi1. These results were in agreement with those presented recently on the interaction of Yfr003c (Ypi1) and Glc7 by affinity precipitation (
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