FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2092116013> ?p ?o ?g. }

• W2092116013 endingPage "34430" @default.
• W2092116013 startingPage "34421" @default.
• W2092116013 abstract "The yeast gene VHS3 (YOR054c) has been recently identified as a multicopy suppressor of the G1/S cell cycle blockade of a conditional sit4 and hal3 mutant. Vhs3 is structurally related to Hal3, a negative regulatory subunit of the Ser/Thr protein phosphatase Ppz1 important for cell integrity, salt tolerance, and cell cycle control. Phenotypic analyses using vhs3 mutants and overexpressing strains clearly show that Vhs3 has functions reminiscent to those of Hal3 and contrary to those of Ppz1. Mutation of Vhs3 His459, equivalent to the supposedly functionally relevant His90 in the plant homolog AtHal3a, did not affect Vhs3 functions mentioned above. Similarly to Hal3, Vhs3 binds in vivo to the C-terminal catalytic moiety of Ppz1 and inhibits in vitro its phosphatase activity. Therefore, our results indicate that Vhs3 plays a role as an inhibitory subunit of Ppz1. We have found that the vhs3 and hal3 mutations are synthetically lethal. Remarkably, lethality is not suppressed by deletion of PPZ1, PPZ2, or both phosphatase genes, indicating that it is not because of an excess of Ppz phosphatase activity. Furthermore, a Vhs3 version carrying the H459A mutation did not rescue the synthetically lethal phenotype. A conditional vhs3 tetO:HAL3 double mutant displays, in the presence of doxycycline, a flocculation phenotype that is dependent on the presence of Flo8 and Flo11. These results indicate that, besides its role as Ppz1 inhibitory subunit, Vhs3 (and probably Hal3) might have important Ppz-independent functions. The yeast gene VHS3 (YOR054c) has been recently identified as a multicopy suppressor of the G1/S cell cycle blockade of a conditional sit4 and hal3 mutant. Vhs3 is structurally related to Hal3, a negative regulatory subunit of the Ser/Thr protein phosphatase Ppz1 important for cell integrity, salt tolerance, and cell cycle control. Phenotypic analyses using vhs3 mutants and overexpressing strains clearly show that Vhs3 has functions reminiscent to those of Hal3 and contrary to those of Ppz1. Mutation of Vhs3 His459, equivalent to the supposedly functionally relevant His90 in the plant homolog AtHal3a, did not affect Vhs3 functions mentioned above. Similarly to Hal3, Vhs3 binds in vivo to the C-terminal catalytic moiety of Ppz1 and inhibits in vitro its phosphatase activity. Therefore, our results indicate that Vhs3 plays a role as an inhibitory subunit of Ppz1. We have found that the vhs3 and hal3 mutations are synthetically lethal. Remarkably, lethality is not suppressed by deletion of PPZ1, PPZ2, or both phosphatase genes, indicating that it is not because of an excess of Ppz phosphatase activity. Furthermore, a Vhs3 version carrying the H459A mutation did not rescue the synthetically lethal phenotype. A conditional vhs3 tetO:HAL3 double mutant displays, in the presence of doxycycline, a flocculation phenotype that is dependent on the presence of Flo8 and Flo11. These results indicate that, besides its role as Ppz1 inhibitory subunit, Vhs3 (and probably Hal3) might have important Ppz-independent functions. The Saccharomyces cerevisiae Ppz Ser/Thr protein phosphatases, encoded by genes PPZ1 and PPZ2 (1Posas F. Casamayor A. Morral N. Arino J. J. Biol. Chem. 1992; 267: 11734-11740Abstract Full Text PDF PubMed Google Scholar, 2Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (116) Google Scholar, 3Hughes V. Muller A. Stark M.J. Cohen P.T. Eur. J. Biochem. 1993; 216: 269-279Crossref PubMed Scopus (43) Google Scholar), are characterized by a C-terminal half closely related to type 1 phosphatases (see Ref. 4Arino J. Eur. J. Biochem. 2002; 269: 1072-1077Crossref PubMed Scopus (35) Google Scholar for a recent review). These phosphatases are involved in several cell processes. They interact functionally with the protein kinase C-activated MAP 1The abbreviations used are: MAP, mitogen-activated protein; GST, glutathione S-transferase; 5-FOA, 5-fluoro-orotic acid; ORF, open reading frame. kinase pathway and thus play a role in cell wall integrity (2Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (116) Google Scholar, 5Posas F. Casamayor A. Arino J. FEBS Lett. 1993; 318: 282-286Crossref PubMed Scopus (73) Google Scholar), regulate salt tolerance (6Posas F. Camps M. Arino J. J. Biol. Chem. 1995; 270: 13036-13041Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and control cell cycle at the G1/S transition (7Clotet J. Posas F. de Nadal E. Arino J. J. Biol. Chem. 1996; 271: 26349-26355Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Clotet J. Gari E. Aldea M. Arino J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (70) Google Scholar). In all these cases, Ppz1 has a more prominent role, as denoted by the observation that cells lacking Ppz2 display a wild-type phenotype, unless PPZ1 has been also deleted. Recent evidence indicates that most of the phenotypes associated with the absence or the overexpression of the Ppz phosphatases are a consequence of the inhibitory effect that these phosphatases exert on the function of the Trk1/Trk2 potassium transporters (9Yenush L. Mulet J.M. Arino J. Serrano R. EMBO J. 2002; 21: 920-929Crossref PubMed Scopus (127) Google Scholar). In addition, it has been postulated a negative role for Ppz1 on the calcineurin pathway, which would explain the increased expression of the ENA1 Na+-ATPase in ppz1 cells and, at least in part, the salt tolerant phenotype of the mutant strain (10Ruiz A. Yenush L. Arino J. Eukaryotic Cell. 2003; 2: 937-948Crossref PubMed Scopus (59) Google Scholar). Ppz1 is negatively regulated by Hal3/Sis2, which binds to the C-terminal catalytic moiety of the phosphatase and inhibits its activity (11de 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 (93) Google Scholar), although the mechanism of inhibition is still unknown. HAL3/SIS2 was identified independently by two laboratories: as a gene able to confer saline tolerance when overexpressed (12Ferrando A. Kron S.J. Rios G. Fink G.R. Serrano R. Mol. Cell. Biol. 1995; 15: 5470-5481Crossref PubMed Scopus (126) Google Scholar), and as a multicopy suppressor of the growth defect of a sit4 mutant (13Di Como C.J. Bose R. Arndt K.T. Genetics. 1995; 139: 95-107Crossref PubMed Google Scholar). Sit4 is a Ser/Thr protein phosphatase (14Arndt K.T. Styles C.A. Fink G.R. Cell. 1989; 56: 527-537Abstract Full Text PDF PubMed Scopus (177) Google Scholar) required for proper passage from G1 to S phase (15Sutton A. Immanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (272) Google Scholar, 16Fernandez-Sarabia M.J. Sutton A. Zhong T. Arndt K.T. Genes Dev. 1992; 6: 2417-2428Crossref PubMed Scopus (115) Google Scholar). The current evidence suggests that Hal3 regulates most (if not all) the functions of Ppz1. Therefore, overexpression of HAL3 in an slt2/mpk1 MAP kinase mutant aggravates the lytic defect of this strain (11de 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 (93) Google Scholar), thus mimicking the effect of deletion of PPZ1. Similarly, overexpression of HAL3 confers salt tolerance (and increases ENA1 expression), and mutation of the gene results in salt sensitivity, in a Ppz-dependent fashion (10Ruiz A. Yenush L. Arino J. Eukaryotic Cell. 2003; 2: 937-948Crossref PubMed Scopus (59) Google Scholar, 11de 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 (93) Google Scholar). Finally, it is known that the sit4 and hal3 mutations are synthetically lethal because of a G1/S blockade (13Di Como C.J. Bose R. Arndt K.T. Genetics. 1995; 139: 95-107Crossref PubMed Google Scholar, 17Simon E. Clotet J. Calero F. Ramos J. Arino J. J. Biol. Chem. 2001; 276: 29740-29747Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), and this phenotype is suppressed by disruption of PPZ1 (8Clotet J. Gari E. Aldea M. Arino J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (70) Google Scholar). In a recent work (18Munoz I. Simon E. Casals N. Clotet J. Arino J. Yeast. 2003; 20: 157-169Crossref PubMed Scopus (39) Google Scholar) we reported the use of a conditional sit4Δ tetO:HAL3 strain to screen for multicopy suppressors of the G1 blockade suffered by this strain under non permissive conditions (presence of doxycycline). Among the several ORFs identified, YOR054c (renamed as VHS3) has our immediate attention because it encoded an acidic, 674 residue protein displaying a substantial sequence similarity (49% identity) with Hal3. In this work we characterize the biological role of Vhs3 and demonstrate that in addition to acting as a negative regulatory subunit of the Ppz1 protein phosphatase, this protein may also have other important functions. Strains and Growth Conditions—Yeast cells were grown at 28 °C in YPD medium (10 g/liter yeast extract, 20 g/liter peptone, and 20 g/liter dextrose) or, when indicated, in synthetic minimal or complete minimal medium (19Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 147-148Google Scholar). The relevant genotype of the strains described in this work can be found in Table I.Table IYeast strains used in this workNameRelevant genotypeSource/Ref.JA100MATa ura3-52 leu2-3,112 his4 trp1-1 can-1r11de 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 (93) Google ScholarJA110JA100 sit4::TRP18Clotet J. Gari E. Aldea M. Arino J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (70) Google ScholarJC002JA100 sit4::TRP1promtet0::HAL317Simon E. Clotet J. Calero F. Ramos J. Arino J. J. Biol. Chem. 2001; 276: 29740-29747Abstract Full Text Full Text PDF PubMed Scopus (46) Google ScholarJC133JA100 sit4::TRP1 vhs3::URA3This workJC010JA100 mpk1::LEU260Vissi E. Clotet J. de Nadal E. Barcelo A. Bako E. Gergely P. Dombradi V. Arino J. Yeast. 2001; 18: 115-124Crossref PubMed Scopus (22) Google ScholarMAR20JA100 mpk1::LEU2 hal3::LEU2This workMAR9JA100 mpk1::LEU2 vhs3::URA3This workJA104JA100 hal3::LEU211de 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 (93) Google ScholarMAR88JA100 hal3::kanMXThis workMAR79JA100 vhs3::URA3This workMAR107JA100 hal3::kanMX vhs3::nat1 [YEplac195-HAL3]This workMAR109JA100 hal3::kanMX vhs3::nat1 ppz1::LEU2 [YEplac195-HAL3]This workMAR110JA100 hal3::kanMX vhs3::nat1 ppz1::LEU2 ppz2::TRP1 [YEplac195-HAL3]This workEDN75JA100 ppz1::KANMx24de Nadal E. Fadden R.P. Ruiz A. Haystead T. Arino J. J. Biol. Chem. 2001; 276: 14829-14834Abstract Full Text Full Text PDF PubMed Scopus (29) Google ScholarJC001JA100 promtet0::HAL3This workMAR24JA100 promtet0::HAL3 vhs3::URA3This workMAR80JA100 promtet0::HAL3 flo11::TRP1This workMAR81JA100 promtet0::HAL3 vhs3::URA3 flo11::TRP1This workMAR82JA100 promtet0::HAL3 flo8::TRP1This workMAR83JA100 promtet0::HAL3 vhs3::URA3 flo8::TRP1This workMAR84JA100 promtet0::HAL3 ste12::TRP1This workMAR85JA100 promtet0::HAL3 vhs3::URA3 ste12::TRP1This workMAR86JA100 promtet0::HAL3 tec1::TRP1This workMAR87JA100 promtet0::HAL3 vhs3::URA3 tec1::TRP1This workAGS1JA100 promtet0::HAL3 mss11::TRP1This workAGS2JA100 promtet0::HAL3 vhs3::URA3 mss11::TRP1This work1788MATa/a ura3-52 leu2-3,112 his4 trp1-1 can-1rD. LevinMAR61788 hal3::LEU2/HAL3 vhs3::URA3/VHS3This workAGS41788 hal3::LEU2/HAL3 vhs3::kanMX/VHS3This workMAR261788 hal3::LEU2/HAL3 vhs3::URA3/VHS3 ppz1::KAN/PPZ1This workMAR231788 hal3::LEU2/HAL3 vhs3::URA3/VHS3 ppz2::TRP1/PPZ2This workMAR271788 hal3::LEU2/HAL3 vhs3::URA3/VHS3 ppz1::KAN/PPZ1 ppz2::TRP1/PPZ2This workMAR1121788 hal3::LEU2/HAL3 vhs3::nat1/VHS3 ppz1::KAN/PPZ1 ppz2::TRP1/PPZ2This workMAR113MATa hal3::LEU2 vhs3::nat1 ppz1::KAN/ ppz2::TRP1 [YEplac 195-HAL3]This workMAR115MATa hal3::LEU2/HAL3 vhs3::kanMX/VHS3 [YEplac 195-VHS3]This workMCY3000FY250 glc7-T152K32Sanz P. Alms G.R. Haystead T.A. Carlson M. Mol. Cell. Biol. 2000; 20: 1321-1328Crossref PubMed Scopus (180) Google ScholarDBY746MATα ura3-52 leu2-3,112 his3-Δ1 trp1-Δ239D. BotsteinEDN4DBY746 hal3::LEU261de Nadal E. Calero F. Ramos J. Arino J. J. Bacteriol. 1999; 181: 6456-6462Crossref PubMed Google ScholarEDN2DBY746 ppz1::TRP110Ruiz A. Yenush L. Arino J. Eukaryotic Cell. 2003; 2: 937-948Crossref PubMed Scopus (59) Google ScholarEDN85DBY746 ppz1::TRP1 ppz2::KAN10Ruiz A. Yenush L. Arino J. Eukaryotic Cell. 2003; 2: 937-948Crossref PubMed Scopus (59) Google ScholarMAR11DBY746 vhs3::URA3This work Open table in a new tab Gene Disruption and Plasmid Construction—Disruption of the VHS3 gene was done by cloning a 3.6-kbp KpnI/EcoRI genomic fragment, encompassing the entire gene, into the same sites of plasmid pUC19, to yield pUC19-VHS3 and replacing a 1.05-kbp NheI/SnaBI fragment with a NheI/SmaI 1.1-kbp fragment from plasmid YDp-U (20Berben G. Dumont J. Gilliquet V. Bolle P.A. Hilger F. Yeast. 1991; 7: 475-477Crossref PubMed Scopus (317) Google Scholar), containing the URA3 marker. The disruption cassette is recovered by digestion with KpnI/EcoRI and used to transform yeast cells. Disruption of VHS3 with the nat1 marker was accomplished as follows. The 1.29-kbp PvuII/SpeI fragment from plasmid pAG25 (21Goldstein A.L. McCusker J.H. Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1389) Google Scholar), containing the nat1 gene from Streptomyces noursei was used to replace the 1.05-kbp SnaBI/NheI fragment of plasmid pUC19-VHS3. The deletion cassette was released by digestion with AfeI/PvuII and used to transform the appropriate strains. Positive clones were selected in the presence of nourseothricin (21Goldstein A.L. McCusker J.H. Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1389) Google Scholar). Disruption of VHS3 with the kanMX marker (from nucleotides –40 to 2050, relative to the initiating Met codon) was accomplished by the short flanking gene replacement technique (22Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2241) Google Scholar). The HAL3 gene was disrupted with the LEU2 marker as previously described (12Ferrando A. Kron S.J. Rios G. Fink G.R. Serrano R. Mol. Cell. Biol. 1995; 15: 5470-5481Crossref PubMed Scopus (126) Google Scholar). To construct strain MAR88 (hal3::kanMX), genomic DNA from the appropriate deletion mutant in the BY4741 background (23Giaever G. Chu A.M. Ni L. Connelly C. Riles L. Veronneau S. Dow S. Lucau-Danila A. Anderson K. Andre B. Arkin A.P. Astromoff A. El Bakkoury M. Bangham R. Benito R. Brachat S. Campanaro S. Curtiss M. Davis K. Deutschbauer A. Entian K.D. Flaherty P. Foury F. Garfinkel D.J. Gerstein M. Gotte D. Guldener U. Hegemann J.H. Hempel S. Herman Z. Jaramillo D.F. Kelly D.E. Kelly S.L. Kotter P. LaBonte D. Lamb D.C. Lan N. Liang H. Liao H. Liu L. Luo C. Lussier M. Mao R. Menard P. Ooi S.L. Revuelta J.L. Roberts C.J. Rose M. Ross-Macdonald P. Scherens B. Schimmack G. Shafer B. Shoemaker D.D. Sookhai-Mahadeo S. Storms R.K. Strathern J.N. Valle G. Voet M. Volckaert G. Wang C.Y. Ward T.R. Wilhelmy J. Winzeler E.A. Yang Y. Yen G. Youngman E. Yu K. Bussey H. Boeke J.D. Snyder M. Philippsen P. Davis R.W. Johnston M. Nature. 2002; 418: 387-391Crossref PubMed Scopus (3267) Google Scholar) was used to amplify the HAL3 genomic locus using oligonucleotides spanning from positions –108 to +2150, and the amplification fragment used to transform the wild-type strain JA100. Deletion of the PPZ1 gene with the kanMX marker has been reported previously (24de Nadal E. Fadden R.P. Ruiz A. Haystead T. Arino J. J. Biol. Chem. 2001; 276: 14829-14834Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Deletion of PPZ1 with the LEU2 marker was done by replacing a 1.6-kbp XhoI/StuI fragment from plasmid YEp181-Ppz1 (7Clotet J. Posas F. de Nadal E. Arino J. J. Biol. Chem. 1996; 271: 26349-26355Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) with the SmaI/SalI 1.7 kbp LEU2 marker from plasmid YDp-L (20Berben G. Dumont J. Gilliquet V. Bolle P.A. Hilger F. Yeast. 1991; 7: 475-477Crossref PubMed Scopus (317) Google Scholar). The PstI/NdeI 2.5 kbp cassette is used to transform cells. Deletion of PPZ2 with a TRP1 marker was performed as described in Ref. 7Clotet J. Posas F. de Nadal E. Arino J. J. Biol. Chem. 1996; 271: 26349-26355Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar. The use of the cassette tetO:HAL3 was described previously in Ref. 17Simon E. Clotet J. Calero F. Ramos J. Arino J. J. Biol. Chem. 2001; 276: 29740-29747Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar. Deletion of the FLO11/MUC1 gene was performed as follows. A 5.2-kbp region of the FLO11 genomic locus spanning the entire ORF, plus 484 nucleotides upstream and 761 nucleotides downstream, was amplified by PCR and cloned into the XbaI/EcoRV sites of pBluescript to give plasmid pBS-FLO11. Then, a 3.6-kbp HincII/PstI fragment of the ORF was replaced by the 0.85-kbp TRP1 marker, recovered from plasmid YDp-W (20Berben G. Dumont J. Gilliquet V. Bolle P.A. Hilger F. Yeast. 1991; 7: 475-477Crossref PubMed Scopus (317) Google Scholar) by digestion with SmaI/PstI, to yield plasmid pBS-FLO11::TRP1. This plasmid was digested with ApaI/SacI, and the resulting 2.5-kbp fragment was used to transform cells. To construct the flo8::TRP1 cassette, a 3.99-kbp fragment spanning from –998 to +2987 (referred to the ATG of the FLO8 ORF) was amplified by PCR, digested with XbaI (an artificially added site) and EcoRV, and then cloned into the XbaI and HincII sites of pBluescript to give plasmid pBS-FLO8. An EcoRI/SalI 1.8-kbp fragment of the FLO8 gene was replaced by the marker TRP1 (0.85 kbp), recovered from plasmid YDp-W. The resulting construct, pBS-flo8::TRP1, was digested with NdeI, and cells were transformed with the 2.65-kbp fragment released. To disrupt the STE12 gene, a 3.7-kbp fragment (from –970 to +2700 relative to the initiating codon) was amplified by PCR including added PstI and EcoRI sites. After digestion with the indicated enzymes, it was inserted into pUC19 generating pUC19-STE12. Then, a HincII/BamHI 1.6-kbp fragment was replaced by the marker TRP1 (obtained by digestion of YDp-W with SmaI/BamHI). This plasmid, called pUC19-ste12::TRP1 was digested with SacI, and the 1.75-kbp ste12::TRP1 cassette used to transform the appropriate strains. Disruption of the TEC1 gene was made as follows. First, a 3.0-kbp fragment from positions –1050 to +1950 (referred to the ATG codon) was amplified by PCR using a 3′-primer with a KpnI artificial site. The fragment obtained by digestion with PstI and KpnI was cloned into pUC19, yielding plasmid pUC19-TEC1. Then, the 0.92-kbp XbaI/StuI fragment was replaced by the SmaI/NheI fragment of YDp-W, which contains the TRP1 marker and the resulting construct (pUC19-tec1::TRP1) digested with SphI and XhoI to produce the tec1::TRP1 cassette (2.95 kbp) employed for yeast transformation. The MSS11 gene was disrupted with the TRP1 marker as follows. A fragment from –950 to +2742 was amplified by PCR with an added HindIII restriction site, digested with BamHI/HindIII and cloned into the same sites of plasmid pUC19 to give pUC19-MSS11. The construct was cleaved by XhoI/HincII and the 0.89-kbp DNA fragment obtained was replaced with a 0.85-kbp TRP1 marker, previously released from plasmid YDp-W by digestion with SalI/SmaI. The final construct, pUC19-mss11::TRP1, was digested with SnaBI/KpnI and the 2.47 kbp fragment released was used for yeast transformation. All gene deletions generated in this work were confirmed by PCR. For high copy expression of HAL3, the gene was recovered from plasmid YEp351-HAL3 (12Ferrando A. Kron S.J. Rios G. Fink G.R. Serrano R. Mol. Cell. Biol. 1995; 15: 5470-5481Crossref PubMed Scopus (126) Google Scholar) by digestion with EcoRI/HindIII and cloned into these sites of YEplac112 (TRP1 marker) and YEplac181 (LEU2 marker) vectors (25Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2528) Google Scholar). High copy expression of VHS3 was achieved by cloning a 3566-bp insert, starting from a KpnI site at position –776 from the ATG codon of VHS3 and ending at the EcoRI site located 765 bp after the stop codon, at these sites of plasmids YEplac195 (URA3 marker), YEplac181, or YEplac112 (TRP1 marker). An identical cloning strategy using plasmid YCplac22 (TRP1 marker) allowed low copy, centromeric expression. The construction of a version of VHS3 carrying a C-terminal 3×FLAG tag was as follows. An artificial SacI site right in front of the stop codon (which introduces the residues EL) was created by sequential PCR using external oligonucleotides that encompassed the ClaI and AfeI sites. The amplification fragment was digested with these enzymes and used to replace the 1.13-kbp ClaI/AfeI fragment of YEp195-VHS3. A 3×FLAG tag, with added SacI sites, was amplified by PCR from plasmid pCM220 (a gift of M. Aldea, Universitat de Lleida, Spain) and then cloned into the previous construct in the appropriate orientation to yield YEp195-VHS3(3×FLAG). Mutation of Vhs3 His459 to Ala was made by sequential PCR. In a first step, the 1.13-kbp ClaI/AfeI fragment of VHS3 gene was amplified in two separate reactions by using primers with the modification introduced to change His459 to Ala (CA to GC). In the second step the entire ClaI/AfeI fragment was amplified, digested, and the product cloned into the ClaI/AfeI sites of YEp195-VHS3, to yield YEp195-VHS3(H459A). The mutated version of the gene was also cloned in the centromeric plasmid YCplac22 as a KpnI/EcoRI fragment. To express in bacteria GST-Hal3 and GST-Vhs3 fusion proteins the HAL3 and VHS3 genes were amplified by PCR, with added EcoRI/XhoI sites, and cloned into plasmid pGEX6P-1 (Amersham Biosciences). The catalytic domain of Ppz1 (Δ1–344) amplified by PCR as described previously (7Clotet J. Posas F. de Nadal E. Arino J. J. Biol. Chem. 1996; 271: 26349-26355Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) was cloned into the SalI and HindIII sites of the pSP72 vector (Promega) to yield plasmid pSC2. The insert was recovered by digestion with BamHI and PvuII and cloned into the BamHI and SmaI sites of plasmid pGEX6P-1. The constructs for bacterial expression of GST-Ypi1 and GST-Glc7 have been previously described (26Garcia-Gimeno M.A. Munoz I. Arino J. Sanz P. J. Biol. Chem. 2003; 278: 47744-47752Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). A FLO11-lacZ reporter plasmid (pFLO11-LacZ) was constructed by PCR amplification of the 3-kbp region 5′ of the ATG using primers with added BglII sites, following by cloning into the BamHI site of YEp367R (27Myers A.M. Tzagoloff A. Kinney D.M. Lusty C.J. Gene (Amst.). 1986; 45: 299-310Crossref PubMed Scopus (513) Google Scholar). All fragments generated by PCR reactions (with the only exception of the FLO11 promoter) were fully sequenced to confirm the absence of unwanted changes. β-Galactosidase Assays—To evaluate the influence of high levels of HAL3 or VHS3 on ENA1 expression, the different strains tested were transformed with plasmids YEpHAL3 or YEpVHS3, and then with plasmid pKC201, which contains the entire ENA1 promoter fused to the β-galactosidase gene (28Alepuz P.M. Cunningham K.W. Estruch F. Mol. Microbiol. 1997; 26: 91-98Crossref PubMed Scopus (95) Google Scholar). β-Galactosidase assays were carried out as described in Ref. 10Ruiz A. Yenush L. Arino J. Eukaryotic Cell. 2003; 2: 937-948Crossref PubMed Scopus (59) Google Scholar. Analysis of the FLO11 promoter activity was carried out by introducing into the appropriate strains the FLO11-lacZ reporter described above. Cultures were grown overnight in selective medium, diluted up to an OD660 of 0.005 and then grown for 15 h at 28 °C in YPD medium, in the presence or the absence of 100 μg/ml doxycycline (control cells received the same volume of the vehicle, a 50% ethanol solution). Then cells were collected and processed for β-galactosidase assay as above. In Vitro and in Vivo Binding Assays—In vitro binding assays were performed as follows. GST-Ppz1(Δ1–344) was expressed in bacteria and bound to glutathione-agarose beads essentially as described previously (26Garcia-Gimeno M.A. Munoz I. Arino J. Sanz P. J. Biol. Chem. 2003; 278: 47744-47752Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Cell extracts from strain EDN75 (ppz1Δ) were prepared as described (11de 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 (93) Google Scholar) and 1 mg of total protein incubated with 50 μl of the affinity beads for 1 h at 4 °C. Washing and subsequent procedures were as in Ref. 11de 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 (93) Google Scholar except that after extensive washing the beads were resuspended in 100 μl of 2× SDS sample buffer and boiled. After a brief centrifugation, the sample (10 μl) was electrophoresed, transferred to membranes, and probed with anti-FLAG-antibodies (Sigma). Expression in yeast of the GST fusion versions of the full-length and the catalytic domain of Ppz1 from the native PPZ1 promoter was accomplished by transformation of strain EDN75 (ppz1::kanMX) with plasmids pYGST-C1Z1 and pYGST-C2Z1, respectively (11de 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 (93) Google Scholar). Identification of in vivo binding of Vhs3 with the different Ppz1 versions was carried out essentially as previously described for Hal3 (11de 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 (93) Google Scholar) except that cells were transformed in this case with plasmid YEp195-VHS3(3×FLAG) and bound proteins revealed with anti-FLAG antibodies. In Vitro Phosphatase Assays—The effect of the diverse inhibitors on Ppz1 and Glc7 phosphatase activities were evaluated using bacterially expressed proteins. Conditions for expression and purification of the Ypi1, Ppz1(Δ1–344), and Glc7 fusion proteins have been previously reported (26Garcia-Gimeno M.A. Munoz I. Arino J. Sanz P. J. Biol. Chem. 2003; 278: 47744-47752Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). For expression of the phosphatases the growth medium did include 0.5 mm MnCl2. GST-Hal3 and GST-Vhs3 were expressed in bacteria by induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h at 30 °C and purified essentially as described previously (26Garcia-Gimeno M.A. Munoz I. Arino J. Sanz P. J. Biol. Chem. 2003; 278: 47744-47752Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Ppz1 and Glc7 phosphatase activities were measured using p-nitrophenyl phosphate as in Ref. 26Garcia-Gimeno M.A. Munoz I. Arino J. Sanz P. J. Biol. Chem. 2003; 278: 47744-47752Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar with the following modifications: the concentration of substrate was 10 mm, 1 μg of Ppz1 and 2 μg of Glc7 were used, and the assays were carried out for 20 min. When inhibitors were included, they were incubated with the phosphatase at 30 °C for 5 min. The reactions were carried out in a final volume of 300 μl. The protein phosphatase activity of Ppz1 was assayed as using the HA-tagged N-terminal domain of Reg1 as endogenous substrate. Crude yeast extracts were prepared as described (11de 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 (93) Google Scholar), and the assay was performed essentially as described in Ref. 26Garcia-Gimeno M.A. Munoz I. Arino J. Sanz P. J. Biol. Chem. 2003; 278: 47744-47752Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, except that 2 μg of GST-Ppz1(Δ1–344) was used in the assay. Other Techniques—Growth on plates (drop tests) or in liquid cultures was assessed as in Refs. 6Posas F. Camps M. Arino J. J. Biol. Chem. 1995; 270: 13036-13041Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar and 29Calero F. Gomez N. Arino J. Ramos J. J. Bacteriol. 2000; 182: 394-399Crossref PubMed Scopus (40) Google Scholar, respectively. Yeast cells were arrested in G1 with α-factor, and DNA content monitored by flow cytometry as in Ref. 8Clotet J. Gari E. Aldea M. Arino J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (70) Google Scholar. Budding index was determined by microscopic counting of at least 400 cells per point. Sporulation and tetrad dissection were done essentially as described in Ref. 19Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 147-148Google Scholar. Random spore analysis was performed as in Ref. 30Treco D.A. Winston F. Current Protocols in Molecular Biology. John Wiley & Sons, New York1998: 13.2.10-13.2.11Google Scholar. For 5-fluoro-orotic acid (5-FOA) selection, cells were grown overnight on YPD and around 1000 colony forming units plated in complete minimal medium (containing 50 μg/ml uracil) with 1 mg/ml 5-FOA. Flocculation assays were performed as described by Bony et al. (31Bony M. Barre P. Blondin B. Yeast. 1998; 14: 25-35Crossref PubMed Scopus (61) Google Scholar) with several modifications. Basically, yeast cells were inoculated in minimal medium at OD660 0.01 with or without 50 μg/ml of doxycycline and were incubated for 2 days. Then, ce" @default.
• W2092116013 created "2016-06-24" @default.
• W2092116013 creator A5000903678 @default.
• W2092116013 creator A5069205845 @default.
• W2092116013 creator A5074601374 @default.
• W2092116013 creator A5076826212 @default.
• W2092116013 creator A5079404424 @default.
• W2092116013 creator A5043776896 @default.
• W2092116013 date "2004-08-01" @default.
• W2092116013 modified "2023-09-30" @default.
• W2092116013 title "Functional Characterization of the Saccharomyces cerevisiae VHS3 Gene" @default.
• W2092116013 cites W1578409740 @default.
• W2092116013 cites W1609000300 @default.
• W2092116013 cites W1856170453 @default.
• W2092116013 cites W1865342897 @default.
• W2092116013 cites W1873737515 @default.
• W2092116013 cites W1909304621 @default.
• W2092116013 cites W1927541211 @default.
• W2092116013 cites W1942756820 @default.
• W2092116013 cites W1959499907 @default.
• W2092116013 cites W1967390330 @default.
• W2092116013 cites W1970921900 @default.
• W2092116013 cites W1970939369 @default.
• W2092116013 cites W1983482381 @default.
• W2092116013 cites W1995378150 @default.
• W2092116013 cites W1997859335 @default.
• W2092116013 cites W1998368029 @default.
• W2092116013 cites W1998911006 @default.
• W2092116013 cites W2004432410 @default.
• W2092116013 cites W2010141757 @default.
• W2092116013 cites W2014449180 @default.
• W2092116013 cites W2017474145 @default.
• W2092116013 cites W2020733599 @default.
• W2092116013 cites W2046139052 @default.
• W2092116013 cites W2050748701 @default.
• W2092116013 cites W2052228371 @default.
• W2092116013 cites W2057233307 @default.
• W2092116013 cites W2060309008 @default.
• W2092116013 cites W2060742513 @default.
• W2092116013 cites W2066628094 @default.
• W2092116013 cites W2074258205 @default.
• W2092116013 cites W2079158026 @default.
• W2092116013 cites W2086285521 @default.
• W2092116013 cites W2087535570 @default.
• W2092116013 cites W2088206337 @default.
• W2092116013 cites W2091385671 @default.
• W2092116013 cites W2094236966 @default.
• W2092116013 cites W2096258273 @default.
• W2092116013 cites W2100057937 @default.
• W2092116013 cites W2109000365 @default.
• W2092116013 cites W2117532657 @default.
• W2092116013 cites W2124902591 @default.
• W2092116013 cites W2130236675 @default.
• W2092116013 cites W2136555294 @default.
• W2092116013 cites W2137057508 @default.
• W2092116013 cites W2141066474 @default.
• W2092116013 cites W2142437744 @default.
• W2092116013 cites W2142782589 @default.
• W2092116013 cites W2153352656 @default.
• W2092116013 cites W2163996265 @default.
• W2092116013 cites W2170308357 @default.
• W2092116013 cites W2170875194 @default.
• W2092116013 cites W2171510860 @default.
• W2092116013 cites W2178458220 @default.
• W2092116013 cites W4232922293 @default.
• W2092116013 cites W4240629327 @default.
• W2092116013 doi "https://doi.org/10.1074/jbc.m400572200" @default.
• W2092116013 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15192104" @default.
• W2092116013 hasPublicationYear "2004" @default.
• W2092116013 type Work @default.
• W2092116013 sameAs 2092116013 @default.
• W2092116013 citedByCount "46" @default.
• W2092116013 countsByYear W20921160132012 @default.
• W2092116013 countsByYear W20921160132013 @default.
• W2092116013 countsByYear W20921160132014 @default.
• W2092116013 countsByYear W20921160132015 @default.
• W2092116013 countsByYear W20921160132016 @default.
• W2092116013 countsByYear W20921160132017 @default.
• W2092116013 countsByYear W20921160132018 @default.
• W2092116013 countsByYear W20921160132019 @default.
• W2092116013 countsByYear W20921160132020 @default.
• W2092116013 countsByYear W20921160132021 @default.
• W2092116013 countsByYear W20921160132022 @default.
• W2092116013 countsByYear W20921160132023 @default.
• W2092116013 crossrefType "journal-article" @default.
• W2092116013 hasAuthorship W2092116013A5000903678 @default.
• W2092116013 hasAuthorship W2092116013A5043776896 @default.
• W2092116013 hasAuthorship W2092116013A5069205845 @default.
• W2092116013 hasAuthorship W2092116013A5074601374 @default.
• W2092116013 hasAuthorship W2092116013A5076826212 @default.
• W2092116013 hasAuthorship W2092116013A5079404424 @default.
• W2092116013 hasBestOaLocation W20921160131 @default.
• W2092116013 hasConcept C104317684 @default.
• W2092116013 hasConcept C185592680 @default.
• W2092116013 hasConcept C2777576037 @default.
• W2092116013 hasConcept C54355233 @default.
• W2092116013 hasConcept C70721500 @default.
• W2092116013 hasConcept C86803240 @default.

• next