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Transcriptional Profiling of the Protein Phosphatase 2C Family in Yeast Provides Insights into the Unique Functional Roles of Ptc1

2006; Elsevier BV; Volume: 281; Issue: 46 Linguagem: Inglês

10.1074/jbc.m607919200

ISSN

1083-351X

Autores

Asier González, Amparo Ruiz, Raquel Serrano, Joaquı́n Ariño, Antonio Casamayor,

Tópico(s)

Genetics, Aging, and Longevity in Model Organisms

Resumo

Type 2C protein phosphatases are encoded in Saccharomyces cerevisiae by several related genes (PTC1-5 and PTC7). To gain insight into the functions attributable to specific members of this gene family, we have investigated the transcriptional profiles of ptc1-5 mutants. Two main patterns were obtained as follows: the one generated by the ptc1 mutation and the one resulting from the lack of Ptc2-5. ptc4 and ptc5 profiles were quite similar, whereas that of ptc2 was less related to this group. Mutation of PTC1 resulted in increased expression of numerous genes that are also induced by cell wall damage, such as YKL161c, SED1, or CRH1, as well as in higher amounts of active Slt2 mitogen-activated protein kinase, indicating that lack of the phosphatase activates the cell wall integrity pathway. ptc1 cells were even more sensitive than slt2 mutants to a number of cell wall-damaging agents, and both mutations had additive effects. The sensitivity of ptc1 cells was not dependent on Hog1. Besides these phenotypes, we observed that calcineurin was hyperactivated in ptc1 cells, which were also highly sensitive to calcium ions, heavy metals, and alkaline pH, and exhibited a random haploid budding pattern. Remarkably, many of these traits are found in certain mutants with impaired vacuolar function. As ptc1 cells also display fragmented vacuoles, we hypothesized that lack of Ptc1 would primarily cause vacuolar malfunction, from which other phenotypes would derive. In agreement with this scenario, overexpression of VPS73, a gene of unknown function involved in vacuolar protein sorting, largely rescues not only vacuolar fragmentation but also sensitivity to cell wall damage, high calcium, alkaline pH, as well as other ptc1-specific phenotypes. Type 2C protein phosphatases are encoded in Saccharomyces cerevisiae by several related genes (PTC1-5 and PTC7). To gain insight into the functions attributable to specific members of this gene family, we have investigated the transcriptional profiles of ptc1-5 mutants. Two main patterns were obtained as follows: the one generated by the ptc1 mutation and the one resulting from the lack of Ptc2-5. ptc4 and ptc5 profiles were quite similar, whereas that of ptc2 was less related to this group. Mutation of PTC1 resulted in increased expression of numerous genes that are also induced by cell wall damage, such as YKL161c, SED1, or CRH1, as well as in higher amounts of active Slt2 mitogen-activated protein kinase, indicating that lack of the phosphatase activates the cell wall integrity pathway. ptc1 cells were even more sensitive than slt2 mutants to a number of cell wall-damaging agents, and both mutations had additive effects. The sensitivity of ptc1 cells was not dependent on Hog1. Besides these phenotypes, we observed that calcineurin was hyperactivated in ptc1 cells, which were also highly sensitive to calcium ions, heavy metals, and alkaline pH, and exhibited a random haploid budding pattern. Remarkably, many of these traits are found in certain mutants with impaired vacuolar function. As ptc1 cells also display fragmented vacuoles, we hypothesized that lack of Ptc1 would primarily cause vacuolar malfunction, from which other phenotypes would derive. In agreement with this scenario, overexpression of VPS73, a gene of unknown function involved in vacuolar protein sorting, largely rescues not only vacuolar fragmentation but also sensitivity to cell wall damage, high calcium, alkaline pH, as well as other ptc1-specific phenotypes. Ser/Thr protein phosphatases have been classically classified in four groups as follows: PP1, PP2A, PP2B, and PP2C. PP1, PP2A, and PP2B catalytic subunits are closely related in their primary sequence and define the PPP family. Type 2C phosphatases, which constitute the PPM family, are not related in sequence with PPP members, although their three-dimensional structures and catalytic mechanism appear to be very similar (1Cohen P.T. Arino J. Alexander D.R. Protein Phosphatases. Springer-Verlag, Heidelberg2004: 1-20Google Scholar). Protein phosphatase 2C represents an evolutionary conserved group of proteins that, in contrast with most members of the PPP family, are monomeric enzymes that apparently lack regulatory subunits. Five type 2C phosphatase genes (PTC1-5) have been classically defined in the budding yeast Saccharomyces cerevisiae (2Stark M.J. Yeast. 1996; 12: 1647-1675Crossref PubMed Scopus (170) Google Scholar), although a sixth member (YHR076w/PTC7) was recently added to the list (3Jiang L. Whiteway M. Ramos C. Rodriguez-Medina J.R. Shen S.H. FEBS Lett. 2002; 527: 323-325Crossref PubMed Scopus (29) Google Scholar). A putative seventh member (YCR079w) was reported some time ago, although its phosphatase activity in vitro has not been demonstrated (4Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2946-2957Crossref PubMed Scopus (127) Google Scholar). Although the first reports on the characterization and biological role of type 2C phosphatases in yeast appeared more than 15 years ago, our knowledge on the specific functions of each isoform and how they are regulated is still very limited. It is commonly accepted that a major role for type 2C phosphatases in yeast is to negatively regulate the osmotically activated HOG pathway by dephosphorylating and inactivating the Hog1 MAP 7The abbreviations used are: MAP, mitogen-activated protein; CFW, Calcofluor white; CWI, cell wall integrity; MAPK, MAP kinase. 7The abbreviations used are: MAP, mitogen-activated protein; CFW, Calcofluor white; CWI, cell wall integrity; MAPK, MAP kinase. kinase (5Warmka J. Hanneman J. Lee J. Amin D. Ota I. Mol. Cell. Biol. 2001; 21: 51-60Crossref PubMed Scopus (146) Google Scholar, 6Maeda T. Tsai A.Y. Saito H. Mol. Cell. Biol. 1993; 13: 5408-5417Crossref PubMed Scopus (150) Google Scholar, 7Young C. Mapes J. Hanneman J. Al Zarban S. Ota I. Eukaryot. Cell. 2002; 1: 1032-1040Crossref PubMed Scopus (61) Google Scholar, 8Saito H. Tatebayashi K. J. Biochem. (Tokyo). 2004; 136: 267-272Crossref PubMed Scopus (181) Google Scholar, 9Martin H. Flandez M. Nombela C. Molina M. Mol. Microbiol. 2005; 58: 6-16Crossref PubMed Scopus (119) Google Scholar). Most PP2C isoforms have been associated with this function, although with slightly different roles. Thus, it has been proposed that although Ptc1 would play a role in maintaining low levels of Hog1 activity under basal conditions and adaptation to osmotic stress (5Warmka J. Hanneman J. Lee J. Amin D. Ota I. Mol. Cell. Biol. 2001; 21: 51-60Crossref PubMed Scopus (146) Google Scholar), Ptc2 and Ptc3 would be necessary to limit an excessive activation of the kinase during stress (7Young C. Mapes J. Hanneman J. Al Zarban S. Ota I. Eukaryot. Cell. 2002; 1: 1032-1040Crossref PubMed Scopus (61) Google Scholar). Ptc1 would be recruited to the scaffold upstream Hog1 kinase, Pbs2, through its interaction with Nbp2 (10Mapes J. Ota I.M. EMBO J. 2004; 23: 302-311Crossref PubMed Scopus (62) Google Scholar). In addition to Ptc1-3, a role for Ptc4 in dephosphorylating Hog1 has been proposed recently (11Shitamukai A. Hirata D. Sonobe S. Miyakawa T. J. Biol. Chem. 2004; 279: 3651-3661Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Besides its regulatory role in the HOG pathway, diverse type 2C phosphatase isoforms have been related to a variety of specific functions. Thus, Ptc1 has been involved in the regulation of tRNA splicing (12Robinson M.K. van Zyl W.H. Phizicky E.M. Broach J.R. Mol. Cell. Biol. 1994; 14: 3634-3645Crossref PubMed Scopus (53) Google Scholar) and in mitochondrial inheritance (13Roeder A.D. Hermann G.J. Keegan B.R. Thatcher S.A. Shaw J.M. Mol. Biol. Cell. 1998; 9: 917-930Crossref PubMed Scopus (48) Google Scholar). Ptc2 and Ptc3 have been postulated as responsible for the dephosphorylation of cyclin-dependent kinases (4Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2946-2957Crossref PubMed Scopus (127) Google Scholar) and to be required for checkpoint inactivation after a DNA double strand break, which would confer to these specific isoforms an important role in regulating DNA checkpoint pathways (14Leroy C. Lee S.E. Vaze M.B. Ochsenbien F. Guerois R. Haber J.E. Marsolier-Kergoat M.C. Mol. Cell. 2003; 11: 827-835Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 15Marsolier M.C. Roussel P. Leroy C. Mann C. Genetics. 2000; 154: 1523-1532Crossref PubMed Google Scholar). Ptc2 has been proposed to negatively regulate the unfolded protein response through dephosphorylation of the Ire1 protein kinase (16Welihinda A.A. Tirasophon W. Green S.R. Kaufman R.J. Mol. Cell. Biol. 1998; 18: 1967-1977Crossref PubMed Scopus (97) Google Scholar). Overexpression of PTC2 and PTC3 (but not PTC1) is able to rescue the synthetically lethal phenotype of sit4 hal3 mutants, whereas overexpression of all three genes rescues the growth defect of an slt2/mpk1 MAP kinase mutant strain at 37 °C (17Munoz I. Simon E. Casals N. Clotet J. Arino J. Yeast. 2003; 20: 157-169Crossref PubMed Scopus (39) Google Scholar). Interestingly, the ptc1 mutation was found to be synthetically lethal with that of slt2 (18Huang K.N. Symington L.S. Genetics. 1995; 141: 1275-1285Crossref PubMed Google Scholar), whereas overexpression of PTC1-4 suppressed the lethality of a cnb1 slt2 strain (11Shitamukai A. Hirata D. Sonobe S. Miyakawa T. J. Biol. Chem. 2004; 279: 3651-3661Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Recent work in our laboratory has demonstrated that mutation of PTC1 (but not that of PTC2-5) confers sensitivity to lithium cations to yeast cells (19Ruiz A. Gonzalez A. García-Salcedo R. Ramos J. Arino J. Mol. Microbiol. 2006; 62: 263-277Crossref PubMed Scopus (40) Google Scholar). Therefore, the current evidence defines a scenario in which type 2C phosphatases control a large number of processes in yeast cells, probably through a complex interplay of functions that in some cases could be rather specific but in many other cases appear largely overlapping. The available information, however, is rather fragmentary and does not provide a comprehensive understanding of the biological role of these important enzymes. We considered that a broader and more systematic overview could be obtained by comparative analysis of the transcriptomic profiles from cells deficient in each of these phosphatases. We have observed that cells lacking Ptc1 present a distinct and very specific expression pattern, reminiscent to that of cells suffering some kind of cell wall damage. Further characterization of ptc1 mutants revealed multiple, apparently unrelated phenotypic defects, suggesting a large variety of cellular functions. However, our results allow proposing a simple model that would explain most of the functions attributed to Ptc1. Yeast Strains and Culture Conditions—Yeast strain BY4741 was used as a wild type, and unless otherwise stated, the deletion mutants studied (Table 1) were in this same genetic background.TABLE 1Yeast strains used in this workNameRelevant genotypeSource/Ref.BY4741MATa his3Δ1 leu2Δ met15Δ ura3Δ69Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarMAR143BY4741 ptc1::nat119Ruiz A. Gonzalez A. García-Salcedo R. Ramos J. Arino J. Mol. Microbiol. 2006; 62: 263-277Crossref PubMed Scopus (40) Google ScholarBY4741 ptc2::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarBY4741 ptc3::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarBY4741 ptc4::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarBY4741 ptc5::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarBY4741 slt2::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarMAR154BY4741 slt2::kanMX4 ptc1::nat1This workBY4741 wsc1::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarMAR210BY4741 wsc1::kanMX4 ptc1::nat1This workBY4741 mid2::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarMAR211BY4741 mid2::kanMX4 ptc1::nat1This workBY4741 rom2::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarMAR212BY4741 rom2::kanMX4 ptc1::nat1This workBY4741 bck1::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarMAR213BY4741 bck1::kanMX4 ptc1::nat1This workBY4741 rlm1::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarMAR214BY4741 rlm1::kanMX4 ptc1::nat1This workBY4741 ykl161c::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3169) Google ScholarAGS32BY4741 ykl161c::kanMX4 ptc1::nat1This workBY4741 hog1::kanMX469Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. 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Sci. U. S. A. 1998; 95: 7357-7362Crossref PubMed Scopus (92) Google ScholarMAR161JA100 ptc1::nat1This workJC10JA100 slt2::LEU271Vissi 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 ScholarMAR215JA100 slt2::LEU2 ptc1::nat1This workaThis strain was only viable in the presence of 1 m sorbitola This strain was only viable in the presence of 1 m sorbitol Open table in a new tab Preparation of Yeast Extracts and Immunoblot Analysis— For immunodetection of the MAP kinase Slt2, saturated cell cultures of the indicated strains in YPD medium were diluted to an A660 of 0.2 in fresh YPD and grown until A660 of 1.3-1.4. Cell cultures (5 ml) were harvested by filtration, washed once with pre-chilled water, snap-frozen, and kept at -80 °C. Total cell proteins were purified basically as described previously (20Martin H. Rodriguez-Pachon J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Briefly, cell pellets were resuspended in 150 μl of lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, and 0.1% SDS) containing phosphatase and kinase inhibitors (50 mm NaF, 1 mm sodium orthovanadate, 50 mm β-glycerol phosphate, 5 mm sodium pyrophosphate, 0.5 mm EDTA, pH 8.0), 1 mm phenylmethylsulfonyl fluoride, and a protease inhibitor mixture (Complete EDTA-free protease inhibitor mixture tablets; Roche Applied Science). One volume of acid-washed glass beads was added, and cells were broken at 4 °C by vigorous shaking in a Fast Prep cell breaker (Bio 101, Inc., Vista, CA; setting 5.5 for 25 s). After sedimentation at 16,000 × g, the cleared lysate was recovered and the protein concentration quantified by Bradford assay. Forty μg of total protein were fractionated by SDS-PAGE (using 10% polyacrylamide gels) and transferred to nitrocellulose membranes (Hybond C-Extra; Amersham Biosciences). Membranes were incubated for 2 h with either anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (New England Biolabs), at 1:2000 dilution, or anti-GST-Slt2 antibody (21Martin H. Arroyo J. Sanchez M. Molina M. Nombela C. Mol. Gen. Genet. 1993; 241: 177-184Crossref PubMed Scopus (114) Google Scholar), at 1:10,000 dilution, to detect dually phosphorylated Slt2 or total Slt2, respectively. A 1:25,000 dilution of horseradish peroxidase-conjugated anti-rabbit antibody was used to detect the primary antibodies. ECL Advance Western blotting detection kit (Amersham Biosciences) was used to visualize the immunocomplexes. Chemiluminescence was detected using an LAS-3000 equipment (Fuji) and quantified using the Multi Gauge version 3.0 software. β-Galactosidase Reporter Assays—Wild type strain BY4741 and its isogenic mutants (22Giaever 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 (3222) Google Scholar) were co-transformed with the diverse β-galactosidase reporter constructs. Cells were grown to saturation on synthetic medium lacking uracil and then inoculated into YPD medium to give an A660 of 0.15. Growth was resumed until A660 of 0.8 was reached, and cells were then recovered by centrifugation, and β-galactosidase was measured as described previously (23Ruiz A. Yenush L. Arino J. Eukaryot. Cell. 2003; 2: 937-948Crossref PubMed Scopus (57) Google Scholar). RNA Purification—For RNA purification, 30 ml of yeast cultures were grown at 28 °C in YPD medium until A660 0.6-0.8. Yeast cells were harvested by centrifugation and washed with cold water. Dried cell pellets were kept at -80 °C until RNA purification. Total RNA was extracted using the RiboPure-Yeast kit (Ambion) following the manufacturer's instructions. RNA quality was assessed by electrophoresis in denaturing 0.8% agarose gel and quantified by measuring A260 in a BioPhotometer (Eppendorf). cDNA Synthesis and DNA Microarray Experiments—Transcriptional analyses were performed using DNA microarrays containing PCR-amplified fragments from 6014 S. cerevisiae open reading frames (24Viladevall L. Serrano R. Ruiz A. Domenech G. Giraldo J. Barcelo A. Arino J. J. Biol. Chem. 2004; 279: 43614-43624Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 25Alberola T.M. Garcia-Martinez J. Antunez O. Viladevall L. Barcelo A. Arino J. Perez-Ortin J.E. Int. Microbiol. 2004; 7: 199-206PubMed Google Scholar). Amplified DNA was resuspended in 50% dimethyl sulfoxide and arrayed onto aminosilane-coated glass slides (UltraGAPS™; Corning Glass) using a MicroGrid II spotter (BioRobotics). Fluorescent Cy3- and Cy5-labeled cDNA was prepared from 8 μg of purified total RNA by the indirect dUTP-labeling method, using the CyScribe post-labeling kit (Amersham Biosciences). DNA fragments from 6014 open reading frames were PCR-amplified from yeast genomic DNA (25Alberola T.M. Garcia-Martinez J. Antunez O. Viladevall L. Barcelo A. Arino J. Perez-Ortin J.E. Int. Microbiol. 2004; 7: 199-206PubMed Google Scholar). Pre-hybridization, hybridization, and washes were carried out as recommended by The Institute for Genomic Research with minor modifications. Briefly, prehybridizations of the DNA microarrays were carried out at 42 °C for 1 h in a solution containing 5× SSC, 0.1% SDS, 1% bovine serum albumin. For hybridization, dried Cy3- and Cy5-labeled probes were resuspended in 35 μl of hybridization solution (50% formamide, 5× SSC, 0.1% SDS) each and mixed. Five μg of salmon sperm DNA was added to the mix before denaturation for 3 min at 95 °C. DNA microarrays were hybridized in an ArrayBooster hybridization station (Sunergia Group) for 14 h at 42 °C. For each experimental condition (mutant versus wild type strain) a dye swapping was performed. The scanner ScanArray 4000 (Packard Instrument Co.) was used to obtain the Cy3 and Cy5 images with a resolution of 10 μm. The fluorescent intensity of the spots was measured and processed using the GenePix Pro 6.0 software (Molecular Devices). Spots with either a diameter smaller than 120 μm or a fluorescence intensity for Cy3 and Cy5 lower than 150 units were not considered for further analysis. A given gene was considered to be induced or repressed when the ratio ptc mutant versus wt was higher than 1.80 or lower than 0.50, respectively. Genes whose expression was considered changed in any of the tested mutants were selected for further analyses. The GEPAS server was used to preprocess the data and to establish correlations between expression patterns (26Herrero J. Al Shahrour F. Diaz-Uriarte R. Mateos A. Vaquerizas J.M. Santoyo J. Dopazo J. Nucleic Acids Res. 2003; 31: 3461-3467Crossref PubMed Scopus (159) Google Scholar). Expression profile analysis of the selected genes was determined with EPCluster (27Brazma A. Vilo J. FEBS Lett. 2000; 480: 17-24Crossref PubMed Scopus (441) Google Scholar). Other Techniques—Sensitivity of the different yeast strains to alkaline pH, high temperature, or to different compounds or cations was assayed by drop test on YPD plates as described previously (28Serrano R. Bernal D. Simon E. Arino J. J. Biol. Chem. 2004; 279: 19698-19704Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). When needed, 1 m sorbitol was added to the medium prior to sterilization by autoclaving. Growth in liquid medium was performed in 96-well plates. Two hundred fifty-μl cultures at initial A660 of 0.01 were grown at 28 °C in YPD in the presence of the specified conditions for 12-14 h. Growth was monitored in an iEMS Reader MF (Labsystems) at 620 nm. Vacuole morphology was assessed as described previously (29Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Crossref PubMed Scopus (1132) Google Scholar). Ten ml of yeast cultures at A660 of 1.0 were harvested, washed, and resuspended in 0.5 ml of fresh YPD. The fluorescent dye FM4-64 (Molecular Probes) was added at a final concentration of 20 μm, and cells were incubated for 15 min at 30 °C. The cells were then washed, resuspended in 3 ml of YPD, and incubated for 30-60 min to allow the internalization by endocytosis and accumulation of the dye within the vacuole. Identification of the carboxypeptidase Y processed forms was achieved by immunodetection as follows. Extracts were prepared from 10 ml of yeast cultures (A660 of 1.0) in TEPI buffer (50 mm Tris, pH 7.5, 5 mm EDTA, 0.5% SDS, plus protease inhibitors). Two hundred μl of extracts were incubated for 5 min at 95 °C. Then 800 μl of TNET buffer (30 mm Tris, pH 7.5, 120 mm NaCl, 5 mm EDTA, 1% Triton X-100) were added, mixed, and then centrifuged for 10 min at 16000 × g. Supernatants were resolved by 10% SDS-PAGE before detection of carboxypeptidase Y by immunoblot. Staining of bud scars for determination of budding pattern was performed in exponential cultures that were grown at either 30 or 37 °C, for 6-8 h. Cells were fixed with 3.7% formaldehyde for 1 h, washed with phosphate-buffered saline, and stained with 0.02 mg/ml CFW (Fluorescent Brightener F-6259; Sigma). Expression Profile Analysis of PP2C Mutant Yeast Strains—In this work we have focused on the study of Ptc1-5 phosphatases. YCR079w has not been included because the encoded protein failed to show any phosphatase activity (4Cheng A. Ross K.E. Kaldis P. Solomon M.J. Genes Dev. 1999; 13: 2946-2957Crossref PubMed Scopus (127) Google Scholar), and Ptc7 (YHR076w) was not considered because of its relatively remote similarity with other members of this family. Ptc1-5, as shown in Fig. 1A, share a common catalytic domain that has specific amino-terminal or carboxyl-terminal extensions (in Ptc5 and Ptc2/Ptc3, respectively). Amino acid sequence alignment of these proteins shows that Ptc2 and Ptc3 have the highest degree of identity, whereas Ptc5 is only distantly related (Fig. 1B). To identify novel and possibly specific functions of the members of this family, we decided to analyze the alterations provoked in the expression pattern by the absence of every single PP2C gene. To this end, we compared the expression profile of the mutant strains with that obtained from wild type cells, and we considered a given gene to be induced when its expression was at least 1.8-fold higher in a mutant strain than in the wild type strain. A gene was catalogued as repressed when its expression level decreased by at least 0.5-fold for a given mutant. A functional classification of the PTC family was obtained by clustering the different expression profiles obtained for each mutant strain. Fig. 1B shows the dendrogram generated from the DNA microarray experiments. It is remarkable that the classifications obtained by sequence analysis and by expression profiling were substantially different. For instance, the structural similarity between Ptc2 and Ptc3 did not translate in a similar transcriptional response, as the expression profile obtained from the ptc3 mutant was more related to the profile from the ptc4 and ptc5 strains than to the one from ptc2. Similarly, according to their amino acid sequence, it could be predicted that the ptc5 profile should be relatively unrelated from the rest of the ptc mutants. Lack of PTC5, however, induces a series of transcriptional modifications very similar to the ones provoked by the ptc4 deletion. Surprisingly, the expression profile of cells lacking Ptc1, a protein structurally related to Ptc2 and Ptc3, was largely different from any of the mutants studied here. In fact, when the correlation coefficient (r) was calculated for each pairwise comparison of the transcriptional profiles, the correlation between ptc1 and the rest of mutants was near zero in all cases (Table 2).TABLE 2Correlation coefficients (r) obtained after pairwise comparison of the changes in the expression patterns induced by the lack of the indicated PTC genes, using the PlotCorr Server at GEPAS (26Herrero J. Al Shahrour F. Diaz-Uriarte R. Mateos A. Vaquerizas J.M. Santoyo J. Dopazo J. Nucleic Acids Res. 2003; 31: 3461-3467Crossref PubMed Scopus

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