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Essential Regulatory Subunits of the Protein Phosphatase Glc7 in the Yeast Saccharomyces Cerevisiae

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Essential Regulatory Subunits of the Protein Phosphatase Glc7 in the Yeast Saccharomyces Cerevisiae Doctoral Thesis Study of the biological functions of the non- essential regulatory subunits of the protein phosphatase Glc7 in the yeast Saccharomyces cerevisiae Jofre Ferrer-Dalmau Falgueras Dept. Bioquímica i Biologia Molecular Universitat Autònoma de Barcelona November 2013 Study of the biological functions of the non- essential regulatory subunits of the protein phosphatase Glc7 in the yeast Saccharomyces cerevisiae Thesis disertation for the degree of Doctor in Biochemistry, Molecular Biology and Biomedicine, presented by Jofre Ferrer-Dalmau Falgueras. This work was performed at the Veterinary Unit of the Department of Biochemistry and Molecular Biology of the ‘Universitat Autònoma de Barcelona’ under the direction of Dr. Antonio Casamayor Gracia. Jofre Ferrer-Dalmau Falgueras Antonio Casamayor Gracia November 2013 Universitat Autònoma de Barcelona Departament de Bioquímica i Biologia Molecular Facultat de Veterinària Amb aquestes línies vull agrair a totes les persones, sense les quals, aquest treball no s’hagués pogut realitzar. També, vull anticipar-me a les queixes disculpant-me per si en l’anhel d’en recordar-me de tothom, m’he deixat alguna persona sense nombrar. Primer de tot, agrair molt especialment al Dr. Antonio Casamayor Gracia no només per acollir- me en el seu laboratori i donar-me l’oportunitat d’aprendre sobre la ciència del llevat sinó també per transmetrem part dels seus coneixements i passió per la ciència. També, als membres del grup que han anat passant com en Martí Boleda, el Dr. Daniel Pastor (demostrant-me que amb tofu es pot sobreviure !) i la Dra. Anna Bahí (sort que hi era per mantenir-me a ratlla i l’ordre en el caos que organitzava a la seva poiata quan la intentava envair!). Agrair molt cordialment al Dr. Joaquín Ariño pel suport logístic del seu laboratori i poder-me beneficiar dels avantatges del seu grup. Avantatges no només d’equipament i material, sinó també dels membres veterans del grup sempre ajudant-me en tot moment. El Dr. Asier Gonzàlez per ser el meu “jefe” durant una setmana fent tot l’estudi fenotípic (mai oblidaré aquells dots!). La Dra. Maria Platara que em va ensenyar la tècnica més nostrada del laboratori: el β-gal !. El Dr. Carlos Casado per creure en l’aneuploïdia des d’un principi. La Dra. Amparo Ruiz per ensenyar-me que és possible fer β-gals de més de 300 tubs i pels plàsmidis Snf1 enviats des de NYC. I, la Dra. Raquel Serrano per ensenyar-me a manejar l’Antonio. També, a la Dra. Lina Barreto i en David Canadell pels creixements en baix potassi. No em vull oblidar dels altres membres del grup com l’Albert, la Laura, la Sylvia, en Boris i la Cristina. Tota aquesta tesis no hagués estat possible sense el treball en la preparació de medis, plaques, comandes, o sigui el dia a dia en el laboratori de l’Anna Vilalta i la Montserrat Robledo. A més, de la Rosa i la Lourdes per tots els tràmits burocràtics que m’han fet. També al laboratori de la Dra. Anna Bassols, la Laia, l’Anna, en Dani, la Maria José i la Laura per fer que bioquímica de veterinària estigués plena de vida. En Nèstor pel seu material gràfic i en Gerard. També, els consells en els microarrays i la seqüenciació de mostres de la Dr. Anna Barceló, en Xavi i en Roger Lahoz del Servei de Seqüenciació de la UAB. En anglès, I thank H. Sychrovà and G. Wiesenberger for help in developing and testing the YNB- based, K-free medium. We also thank C. Moore, B. Dichtl, K. Tatchell, A. Amon, K. Kimata and M. Marquina for provision of strains and plasmids provided for cation homeostasis, cell cycle and unfolded protein response experiments. Agradecer a Clara Navarrete i José L. Martínez del laboratorio del Dr. José Ramos de la Universidad de Córdoba por los experimentos de determinación del contenido y flujos de litio. A los Dra. M. Francisca Randez Gil i Dr. José Antonio Prieto Alamán l (IATA, València) no sólo por los experimentos que nos han hecho sobre UPR, sino también por toda la asistencia en el diseño, planificación y conocimientos sobre la UPR. Gracias a ellos, hemos indagado en un tema totalmente desconocido en nuestro laboratorio que nosotros solos no nos hubiésemos atrevido a investigar. A la Dra. Bàrbara Baro del laboratori de la Dra. Ethel Queralt (IDIBELL) per ajudar-nos en els experiments de FACS en els mutants ref2. No em vull oblidar dels membres de la meva comissió de seguiment: el Dr. David Garcia Quintana i el Dr. Josep Biosca, que va ser la primera persona que em va introduir en el món dels llevats aguantant-me durant tot un any. Agrair a la família Alonso per les reunions familiars que em permetien desconnectar dels llevats. Els meus pares per sempre transmetre’m tot el suport per fer la tesi i sempre està al meu costat quan se’ls necessitava. I, finalment, a la Carol.....en mots no puc explicar tot el que significa per mi. Per sort, sempre a estat i estarà al meu costat.... Table of contents Table of contents ABBREVIATIONS .......................................................................................................... 5 SUMMARY ..................................................................................................................... 7 INTRODUCTION............................................................................................................ 9 1. Saccharomyces cerevisiae as a model for the investigation ............................................ 9 2. The regulation by protein phosphorylation ................................................................... 9 2.1 General description of phosphorylation properties ..................................................... 9 2.2 Properties of Ser / Thr phosphatase ........................................................................... 10 2.2.1 PhosphoProtein Phosphatases (PPPs) ................................................................. 11 2.2.2 Metal-dependent Protein Phosphatase (PPMs) ................................................. 11 2.2.3 Aspartate-based phosphatases: FCP/SCP ........................................................... 12 2.3 Protein Phosphatase-1 (PP1) ....................................................................................... 12 2.3.1 Mechanisms of interaction for the PP1c regulatory subunit. Binding motifs ..... 14 2.3.2 Glc7, the catalytic subunit of yeast PP1 .............................................................. 16 2.3.3 The Glc7 regulatory subunit Ref2 ........................................................................ 20 2.3.4 The Glc7 regulatory subunit Reg1 ....................................................................... 22 3. The stress response in S. cerevisiae ............................................................................. 24 3.1 Ionic homeostasis ........................................................................................................ 25 3.1.1 The ATPases Ena ......................................................................................................... 26 3.2 Calcium homeostasis ................................................................................................... 28 3.3 The Cell Wall Integrity signaling pathway ................................................................... 31 3.4 Efflux pump-mediated drug resistance as a stress response ...................................... 35 3.5 Unfolded protein response ......................................................................................... 38 4. Aneuploidy ................................................................................................................ 43 OBJECTIVES ................................................................................................................. 46 EXPERIMENTAL PROCEDURES ............................................................................... 47 1. Growth of Escherichia coli and yeast strains ................................................................ 47 2. DNA recombinant techniques ..................................................................................... 47 3. Deletion cassettes, gene disruptions ........................................................................... 47 4. Diploid generation and obtaining of haploid cells ........................................................ 55 5. Plasmids construction ................................................................................................ 56 6. Growth test ................................................................................................................ 60 1 Table of contents 7. Total RNA Extraction .................................................................................................. 61 8. cDNA synthesis and DNA microarrays ......................................................................... 61 9. Yeast DNA genomic extraction .................................................................................... 62 10. Comparative Genomic Hybridization ........................................................................... 62 11. Quantitative Real-Time PCR (qRT-PCR) ........................................................................ 63 12. Semi-quantitative RT-PCR ........................................................................................... 65 13. Northern Blot Analysis ............................................................................................... 65 14. Preparation of cell lysates, Immunoprecipitation, and Western blotting ...................... 65 15. Glucose measurements .............................................................................................
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