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Ribosomal Asc1p/RACK1 in the Phosphorylation Signaling Network of Saccharomyces Cerevisiae

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Ribosomal Asc1p/RACK1 in the Phosphorylation Signaling Network of Saccharomyces Cerevisiae Ribosomal Asc1p/RACK1 in the phosphorylation signaling network of Saccharomyces cerevisiae Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program “Molecular Biology of Cells” of the Georg-August University School of Science (GAUSS) submitted by Kerstin Schmitt from Göttingen Göttingen 2015 Thesis Committee Dr. Oliver Valerius Department of Molecular Microbiology and Genetics Institute of Microbiology and Genetics, Georg-August-University Göttingen Prof. Dr. Heike Krebber Department of Molecular Genetics Institute of Microbiology and Genetics, Georg-August-University Göttingen Prof. Dr. Rolf Daniel Department of Genomic and Applied Microbiology Institute of Microbiology and Genetics, Georg-August-University Göttingen Members of the Examination Board Referee: Dr. Oliver Valerius Department of Molecular Microbiology and Genetics Institute of Microbiology and Genetics, Georg-August-University Göttingen 2nd Referee: Prof. Dr. Heike Krebber Department of Molecular Genetics Institute of Microbiology and Genetics, Georg-August-University Göttingen Further members of the Examination Board Prof. Dr. Gerhard H. Braus Department of Molecular Microbiology and Genetics Institute of Microbiology and Genetics, Georg-August-University Göttingen Prof. Dr. Rolf Daniel Department of Genomic and Applied Microbiology Institute of Microbiology and Genetics, Georg-August-University Göttingen Prof. Dr. Kai Heimel Department of Molecular Microbiology and Genetics Institute of Microbiology and Genetics, Georg-August-University Göttingen Prof. Dr. Stefanie Pöggeler Department of Genetics of Eukaryotic Microorganisms Institute of Microbiology and Genetics, Georg-August-University Göttingen Date of oral examination: 17th February 2016 I hereby declare that the doctoral thesis entitled “Ribosomal Asc1p/RACK1 in the phosphorylation signaling network of Saccharomyces cerevisiae” has been written independently and with no other sources and aids than quoted. Kerstin Schmitt Parts of this work are published in: Rachfall, N., Schmitt, K., Bandau, S., Smolinski, N., Ehrenreich, A., Valerius, O., and Braus, G.H. (2013). RACK1/Asc1p, a ribosomal node in cellular signaling. Mol Cell Proteomics 12, 87-105. Schmitt, K., Smolinski, N., Neumann, P., Schmaul, S., Hofer-Pretz, V., Braus, G.H., and Valerius, O. (2017). Asc1p/RACK1 Connects Ribosomes to Eukaryotic Phosphosignaling. Mol Cell Biol 37, e00279-16 This work was supported by the Göttingen Graduate School for Neurosciences and Molecular Biosciences (DFG Grant GSC 226/2) and by the DFG Grant VA352/2-1. Acknowledgements - Danksagung Acknowledgements - Danksagung Ich möchte mich an erster Stelle bei Dr. Oliver Valerius für die hervorragende und engagierte Betreuung dieser Arbeit bedanken: Vielen Dank für die zahlreichen gemeinsamen Gespräche und Diskussionen, das entgegengebrachte Vertrauen und die Begeisterung für das Projekt. Prof. Dr. Gerhard H. Braus danke ich für die Möglichkeit meine Arbeit in der Abteilung Molekulare Mikrobiologie und Genetik anfertigen zu können und für die damit verbundene Unterstützung sowie das Interesse an meiner Arbeit. Prof. Dr. Heike Krebber und Prof. Dr. Rolf Daniel danke ich für die hilfreichen Diskussionen im Rahmen der Thesis Committee Meetings und darüber hinaus für die Kooperationen mit ihren Abteilungen. Aus der Arbeitsgruppe Molekulare Genetik danke ich Dr. Bettina Neumann für die Einführung in die Methode der Dichtegradientenzentrifugation und für viele wichtige Hinweise und Ratschläge. Dr. Andrea Thürmer, Kathleen Gollnow und Dr. Sascha Dietrich aus der Abteilung Genomische und Angewandte Mikrobiologie bin ich sehr dankbar für die gemeinsame Versuchsplanung, die Durchführung der cDNA Synthese und Sequenzierung und für die anschließende gemeinsame Datenanalyse. Ein großer Dank gilt allen ehemaligen und aktuellen Mitgliedern des Labors, insbesondere Dr. Britta Herzog, Anika Kühn, Dr. Harald Kusch, Katharina Mucek, Verena Hofer-Pretz, Dr. Nicole Rachfall, Sabrina Sander, Samantha Schmaul, und Nadine Smolinski für die großartige Unterstützung und die schöne Zeit auch außerhalb des Labors. Ein besonders großer und herzlicher Dank gilt Verena Hofer-Pretz für ihre ausgezeichnete und verlässliche Arbeit. Nadine Smolinski, Sabrina Sander, Samantha Schmaul und Katharina Mucek haben durch ihre Praktika und Abschlussarbeiten einen wesentlichen Beitrag zu dieser Arbeit geleistet, für den ich sehr dankbar bin. Nadine Smolinski danke ich für die enge Zusammenarbeit an dem Projekt auch über ihre Masterarbeit hinaus. Besonders möchte ich mich auch bei Anika Kühn bedanken, die vor allem bei methodischen Fragen immer eine ausgezeichnete Ansprechpartnerin war. Der gesamten Abteilung Molekulare Mikrobiologie und Genetik danke ich für die angenehme Arbeitsatmosphäre und hilfreiche Diskussionen. Der Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und Molekulare Biowissenschaften danke ich für die umfassende finanzielle Unterstützung dieser Arbeit. Meiner Familie und insbesondere meinen Eltern danke ich für ihre Unterstützung und das Interesse an meiner Arbeit. Table of Contents Table of Contents List of Figures ......................................................................................................................... IV List of Supplementary Figures ............................................................................................... V List of Tables ............................................................................................................................ V List of Supplementary Tables ................................................................................................ V Abstract ..................................................................................................................................... 1 1. Introduction .......................................................................................................................... 2 1.1 The WD40 protein Asc1 ...................................................................................................... 2 1.2 The Gβ-like protein Asc1 - an integral component of the ribosome .................................... 4 1.3 Asc1p orthologues within the eukaryotic domain ................................................................ 6 1.3.1 Asc1p orthologues in other fungi ............................................................................ 6 1.3.2 Plant RACK1 proteins ............................................................................................. 7 1.3.3 RACK1 in metazoan organisms .............................................................................. 8 1.3.4 ASC1/RACK1 gene expression correlates with ribosomal genes ............................ 9 1.3.5 Orthologous genes complement ASC1/RACK1-deficiency across phyla ................ 9 1.4 Described molecular function of Asc1p/RACK1 ............................................................... 10 1.4.1 Asc1p/RACK1 as a regulator of mRNA translation ............................................. 10 1.4.1.1 S. cerevisiae Asc1p in co-translational quality control and reading frame maintenance ............................................................................................. 11 1.4.1.2 Asc1p/RACK1-dependent phosphorylation of translation factors ........... 12 1.4.1.3 Asc1p/RACK1-dependent recruitment of proteins to the ribosome ......... 13 1.4.1.4 RACK1’s function in IRES-dependent translation initiation and the miRNA pathway ...................................................................................... 14 1.4.2 Asc1p/RACK1 in signal transduction pathways ................................................... 15 1.4.2.1 Role of Asc1p and its orthologues in signal transduction pathways in fungi and plants................................................................................................. 15 1.4.2.2 Metazoan RACK1 - Receptor for activated C kinase 1 ............................ 17 1.4.2.3 RACK1 links insulin-like growth factor 1 with integrin signaling........... 18 1.4.2.4 Metazoan RACK1 in cAMP/PKA signaling ............................................ 19 1.4.2.5 Function of RACK1 in MAPK cascades .................................................. 21 1.4.2.6 RACK1 as a central hub linking signal transduction with translation ...... 24 1.4.3 RACK1-dependent protein-degradation of its interaction partners ...................... 24 1.5 Regulation of Asc1p/RACK1 through post-translational modifications ........................... 26 1.6 Aim of this study ................................................................................................................ 29 I Table of Contents 2. Materials and Methods ...................................................................................................... 30 2.1 S. cerevisiae strains ............................................................................................................ 30 2.2 Bacterial strain and plasmids .............................................................................................. 33 2.3 Cultivation of microorganisms ........................................................................................... 36 2.3.1 Cultivation of S. cerevisiae ................................................................................... 36 2.3.2 Cultivation of E. coli ............................................................................................. 37 2.4 Isolation of DNA
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