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S. Cerevisiae DOCTOR OF PHILOSOPHY Saccharomyces Cerevisiae as a biotechnological tool for ageing research studies on translation and metabolism Stephanie Cartwright 2013 Aston University Some pages of this thesis may have been removed for copyright restrictions. If you have discovered material in AURA which is unlawful e.g. breaches copyright, (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please read our Takedown Policy and contact the service immediately SACCHAROMYCES CEREVISIAE AS A BIOTECHNOLOGICAL TOOL FOR AGEING RESEARCH: STUDIES ON TRANSLATION AND METABOLISM STEPHANIE PATRICIA CARTWRIGHT Doctor of Philosophy ASTON UNIVERSITY July 2013 ©Stephanie Patricia Cartwright, 2013 Stephanie Patricia Cartwright asserts her moral right to be identified as the author of this thesis. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without proper acknowledgment. 1 ASTON UNIVERSITY SACCHAROMYCES CEREVISIAE AS A BIOTECHNOLOGICAL TOOL FOR AGEING RESEARCH: STUDIES ON TRANSLATION AND METABOLISM Stephanie Patricia Cartwright PhD 2013 Thesis summary The yeast Saccharomyces cerevisiae is an important model organism for the study of cell biology. The similarity between yeast and human genes and the conservation of fundamental pathways means it can be used to investigate characteristics of healthy and diseased cells throughout the lifespan. Yeast is an equally important biotechnological tool that has long been the organism of choice for the production of alcoholic beverages, bread and a large variety of industrial products. For example, yeast is used to manufacture biofuels, lubricants, detergents, industrial enzymes, food additives and pharmaceuticals such as anti-parasitics, anti-cancer compounds, hormones (including insulin), vaccines and nutraceuticals. Its function as a cell factory is possible because of the speed with which it can be grown to high cell yields, the knowledge that it is generally recognized as safe (GRAS) and the ease with which metabolism and cellular pathways, such as translation can be manipulated. In this thesis, these two pathways are explored in the context of their biotechnological application to ageing research: (i) understanding translational processes during the high-yielding production of membrane protein drug targets and (ii) the manipulation of yeast metabolism to study the molecule, L-carnosine, which has been proposed to have anti-ageing properties. In the first of these themes, the yeast strains, spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1, were examined since they have been previously demonstrated to dramatically increase the yields of a target membrane protein (the aquaporin, Fps1) compared to wild-type cells. The mechanisms underlying this discovery were therefore investigated. All high yielding strains were shown to have an altered translational state (mostly characterised by an initiation block) and constitutive phosphorylation of the translational initiation factor, eIF2α. The relevance of the initiation block was further supported by the finding that other strains, with known initiation blocks, are also high yielding for Fps1. A correlation in all strains between increased Fps1 yields and increased production of the transcriptional activator protein, Gcn4, suggested that yields are subject to translational control. Analysis of the 5ˊ untranslated region (UTR) of FPS1 revealed two upstream open reading frames (uORFs). Mutagenesis data suggest that high yielding strains may circumvent these control elements through either a leaky scanning or a re- initiation mechanism. In the second theme, the dipeptide L-carnosine (β-alanyl-L-histidine) was investigated: it has previously been shown to inhibit the growth of cancer cells but delay senescence in cultured human fibroblasts and extend the lifespan of male fruit flies. To understand these apparently contradictory properties, the effects of L-carnosine on yeast were studied. S. cerevisiae can respire aerobically when grown on a non-fermentable carbon source as a substrate but has a respiro-fermentative metabolism when grown on a fermentable carbon source; these metabolisms mimic normal cell and cancerous cell metabolisms, respectively. When yeast were grown on fermentable carbon sources, in the presence of L-carnosine, a reduction in cell growth and viability was observed, which was not apparent for cells grown on a non-fermentable carbon source. The metabolism-dependent mechanism was confirmed in the respiratory yeast species Pichia pastoris. Further analysis of S. cerevisiae yeast strains with deletions in their nutrient-sensing pathway, which result in an increase in respiratory metabolism, confirmed the metabolism-dependent effects of L-carnosine. Key words: yeast; Fps1; L-carnosine; translation initiation; metabolism 2 For my Mother, Father and Brother 3 Acknowledgments I would like to thank Professor Roslyn Bill for her support, time and guidance throughout this project. I would also like to thank Dr Stephane Gross, Dr Alan Hipkiss, Dr Cliff Bailey and Dr Nicklas Bonander for their helpful discussions. I thank Dr Richard Darby, Dr Sarah Routledge, and Zharain Bawa for their help learning laboratory skills and Michelle Clare for her help. I would also like to thank Marvin Dilworth and Debasmita Sarka for their help with molecular biology techniques. I also thank Clare Bromley for her assistance with this project. I would also like to thank BBSRC for their funding for this project. 4 Contents Page Abbreviations ...................................................................................................................................10 List of figures ...................................................................................................................................14 List of tables .....................................................................................................................................17 Chapter 1: Introduction .................................................................................................................18 1.1. Saccharomyces cerevisiae ......................................................................................................18 1.1.1. S. cerevisiae growth characteristics ..................................................................................19 1.1.2. Cultivation of S. cerevisiae ..............................................................................................20 1.2. Saccharomyces cerevisiae as a model organism ......................................................................20 1.2.1. The metabolism of S. cerevisiae .......................................................................................21 1.2.2. S. cerevisiae: a model for cancer cells ..............................................................................24 1.2.3. L-Carnosine: a dipeptide with opposing effects on cancer and somatic cells .....................26 1.2.3.1. Biosynthesis and cellular localisation of L-carnosine ................................................26 1.2.3.2. Cellular L-carnosine concentrations throughout the lifespan .....................................30 1.2.3.3. The anti-senescent properties of L-carnosine ............................................................30 1.2.3.4. The anti-cancer properties of L-carnosine .................................................................32 1.3. S. cerevisiae as a biotechnological tool for recombinant protein production ............................33 1.3.1. S. cerevisiae as a cell factory: comparison with other commonly used hosts .....................34 1.3.2. Membrane proteins as recombinant targets .......................................................................36 1.3.3. The challenge of producing recombinant membrane proteins ...........................................36 1.3.4. The production of the recombinant aquaporin, Fps1 .........................................................38 1.3.4.1. Function and regulation of Fps1 ...............................................................................38 1.3.4.2. The production of Fps1 in high-yielding strains ........................................................40 1.3.5. Mechanisms of translation ...............................................................................................42 1.3.5.1. Ribosomes and their biogenesis ................................................................................42 1.3.5.2. Cap-dependent mechanism of translation ..................................................................43 1.3.5.3. Consensus sequence and leaky scanning ...................................................................46 1.3.5.4. uORF affect translation initiation .............................................................................47 1.3.5.5. Translation initiation and stress ................................................................................48 1.3.5.5.1. eIF2α phosphorylation decreases translation initiation ........................................48 1.3.5.5.2. GCN4 translation ................................................................................................48 1.3.5.6. Translocation of membrane proteins .........................................................................51
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