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Dictyostelium Discoideum The role of peroxisomes in sterol biosynthesis by the cellular slime mould Dictyostelium discoideum Mayyadah Abdullah Mohammed Alkuwayti, BSc This thesis is submitted to the University of Sheffield for the degree of Doctor of Philosophy Department of Molecular Biology and Biotechnology University of Sheffield September 2014 ABSTRACT In eukaryotic cells, the mevalonate pathway of isoprenoid biosynthesis provides the cell with essential precursors for several cellular processes. For example, one product, FDP (farnesyl diphosphate) is a fundamental precursor for sterol biosynthesis and it is also used for protein prenylation. In the slime mould Dictyostelium discoideum some of the mevalonate pathway enzymes possess a peroxisomal targeting signal. This suggested that part of the mevalonate pathway may take place in the peroxisomes. In this study, the intracellular locations of the mevalonate pathway enzymes were investigated by transforming D. discoideum amoebae to express each enzyme as a fusion protein with either GFP (green fluorescent protein) or mRFP (monomeric red fluorescent protein). It was found that three of the mevalonate pathway enzymes are peroxisomal: 3-hydroxy-3-methylglutaryl-coenzyme A synthase isozyme B, phosphomevalonate kinase and farnesyl diphosphate synthase. HMG- CoA reductase is associated with the endoplasmic reticulum and the other four enzymes of the mevalonate pathway were most likely to be in the cytosol: HMG- CoA synthase isozyme A, mevalonate kinase, diphosphomevalonate decarboxylase and IDP-isomerase. The intracellular location of the first five enzymes involved in sterol biosynthesis from farnesyl diphosphate was also identified by using the GFP or mRFP tagged enzyme approach. Some of these enzymes possess a strong peroxisomal targeting signal type 1 (PTS1). It was shown that the first four enzymes of the pathway: squalene synthase, squalene epoxidase, oxidosqualene cyclase and cycloartenol - C-24-methyltransferase are peroxisomal whereas the two isozymes of the fifth enzyme, methylsterol monooxygenase, are associated with the endoplasmic reticulum. It was also demonstrated that the first four enzymes on the sterol biosynthesis pathway are strongly associated with the peroxisomal membrane. However, the putative PTS1 present at the C-terminus of squalene synthase, oxidosqualene cyclase and cycloartenol-C-24-methyltransferase would imply that each of these enzymes should be a peroxisomal matrix protein. We therefore investigated whether the putative PTS1s are involved in directing these three enzymes into the peroxisomes. It was found that squalene synthase was largely peroxisomal even when its PTS1 was absent but the PTS1 in oxidosqualene synthase and cycloartenol-C-24-methyltransferase was essential for entry of these enzymes into peroxisomes. It appears that the sterol biosynthesis in D. discoideum is unusual since the enzymes squalene synthase, squalene epoxidase, oxidosqualene cyclase and cycloartenol -C-24-methyltransferase are accumulated in the peroxisomes whereas in all other organisms studied they are in the endoplasmic reticulum. II ACKNOWLEDGEMENTS Praise to Allah the Almighty, Creator and Sustainers of the Universe. Firstly, I would like to express my sincere gratitude and appreciation to my supervisors Dr Donald Wattas and Dr Ewald Hettema for their excellent supervision, constructive guidance, invaluable feedback, and encouragement during my PhD journey. Secondly, special thanks for my sponsor King Faisal University and Ministry for Higher Education in Saudi Arabia, for their financial and academic support, represented by the cultural attaché in London during my PhD study. I am grateful for Dr Patrick Baker for the three dimensional structure pictures. I offer my regards and blessings to the rest of the members of the Hettema lab, Alison, Jimmy, Gemma, Joanne, Nadal, Peter, Mohammed, John and Murtakab. My greatest indebtedness to my husband Mohammed Alsaif for his encouragement, inspiration, patience and support. The thanks extended to my little boy Othman for his patience during my busy days. I owe a special thanks to my Mum and Dad for their emotional, continues prayers and love. I am grateful also to my father in law for his daily phone support and encouragement. Big thanks to my sisters especially Abeer for the video calls and encouragement, brothers and friends for their moral support and endless encouragement. III TABLE OF CONTENTS Abstract ......................................................................................................................... I Acknowledgements .................................................................................................... III Abbreviations ............................................................................................................ IX Amino acids .............................................................................................................. XII Uniprot genes numbers .......................................................................................... XIII List of tables ............................................................................................................ XIV List of figures ............................................................................................................ XV Chapter1 ........................................................................................................................ 1 Introduction ....................................................................................................... 1 1.1 Peroxisomes ............................................................................................. 3 1.2 Peroxisomal diseases ................................................................................ 5 1.3 Peroxisome Biogenesis (Peroxins/PEX genes) ...................................... 6 1.4 Peroxisomal matrix protein import pathway ........................................ 8 1.6 Peroxisomal membrane proteins .......................................................... 12 1.7 Targeting of peroxisomal membrane proteins .................................... 13 1.8 The mevalonate pathway of isoprenoid ............................................... 14 1.9 The subcellular distribution of the mevalonate pathway enzymes among different organisms ......................................................................... 17 1.10 The biosynthesis of sterols ................................................................... 20 1.11 Dictyostelium discoideum cellular and genetic aspects...................... 23 1.12 Aim of the project ................................................................................ 24 Chapter 2 ..................................................................................................................... 25 Materials and Methods ................................................................................... 25 2.1 Media, chemicals and enzymes ........................................................... 25 2.2 Strains and plasmids ............................................................................. 26 2.2.1 Strains ............................................................................................. 26 2.2.2 Plasmids .......................................................................................... 27 2.2.3 Plasmid design ................................................................................ 29 IV 2.2.4 Dictyostelium discoideum plasmids ............................................... 29 2.2.5 Plasmid design for homologous recombination in S. cerevisiae . 31 2. 3 Primers ................................................................................................... 33 2.4 Cell Culture ............................................................................................ 39 2.5 Growth of Escherichia coli .................................................................... 40 2.5.1 Growth and maintenance of E. coli ............................................... 40 2.5.2 Preparing chemical-competent cells of E.coli DH5α .................. 40 2.5.3 Transformation of chemical-competent E.coli by the heat-shock method ........................................................................................................... 41 2.5.4 Transformation of E.coli by electroporation ................................ 41 2.6 Axenic growth of Dictyostelium discoideum ......................................... 42 2.6.1 Growth and maintenance of D.discoideum ................................... 42 2.6.2 Isolation of D. discoideum genomic DNA by using a QIAamp DNA Mini Kit ............................................................................................... 42 2.6.3 Isolation of D. discoideum cDNA by using a QIAamp DNA Mini Kit .................................................................................................................. 42 2.6.4 Transformation of D. discoideum by electroporation ................. 43 2.6.5 Fluorescence microscopy (D.discoideum) ..................................... 43 2.7.1 Growth and maintenance of S.cerevisiae .................................... 44 2.7.2 Yeast high efficiency transformation procedure ......................... 44 2.7.3 Yeast one step transformation procedure .................................... 45 2.7.4 Isolation of yeast total DNA ......................................................... 45 2.7.5 Fluorescence microscopy (S.cerevisiae) ......................................... 46 2.8 Molecular cloning
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