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3 the Sudcellular Distribution of Carotenoids In / 3 THE SUDCELLULAR DISTRIBUTION OF CAROTENOIDS IN PHYCOMYCES BLAKESLEEANUS T FLR A Thesis submitted by , GRAHAM JOHN PEARSON RILEY 143,05"^ a candidate for the degree of Doctor of Philosophy in BIOCHEMISTRY Department of Biochemistry Royal Holloway College ^ University of London Egham Hill EGHAM Surrey September 1977 ProQuest Number: 10097456 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10097456 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 - 2 - ACKNOWLEDGEMENTS Firstly, I would like to thank my supervisor. Dr. P.M. Bramley, for introducing me to such an interesting topic, for his help, encouragement, and patience throughout my research, and for his guidance in the preparation of this thesis. I would also like to thank all those members of the staff and students of this college who have helped me either practically or theoretically during my postgraduate research, especially Professor J.B. Pridham and Dr. L.R.G. Valadon, both of whom contributed helpful advice and criticism at various stages of this work. I am also very grateful to Ms. Carole Spurdon for advice and assistance with techniques for electron microscopy, and for cutting acceptable sections from my amateurish samples. I thank the Science Research Council for the award of a Research Studentship. - 3 - ABSTRACT In order to study the subcellular distribution of carotenoids in Phycomyces blakesleeanus, homogenates of mycelia of the Cl 15 carS42 mad-1Q7(-) mutant were fractionated by both differential and density gradient centrifugation. Characterisation of the fractions * was achieved by the distribution of marker enzyme activities. Organelles prepared by differential centrifugation were shown to be associated with contaminating lipids, including p-carotene (P, P-carotene). These lipids could not be removed by either repeated washing, or by centrifugation procedures; so a separation technique based on density gradient centrifugation was devised. Only two fractions isolated by the latter method contained carotenoid; the lipid particles, and a particulate fraction. The latter was tentatively identified, on the basis of its density (I.IOg/ml), and its enzyme content, as a vacuole-containing fraction. Both these pigmented fractions exhibited alkaline p-nitro- phenylphosphatase activity, as well as containing sterols and phos­ pholipids. The proteins from these organelles revealed close similarities on amino acid analysis. The relative distribution of P-carotene between these fractions was investigated at various stages of growth. The lipid droplets isolated from P.blakesleeanus resembled those prepared from other plants in both size and lipid content. They contained sufficient protein to form a monolayer around their surf ace. Mycelia were grown in media containing [2-^^c]mevalonic acid (MVA), and the subcellular distribution of radioactive lipids was determined. The rates of incorporation of MVA in various organelles were compared. Organelles were also incubated with [2- ^c ]m VA in vitro. Cytosol enzymes were able to incorporate MVA into terpenols and squalene. No particulate fractions were capable of converting MVA *• t into P-carotene or sterols in the absence of the cytosol. These results are discussed in terms of the possible sites of biosynthesis of carotenoids in the mycelium, and the inter-relation­ ships of the pigmented organelles. - 4 - CONTENTS Page Acknowledgements 2 Abstract 3 Abbreviations 8 Chapter 1 : INTRODUCTION A. The Biosynthesis of Carotenes and Sterols Biogenesis of Isoprene Units 10 Biosynthesis of Polyisoprene Units 14 Squalene Biosynthesis 17 Biosynthesis of Lanosterol 20 Biosynthesis of Ergosterol 20 Estérification of Sterols 27 Sterol Carrier Proteins 28 Biosynthesis of Phytoene 29 Desaturation of Phytoene 31 Formation of Cyclic Carotenes 36 Further Metabolism of Carotenes 40 Cofactor Requirements of Carotene Biosynthesis 40 Control of Carotenogenesis 45 Functions of Sterols and Carotenes 46 B. Fungal Growth and Morphology Taxonomy 50 Phycomyces blakesleeanus 50 Mutants of Phycomyces 53 Genetic Control of Carotenogenesis 53 Fungal Growth 56 C. Fungal Ultrastructure and Organelles The Cell Wall 59 Nuclei 60 Mitochondria 60 ' Endoplasmic Reticulum 61 Cytoplasmic Membranes 62 Microbodies 63 Vacuoles 64 Spherosomes and Lipid Droplets ' 66 - 5 - Page Golgi Complex 73 Other Components of Fungal Cells 74 D. The Subcellular Distribution of Carotenoids and sterols Subcellular Locations of Terpenoid Biosynthesis 75 Subcellular Location of Carotenoids 77 E. Aims of the Present Investigation 79 Chapter 2 : MATERIALS and METHODS A. Materials Solvents 81 Chemicals 81 Microorganisms 81 B. General Experimental Methods Culture Conditions 81 Preparation of Cell Extracts 83 Centrifugation Procedures 83 Marker Enzyme Assays ' 85 Other Determinations 85 Biosynthesis of Terpenoids from [2-^^c]MVA 90 Preparation of Carrier Carotenes 91 Lipid Extraction Procedures 91 Estimation of Lipids 92 Separation of Terpenoids 93 Thin-Layer Chromatography 95 Autoradiography 95 Elution of Bands from Chromatograms 97 Paper Chromatography 97 Liquid Scintillation Counting 97 Amino Acid Analysis 97 ^ Electron Microscopy 98 Chapter 3 : ANALYSIS OF LIPIDS OF CELL ORGANELLES - PRELIMINARY EXPERIMENTS Growth of Phycomyces blakesleeanus 101 - 6 - « Page Electron Microscopy . 104 Preparation of Cell Walls 104 Cell Fractionation by Differential Centrifugation 111 Density Gradient Centrifugation of Mitochondria 117 Chapter 4 : ANALYSIS OF LIPIDS OF PURIFIED CELL ORGANELLES A. Development of Methods for the Preparation of Purified Cell Organelles Preparation of Mitochondria 125 Properties of Mitochondria 125 Separation of Mitochondrial Membranes 128 Constitution of Buffer for the Preparation of Mitochondria 130 Preparation of Nuclei 134 Properties of Isolated Nuclei 138 Separation of Other Organelles 138 B. Lipid Distribution in Purified Subcellular Fractions Methods 144 Results and Conclusions 144 Chapter 5 : CHARACTERIZATION OF p-CAROTENE-CONTAINING ORGANELLES A. Enzymes of Pigmented Fractions Particulate Fraction 150 Lipid Globules 154 B. Chemical Composition of Pigmented Fractions Amino Acid Analyses 157 Distribution of P-Carotene between Globules and Vacuoles 160 Lipid Content of Lipid Droplets 166 Size of Lipid Droplets 166 Protein Content of Lipid Droplets 169 - 7 - Page Chapter 6 : INCORPORATION OF fS-'* "^c]mEVALONIC ACID INTO THE TERPENOIDS OF GROWING MYCELIA Preliminary Experiments 172 Subcellular Distribution of Radioactivity from [2-^^c]MVA 175 Time Course of Incorporation of [2-^^c ]m VA into Lipids 178 Chapter 7 : INCORPORATION OF fZ-**^c Im EVALONIC ACID IN ISOLATED ORGANELLES Incorporation of [2-^^c]MVA in Cell Homogenates 183 Purification of Terpenoids Synthesized from [2-^^c ]m VA 187 Effect of Protein Concentration on Incorporation of MVA by. Cell Homogenates 188 Incorporation of [2-^^c ]m VA in Isolated Cell Fractions 189 Incorporation of MVA into Terpenols 194 Chapter 8 ; CONCLUSIONS 200 BIBLIOGRAPHY 206 - 8 - ABBREVIATIONS The abbreviations used in this thesis are those listed in the Biochemical Journal (1976) 153, 1-21 (revised, 1977, 161, 1 -2 ) with the following additions: D.I. Difference Index of Compositional Relatedness (Metzger ejt , 1968) DMAPP dimethylallyl pyrophosphate E.R. endoplasmic reticulum FP farnesyl phosphate FPP farnesyl pyrophosphate GGP geranylgeranyl phosphate GGPP geranylgeranyl pyrophosphate GPP geranyl pyrophosphate IIMG 3-hydroxy-3-methylglutaric acid' IP isopentenyl phosphate IPP isopentenyl pyrophosphate MVA mevalonic acid PPPP prephytoene pyrophosphate PSPP presqualene pyrophosphate SCP sterol carrier protein INTRODUCTION - 10- A . THE BIOSYNTHESIS OF CAROTENES AND STEROLS Biosynthesis of Isoprene Units Both carotenes and sterols may be derived formally from five- carbon isoprene units, and are hence classified as terpenes (Ruzicka, 1953). The patterns of incorporation of [^^c]acetate into P-carotene (p,p-caroteneJ N Crob and BUtler, 1955, 1956) and cholesterol(cholest- 4'’-enol,y.WLIersch e_t , 1952; Ruzicka, 1953/; Fig. 1.1) are both consistent with the involvement of a five-carbon unit derived from acetate via 3-hydroxy-3-methylglutaryl CoA (HMG-CoA). Yokoyama ejfc (1960) showed that the incorporation of [2-^^c] mevalonic acid (MVA) into p-carotene by a cell-free homogenate of Phycomyces blakesleeanus mycelia was greater than that of [^"*c]HMG. MVA was incorporated into sterols with high efficiency by a rat liver homogenate (Tavormina e_t , 1956). Since Ferguson ejt a^. (1958) demonstrated the reduction of HMG-CoA to MVA by a yeast extract, subsequent studies, involving enzymes purified from a variety of sources (reviewed by Beytia and Porter, 1976), have established that the major pathway of MVA biosynthesis is that shown in Fig. 1.2. Goodwin and Lijinsky (1951) found that the branched-chain amino acids leucine and valine stimulated carotenogenesis in Phycomyces. It has now been established that this is because they are efficient precursors of HMG via an alternative metabolic route
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