Investigation of Dinoflagellate Plastid Protein Transport using Heterologous and Homologous in vivo Systems Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) Vorgelegt dem Fachbereich Biologie der Philipps-Universität Marburg von Andrew Scott Bozarth aus Columbia, Maryland, USA Marburg/Lahn 2010 Vom Fachbereich Biologie der Philipps-Universität als Dissertation angenommen am 26.07.2010 angenommen. Erstgutachter: Prof. Dr. Uwe-G. Maier Zweitgutachter: Prof. Dr. Klaus Lingelbach Prof. Dr. Andreas Brune Prof. Dr. Renate Renkawitz-Pohl Tag der Disputation am: 11.10.2010 Results! Why, man, I have gotten a lot of results. I know several thousand things that won’t work! -Thomas A. Edison Publications Bozarth A, Susanne Lieske, Christine Weber, Sven Gould, and Stefan Zauner (2010) Transfection with Dinoflagellate Transit Peptides (in progress). Bolte K, Bullmann L, Hempel F, Bozarth A, Zauner S, Maier UG (2009) Protein Targeting into Secondary Plastids. J. Eukaryot. Microbiol. 56, 9–15. Bozarth A, Maier UG, Zauner S (2009) Diatoms in biotechnology: modern tools and applications. Appl. Microbiol. Biotechnol. 82, 195-201. Maier UG, Bozarth A, Funk HT, Zauner S, Rensing SA, Schmitz-Linneweber C, Börner T, Tillich M (2008) Complex chloroplast RNA metabolism: just debugging the genetic programme? BMC Biol. 6, 36. Hempel F, Bozarth A, Sommer MS, Zauner S, Przyborski JM, Maier UG. (2007) Transport of nuclear-encoded proteins into secondarily evolved plastids. Biol Chem. 388, 899-906. Table of Contents TABLE OF CONTENTS 1 INTRODUCTION....................................................................................................................................... 1 1.1 THE EVOLUTION OF PHOTOSYNTHETIC EUKARYOTES............................................................................... 1 1.1.1 Primary Endosymbiosis .................................................................................................................. 1 1.1.2 Protein Targeting to Primary Plastids ........................................................................................... 3 1.2 SECONDARY ENDOSYMBIOSIS AND THE CHROMALVEOLATE HYPOTHESIS ............................................... 5 1.3 CONSEQUENCES OF HARBORING EUKARYOTIC ENDOSYMBIONTS ............................................................ 8 1.3.1 Genomic Reorganization ................................................................................................................ 8 1.3.2 Protein Transport into Chromist Plastids....................................................................................... 9 1.4 INFRAKINGDOM ALVEOLATA .................................................................................................................. 11 1.4.1 Alveolates as Such ........................................................................................................................ 11 1.4.2 Protein Transport into Apicomplexan Plastids ............................................................................. 12 1.5 DINOFLAGELLATES ................................................................................................................................. 14 1.5.1 Features of Dinoflagellate ............................................................................................................ 14 1.5.2 Ecological Impetus ....................................................................................................................... 16 1.5.3 Genomic Arrangement of Dinoflagellates .................................................................................... 17 1.5.4 Protein Targeting in Dinoflagellates ............................................................................................ 19 1.5.5 Ceratium horridum ....................................................................................................................... 23 1.5.6 Amphidinium carterae .................................................................................................................. 23 2 AIMS .......................................................................................................................................................... 25 3 RESULTS .................................................................................................................................................. 26 3.1 ANALYSIS OF SYNTHESIZED CERATIUM HORRIDUM ESTS ...................................................................... 26 3.1.1 Analysis of Ceratium horridum cDNA Library ............................................................................. 26 3.1.2 C. horridum EST Sequence Homologies ....................................................................................... 27 3.1.3 C. horridum EST Sequence Functions .......................................................................................... 30 3.1.4 Determination of Ceratium horridum Plastid-Targeted Proteins ................................................ 32 3.1.5 Ceratium horridum Transit Peptide Classification ...................................................................... 34 3.1.6 Ceratium horridum Transit Peptide Variations between Isoforms ............................................... 36 3.1.7 Comparison of Ceratium horridum Transit Peptides with Those of Other Phototrophs .............. 37 3.1.8 Statistical Comparative Analysis of Dinoflagellate Transit Peptides ........................................... 40 3.2 HETEROLOGOUS IN VIVO IMPORT STUDIES ............................................................................................. 43 3.2.1 Heterologous in vivo Import into the Pisum sativum Chloroplasts using Amphidinium carterae PsbO and Prk Transit Peptides ................................................................................................................... 44 3.2.2 Heterologous in vivo Import into the Pisum sativum Chloroplasts using Chromalveolate PsbO and Prk Transit Peptides ............................................................................................................................ 47 3.3 HETEROLOGOUS IN VIVO PROTEIN IMPORT INTO THE PLASTIDS OF PHAEODACTYLUM TRICORNUTUM USING AMPHIDINIUM CARTERAE PSBO AND PRK TARGETING SIGNALS ........................................................... 48 3.3.1 HETEROLOGOUS IN VIVO PROTEIN IMPORT IN PHAEODACTYLUM TRICORNUTUM USING AMPHIDINIUM CARTERAE PSBO AND PRK SIGNAL PEPTIDES .................................................................................................. 48 3.3.2 HETEROLOGOUS IN VIVO PROTEIN IMPORT IN PHAEODACTYLUM TRICORNUTUM USING AMPHIDINIUM CARTERAE PSBO AND PRK SIGNAL, TRANSIT, AND THYLAKOID TARGETING PEPTIDES ................................... 49 3.4 TRACKING HOMOLOGOUS PROTEIN TARGETING IN AMPHIDINIUM CARTERAE ....................................... 52 3.4.1 SUCROSE FRACTIONATION OF AMPHIDINIUM CARTERAE ................................................................... 52 3.4.2 EFFECTS OF BREFELDIN A ON PROTEIN MOBILITY IN SUCROSE GRADIENT FRACTIONATIONS OF AMPHIDINIUM CARTERAE ................................................................................................................................ 55 3.4.3 ELECTRON MICROSCOPIC EXAMINATION OF THE EFFECTS OF BREFELDIN A ON PROTEIN LOCALIZATION IN AMPHIDINIUM CARTERAE .................................................................................................... 59 3.4.3.1 IMMUNOGOLD LABELING WITH Α-PSBO IN AMPHIDINIUM CARTERAE CELLS TREATED WITH BREFELDIN A ................................................................................................................................................... 61 3.4.3.2 IMMUNOGOLD LABELING WITH Α-RBCL IN AMPHIDINIUM CARTERAE CELLS TREATED WITH BREFELDIN A ................................................................................................................................................... 63 4 DISCUSSION ............................................................................................................................................ 66 i 4.1 CERATIUM HORRIDUM EST LIBRARY ..................................................................................................... 66 4.1.1 General Features .......................................................................................................................... 66 4.1.2 Gene Transfers ............................................................................................................................. 67 4.1.3 Plastid Targeting Signals ............................................................................................................. 69 4.2 HETEROLOGOUS IN VIVO IMPORT ASSAYS USING DINOFLAGELLATE TARGETING SIGNALS .................... 72 4.2.1 In vivo transfection of Pisum sativum with Amphidinium carterae PsbO and Prk Transit Peptides 73 4.2.2 Heterologous in vivo Transfections of Phaeodactylum tricornutum ............................................ 74 4.2.2.1 Heterologous in vivo Transfections of Phaeodactylum tricornutum with Amphidinium carterae Signal Peptides ............................................................................................................................. 75 4.2.2.2 Heterologous in vivo Transfections of Phaeodactylum tricornutum with Amphidinium carterae BTSs .............................................................................................................................................. 75 4.2.3 Implications of in vivo Transfections ...........................................................................................