Role of EBAG9 in COPI-Dependent Glycoprotein Maturation and Secretion Processes in Tumor Cells Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin von Diplom-Biologin Jana Wolf, geb. Göttert Präsident der Humboldt-Universität zu Berlin Prof. Dr. Dr. h.c. Christoph Markschies Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Andreas Herrmann Gutachter/innen: 1. Prof. Dr. Thomas Börner 2. Prof. Dr. med. Bernd Dörken 3. PD Dr. Uta Höpken Tag der mündlichen Prüfung: 21.10.2010 Index of contents Index of contents ABSTRACT......................................................................................................................................................... VI ZUSAMMENFASSUNG....................................................................................................................................VII 1 INTRODUCTION......................................................................................................................................... 1 1.1 THE TUMOR ASSOCIATED ANTIGEN EBAG9 ............................................................................................. 1 1.1.1 Features of EBAG9 protein............................................................................................................. 1 1.1.2 EBAG9 and cancer.......................................................................................................................... 2 1.1.3 Biological functions of EBAG9 ...................................................................................................... 4 1.2 THE SECRETORY PATHWAY ....................................................................................................................... 5 1.2.1 From the endoplasmic reticulum to the cell exterior....................................................................... 5 1.2.1.1 Transport from the trans-Golgi-network............................................................................................ 5 1.2.1.2 Polarized transport ......................................................................................................................... 7 1.2.2 Mechanisms of biosynthetic protein transport from the ER towards the Golgi .............................. 9 1.2.3 From budding to fusion: components of the secretory pathway.................................................... 10 1.2.3.1 Coat proteins ............................................................................................................................... 10 1.2.3.2 The vesicle machinery: from Arfs to SNAREs................................................................................. 12 1.2.4 Retrograde transport...................................................................................................................... 15 1.3 BIOSYNTHETIC TRANSPORT AND GLYCOSYLATION ................................................................................. 17 1.3.1 N- and O-glycosylation................................................................................................................. 17 1.3.2 Functional role of glycosylation.................................................................................................... 19 1.4 THE SECRETORY PATHWAY AND DISEASE ................................................................................................ 20 1.5 AIMS ...................................................................................................................................................... 22 2 MATERIALS............................................................................................................................................... 24 2.1 CELLS .................................................................................................................................................... 24 2.2 VIRUSES................................................................................................................................................. 24 2.3 BACTERIAL STRAINS .............................................................................................................................. 25 2.4 MICE STRAINS........................................................................................................................................ 25 2.5 EXPRESSION CONSTRUCTS ..................................................................................................................... 25 2.6 OLIGONUCLEOTIDES .............................................................................................................................. 27 2.7 ANTIBODIES........................................................................................................................................... 27 2.8 REAGENTS ............................................................................................................................................. 30 2.9 BUFFER AND MEDIA ............................................................................................................................... 30 3 METHODS .................................................................................................................................................. 33 3.1 MOLECULAR BIOLOGY ........................................................................................................................... 33 ii Index of contents 3.1.1 Culture of bacteria......................................................................................................................... 33 3.1.2 Transformation of E.coli ............................................................................................................... 33 3.1.3 Isolation of plasmid DNA from E.coli cells .................................................................................. 33 3.1.4 Polymerase chain reaction (PCR).................................................................................................. 34 3.1.5 Restriction digest........................................................................................................................... 34 3.1.6 Agarose gel electrophoresis .......................................................................................................... 34 3.1.7 Isolation of DNA from agarose gels.............................................................................................. 35 3.1.8 Ligation of DNA ........................................................................................................................... 35 3.1.9 Cloning.......................................................................................................................................... 35 3.2 PROTEIN BIOCHEMISTRY ........................................................................................................................ 35 3.2.1 Protein isolation and determination of protein concentration........................................................ 35 3.2.2 SDS-polyacrylamide-gelelectrophoresis (SDS-PAGE)................................................................. 36 3.2.3 Immunoblot (westernblot analysis)............................................................................................... 36 3.2.4 One-dimensional (1d)-isoelectric focusing gel electrophoresis (IEF)........................................... 37 3.2.5 Autoradiography ........................................................................................................................... 37 3.2.6 Quantification of protein bands..................................................................................................... 38 3.3 CELL BIOLOGY ....................................................................................................................................... 38 3.3.1 Cell culture and transfections........................................................................................................ 38 3.3.2 Transfection of mammalian cells .................................................................................................. 38 3.3.3 Generation of stable cell lines ....................................................................................................... 39 3.3.4 Amplification and transduction of cell lines with adenovirus....................................................... 39 3.3.5 Pulse-chase experiments ............................................................................................................... 40 3.3.6 Immunoprecipitation (IP) and co-immunoprecipitation................................................................ 40 3.3.7 Subcellular fractionation ............................................................................................................... 41 3.3.8 Fluorescence activated cell sorting (FACS)-analysis .................................................................... 42 3.3.9 Immunofluorescence..................................................................................................................... 42 3.3.10 Lectin staining............................................................................................................................... 43 3.3.11 Confocal image acquisition and correlation analysis .................................................................... 43 3.3.11.1 Time-lapse imaging .....................................................................................................................