Ghent University Faculty of Sciences Department of Plant Biotechnology and Bioinformatics VIB department of Plant Systems Biology Endomembrane trafficking and brassinosteroid signaling from a small molecule perspective. Wim Dejonghe Promoter: Prof. Dr. Jenny Russinova This thesis was submitted as fulfillment of the requirements to obtain the degree of Doctor (PhD) in sciences: Biochemistry and Biotechnology Academic year 2014-2015 Copyright © 2015 by the Authors All rights reserved. This thesis is meant solely for personal use. This thesis or any portion thereof may not be reproduced or used in any manner whatsoever without the express written permission of the promoter or the first author. This thesis was carried out at the University of Ghent and Flemish Institute for Biotechnology (VIB) department of Plant Systems Biology (PSB). The research was funded by the Flemish institute for Innovation through Science and Technology (IWT) and VIB. Examination committee Chair: Dr Steffen Vanneste Prof. Dr. Geert De Jaeger Department of Plant Systems Biology. VIB, Ghent University Department of Plant Systems Biology. VIB, Ghent University Dr. Erich Kombrink Promoter: Max Planck Institute for Plant Breeding Research. Chemical Biology Laboratory Prof. Dr. Jenny Russinova Department of Plant Systems Biology. VIB, Ghent University Other committee members: Prof. Dr. Kris Gevaert Reading committee: Department of Biochemistry, Ghent University. VIB Department of Medical Prof. Dr. Savvas Savvides Protein Research. Unit for Structural Biology, L-ProBE, Ghent University. Inflammation Research Center, VIB, Ghent University Prof. Dr. Johan Winne Department of Organic and Macromolecular Chemistry. Ghent University Prof. Dr. Patrik Verstreken VIB Center for the Biology of Disease. KU Leuven, Center for Human Genetics, Dr. Daniel Van Damme Laboratory of Neuronal Communication Department of Plant Systems Biology. VIB, Ghent University 3 Contents List of abbreviations ............................................................................................................................. 9 Scope .................................................................................................................................................. 13 Summary ............................................................................................................................................ 15 Samenvatting ...................................................................................................................................... 17 Chapter I: Introduction ....................................................................................................................... 19 Abstract ........................................................................................................................................... 21 1.1. Brassinosteroids and their chemical toolbox* ......................................................................... 22 1.1.2 Small molecules affecting BR biosynthesis ....................................................................... 24 1.1.3. Small molecules affecting BR signaling ........................................................................... 26 1.2. Small molecules for dissecting endomembrane trafficking: a cross‐systems view** .......... 30 1.2.1. Endomembrane trafficking routes in yeasts, plants and mammals: a comparative view . 30 1.2.2. In search for small-molecule modifiers of endomembrane trafficking ............................ 31 1.2.3. Chemical effectors of exocytosis/secretion/anterograde trafficking ................................. 35 1.2.4. Chemical effectors of endocytosis/retrograde trafficking ................................................. 37 1.2.5. Chemical effectors of vacuolar trafficking and autophagy ............................................... 38 1.2.6. Special considerations when analyzing drug-induced endomembrane trafficking phenotypes .................................................................................................................................. 39 1.3. Target Identification Strategies in Plant Chemical Biology*** .............................................. 46 1.3.1. Commonly used target identification strategies in plant chemical biology ...................... 47 1.3.1.1. Forward genetic screen for compound resistance ..................................................... 48 1.3.1.2. Phenotyping approach ............................................................................................... 49 1.3.1.3. In silico-based target identification strategies ........................................................... 51 1.3.2. Emerging target identification strategies in plant chemical biology ................................ 51 1.3.2.1. Activity-based protein profiling (ABPP) .................................................................. 51 1.3.2.2. Yeast 3-hybrid ........................................................................................................... 52 1.3.2.3. Affinity-based technologies ...................................................................................... 54 5 1.3.2.4. Phage display ............................................................................................................. 57 1.3.3. Label-free compound based technologies ......................................................................... 58 1.3.3.1. Chemical denaturation shift ...................................................................................... 59 1.3.3.2. Target identification by chromatographic co-elution (TICC) ................................... 59 1.3.3.3. Drug affinity-responsive target stability (DARTS) ................................................... 59 1.4. Future perspectives .................................................................................................................. 61 1.5. Acknowledgments ................................................................................................................... 64 1.6. References ............................................................................................................................... 65 Chapter II: Small molecule screen for effectors of clathrin-mediated endocytosis and brassinosteroid signaling ............................................................................................................................................. 81 Abstract ........................................................................................................................................... 83 Introduction .................................................................................................................................... 84 Results and Discussion ................................................................................................................... 85 Compound Screen for CME inhibitors ....................................................................................... 85 BR signaling in response to small molecules ............................................................................. 88 Small molecule screen on light-grown and dark-grown seedlings ............................................. 88 Comparative summary of screening results and selection of hit molecules ............................... 90 Materials and methods .................................................................................................................. 101 References .................................................................................................................................... 102 Chapter III: Protonophore uncouplers are potent inhibitors of clathrin-mediated endocytosis in plants .......................................................................................................................................................... 105 Abstract ......................................................................................................................................... 107 Introduction .................................................................................................................................. 108 Results .......................................................................................................................................... 109 ES9 blocks clathrin-mediated endocytosis in different systems ............................................... 109 ES9 affects energy metabolism through uncoupling of oxidative phosphorylation in mitochondria ............................................................................................................................. 110 Cytosolic acidification but not ATP depletion rapidly inhibits CME in Arabidopsis .............. 112 E S9 activity causes an arrest in dynamic behavior of CME machinery at the plasma membrane ................................................................................................................................................... 115 Protonophores affect multiple aspects of endomembrane trafficking ...................................... 116 Discussion ..................................................................................................................................... 120 ES9 and TyrA23 are mitochondrial uncouplers ........................................................................ 120 ATP depletion alone does not efficiently block CME in Arabidopsis root cells and Drosophila neuromuscular junctions ..........................................................................................................