Lipid rafts in Arabidopsis thaliana leaves Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universitat¨ Wurzburg¨ vorgelegt von Fatih Demir aus Mannheim Wurzburg¨ 2010 Pr¨ufungskomission Eingereicht am: 30. September 2010 Vorsitzender: Prof. Dr. Thomas Dandekar 1. Gutachter: Prof. Dr. Rainer Hedrich 2. Gutachter: Prof. Dr. Gregory Harms Tag des Promotionskolloquiums: Doktorurkunde ausgeh¨andigtam: iii Acknowledgments ? First, I would like to thank Prof. Dr. Rainer Hedrich for giving me the opportunity to carry out this research project in a vibrant and stimulating working group with great chances and responsibilities. And for his support whenever I needed it. ? Prof. Dr. Gregory Harms for being my second referee and bearing my contempt in writing an English PhD thesis as a non-native speaker. ? My supervisors Dr. Ines Kreuzer for the supervision of the project and being critical on lipid raft topics and Dr.es J¨org& Yvonne Reinders for teaching me mass spectrometry and the advantages of having a working pre-column which was not stiffed with lipids of all kinds. All the mass spectrometric measurements and data were acquired by a Demir-Reinders cooperation. I also greatly appreciate the technicians and PhD students in the Reinders lab in Regensburg who performed the practical sample preparation for mass spectrometry. ? J¨orgBlachutzik conducted microscopy and revealed all the nice co-localization data which appeared in our publication and also in this thesis { thanks for the bright spots on a dark background. ? My molecular biology capacities, Dr. Dietmar Geiger & his group members for the gener- ation of binary fusion constructs of ABI1, CPK21 and SLAH3. And of course S¨onke Scherzer for conducting electrophysiological measurements of the ABI1-CPK21-SLAH3 interaction in Xenopus laevis oocytes. ? Nazeer Ahmed for being so brave that he proof-read my thesis at first and refused the huge amount of dashes and semicolons. RIP all the dashes and semicolons. ? All the gardeners & technicians of the Botany I, but especially Joachim Rotenh¨ofer.We saw so many fields of Arabidopsis thaliana, Nicotiana benthamiana and Dionaea muscipula growing and being killed by me. Thanks for organizing the plants and keeping an eye on them. I do not want to summarize the amount of biomass I destructed in the last 3.5 years. ? Our cat Medolie who always bited me when I worked too long on this thesis { thanks for that! ? Especially my beloved wife Liliana Demir | I hope, I can compensate all the lost hours, days, weeks which were spent on scientific work somehow, sometime, somewhere. ? Last but not least I would like to acknowledge the financial support from the DFG Graduiertenkolleg 1342 "Lipid signalling" in form of a tax-free stipend. v Contents List of Tables xiii List of Figures xv 1. Introduction 1 1.1. Membrane structure . .2 1.1.1. Components of the membrane . .2 1.1.2. Singer-Nicolson model . .4 1.1.3. Evidence for organization . .6 1.1.4. Lipid modifications . .7 1.1.4.1. Myristoylation . .7 1.1.4.2. Palmitoylation . .8 1.1.4.3. Prenylation . .8 1.1.4.4. GPI-anchor . .9 1.1.4.5. Overview of lipid modifications . .9 1.1.4.6. Lipid modifications in plants . 10 1.2. Lipid rafts . 13 1.2.1. Sizing lipid rafts . 14 1.2.2. Sterols & disruption by MCD . 14 1.2.3. Model membranes . 16 1.2.3.1. Cholesterol & the organizing effect . 16 1.2.3.2. Visualizing lipid rafts . 18 1.2.3.3. Detergent insolubility . 18 1.2.3.4. Lipid modifications . 20 1.2.3.5. Phytosterols & model membranes . 20 1.2.4. Yeast lipid rafts . 21 1.2.4.1. Mating in S. cerevisiae .................... 22 1.2.4.2. Cell cycle control . 23 1.2.5. Lipid rafts in animals . 24 1.2.5.1. Diseases involving lipid rafts . 24 1.2.5.2. Non-sphingolipids & -sterols . 26 1.2.5.3. Raft sizes in animals . 26 1.2.5.4. Caveolae . 27 1.2.5.5. Signaling complexes in animal lipid rafts . 29 1.2.5.6. Activity & affinity regulation via lipid raft localization . 31 vii Contents 1.2.6. Lipid rafts in plants . 32 1.2.6.1. Plant plasma membranes . 32 1.2.6.2. Evidence for organization in the plant PM . 33 1.2.6.3. Previous DRM investigations in plants . 34 1.2.6.4. Identification of a putative plant lipid raft marker . 40 1.3. Aims of the study . 44 2. Methods 45 2.1. Membrane isolation . 45 2.1.1. Plant cultivation . 45 2.1.2. Homogenization of plant material . 45 2.1.3. Isolation of microsomal fraction . 46 2.1.4. Plasma membrane isolation . 46 2.1.5. DRM isolation . 48 2.1.5.1. Sterol depletion by MCD . 48 2.1.5.2. Detergent-treatment . 48 2.1.5.3. Sucrose density centrifugation . 49 2.1.5.4. Fractionation of the sucrose gradient . 49 2.1.5.5. Preparation of DRM samples for mass spectrometry . 49 2.2. Protein biochemistry . 50 2.2.1. Gel electrophoresis . 50 2.2.1.1. Sample preparation . 50 2.2.1.2. SDS-PAGE . 51 2.2.1.3. Gel visualization . 52 2.2.2. Western blot . 53 2.2.2.1. Transfer . 53 2.2.2.2. Antibody detection . 54 2.2.3. Protein quantification . 57 2.2.4. Precipitation methods . 57 2.2.4.1. TCA / Acetone precipitation . 58 2.2.4.2. Chloroform / Methanol precipitation . 58 2.2.4.3. Sodiumcarbonate precipitation . 59 2.2.4.4. Wang precipitation . 59 2.3. Mass spectrometry . 60 2.3.1. Sample preparation . 60 2.3.1.1. Trypsin . 60 viii Contents 2.3.1.2. In-gel digestion . 60 2.3.1.3. Washing of gel pieces . 60 2.3.1.4. In-solution digestion . 60 2.3.1.5. Formic acid Extraction . 62 2.3.2. Data acquisition . 62 2.3.2.1. Quantitative analysis via emPAI . 62 2.3.2.2. Data acquirement . 63 2.3.2.3. Database search parameters . 64 2.3.2.4. Data evaluation . 64 2.3.2.5. Protein data sources & lipidation predictors . 64 2.4. Molecular biology . 65 2.4.1. Bacterial cultivation . 65 2.4.1.1. DNA transformation . 65 2.4.2. DNA gel electrophoresis . 66 2.4.3. DNA purification . 66 2.4.3.1. DNA miniprep . 66 2.4.3.2. DNA midiprep . 67 2.4.3.3. DNA purification from agarose gels . 67 2.4.4. DNA quantification . 68 2.4.5. DNA sequencing . 68 2.4.6. Primer design . 68 2.4.7. PCR Amplification . 68 2.4.7.1. Colony PCR . 69 2.4.7.2. USER PCR . 69 2.4.7.3. PCR Profiles . 70 2.4.8. Restriction digest . 70 2.4.9. Particle Inflow Gun (PIG) . 71 2.4.9.1. Preparation of tungsten particles . 71 2.4.9.2. Coating of tungsten particles with DNA . 71 2.4.9.3. Transient transformation via PIG . 72 2.4.9.4. Fluorescence microscopy . 72 2.4.9.5. Analyzing co-localization experiments . 72 2.4.10. Transient expression in N. benthamiana . 73 2.4.10.1. Used vector constructs . 73 3. Results 75 ix Contents 3.1. Analyzing DRMs from A.th. leaves . 75 3.1.1. Quality control of the PM preparations . 75 3.1.2. Characterization of Triton X-100 & Brij-98 DRMs . 77 3.1.2.1. Quantitative analysis of protein amounts in the DRM isolation 77 3.1.2.2. Characterizing DRM isolations by sucrose gradients . 78 3.1.3. Proteomic analysis of A.th. leaf DRMs . 79 3.1.3.1. Detergent & digestion protocol effects on protein composition 80 3.1.3.2. Functional classification . 81 3.1.3.3. Triton X-100 & Brij-98 specific DRM proteins . 82 3.1.3.4. Molecular weight distribution . 85 3.1.3.5. Transmembrane domains . 86 3.1.3.6. Hydrophobicity properties . 87 3.1.3.7. Identification of putative DRM-specific proteins . 88 3.1.4. MCD effects on Triton X-100 DRMs . 88 3.2. Investigation of candidate DRM / raft proteins . 96 3.2.1. Biochemical characterization of eGFP::StRem 1.3 overexpressor . 96 3.2.2. Biochemical characterization of DRMs / DSF . 97 3.2.3. AtLipocalin & AtSUC1 / 2 localization . 100 3.3. Transient co-expression of ABI1, CPK21 & SLAH3 . 103 3.3.1. Transient expression in N.b. 103 3.3.1.1. Assaying sterol dependency of transiently expressed CPK21 104 3.3.2. Transient co-expression in N.b. 105 4. Discussion 109 4.1. Arabidopsis thaliana DRM protein composition . 110 4.1.1. DRMs enriched in signaling & transport proteins . 110 4.1.2. Correlation with previous DRM studies . ..