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Structure and Architecture of Eisosomes

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Structure and Architecture of Eisosomes Dissertation zur Erlangung des Doktorgrades der Fakultät für Biologie der Ludwig-Maximilians-Universität München Structure and Architecture of Eisosomes vorgelegt von Lena Karotki 2012 Eidesstattliche Versicherung Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation von mir selbstständig und ohne unerlaubte Hilfe angefertigt ist. München, den .............................. ............................................................. (Unterschrift) Erklärung Hiermit erkäre ich, □ dass die Dissertation nicht ganz oder in wesentlichen Teilen einer anderen Prüfungskomission vorgelegt worden ist □ dass ich mich anderweitig einer Doktorprüfung ohne Erfolg nicht unterzogen habe München, den .............................. ............................................................. (Unterschrift) Diese Dissertation wurde von Prof. Dr. Stefan Jentsch betreut. Die Dissertation wurde eingereicht am ................. 1. Gutachter: Prof. Dr. Stefan Jentsch 2. Gutachter: Prof. Dr. Charles David Tag der mündlichen Prüfung: 03.09.2012 1. Table of Contents Structure and Architecture of Eisosomes 1 Table of Contents 1 Table of Contents ................................................................................................ 1 2 List of Publications............................................................................................... 3 3 Abbreviations ....................................................................................................... 4 4 Summary ............................................................................................................. 6 5 Introduction .......................................................................................................... 7 5.1 The composition of the plasma membrane .................................................... 7 5.1.1 The fluid mosaic model ........................................................................... 7 5.1.2 Plasma membrane lipids ......................................................................... 9 5.1.3 Plasma membrane proteins .................................................................. 11 5.2 Plasma membrane organization .................................................................. 13 5.2.1 Macrodomain organization of the plasma membrane ........................... 13 5.2.2 Lipid rafts .............................................................................................. 15 5.2.3 Microdomains ........................................................................................ 16 5.3 Structural aspects of membrane domains ................................................... 18 5.3.1 Lipid packing and asymmetry as driving force for membrane shaping .. 19 5.3.2 Membrane shaping by proteins ............................................................. 20 5.3.3 Membrane deformation by BAR domain containing proteins ................ 21 5.4 Plasma membrane organization in yeast Saccharomyces cerevisiae ......... 26 6 Aim of the thesis ................................................................................................ 30 7 Discussion ......................................................................................................... 32 7.1 Pil1 and Lsp1 assemble into large multimeric complexes ........................... 32 7.2 Pil1 and Lsp1 bind phosphoinositides ......................................................... 36 1 | Page 1. Table of Contents Structure and Architecture of Eisosomes 7.3 Pil1 and Lsp1 sculpt membranes................................................................. 39 7.4 Pil1 and Lsp1 stabilize plasma membrane domains .................................... 44 7.5 Evolutionary conservation of eisosomes ..................................................... 45 8 References ........................................................................................................ 47 9 Acknowledgments ............................................................................................. 54 10 Curriculum vitae .............................................................................................. 55 11 Declaration of Individual Contributions ........................................................... 57 12 Reprints of the Publications ............................................................................ 58 2 | Page 2.List of Publications Structure and Architecture of Eisosomes 2 List of Publications Publication 1: Wang H, Kakaradov B, Collins SR, Karotki L, Fiedler D, Shales M, Shokat KM, Walther TC, Krogan NJ, and Koller D. (2009). A complex-based reconstruction of the Saccharomyces cerevisiae interactome. Mol Cell Proteomics 8, 1361-1381 Publication 2: Ziolkowska NE, Karotki L, Rehman M, Huiskonen JT, and Walther TC (2011). Eisosome-driven plasma membrane organization is mediated by BAR domains. Nat Struct Mol Biol 18, 854-856. Publication 3: Karotki L, Huiskonen JT, Stefan JS, Ziolkowska NE, Roth R, Surma MA, Krogan NJ, Emr SD, Heuser J, Grünewald K, Walther TC (2011). Eisosome Proteins Assemble into a Membrane Scaffold. J Cell Biol 195, 889-902. 3 | Page 3.Abbreviations Structure and Architecture of Eisosomes 3 Abbreviations 3D three dimensional BAR Bin, Amphiphysin, Rvs161/167 CCP clathrin coated pit CCV clathrin-coated vesicles COP coatomer protein cryo-ET cryo-electron tomography DAG Diacylglycerol DEEM deep-etch electron microscopy EM electron microscopy ER endoplasmatic reticulum FCH FES-CIP4 homology F-BAR FCH-BAR GFP green fluorescent protein GPI glycosylphosphatidylinositol GUV giant unilamellar vesicle I-BAR Inverse-BAR IPC inositol phosphate ceramide ld liquid-disordered lo liquid-ordered LPC lysophosphatidylcholine M(IP2)C mannosyl-diinositolphosphate-ceramide MCC membrane compartment containing Can1 4 | Page 3.Abbreviations Structure and Architecture of Eisosomes MCP membrane compartment containing Pma1 MCT membrane compartment of TORC2 MDCK Madin-Darby canine kidney MIPC mannosyl-inositolphosphate-ceramide NBD nitrobenzoxadizole PA phosphatidic acid PC phosphatidylcholine PE phosphatidylethanolamine PH domain Pleckstrin homology domain PI phosphatidylinositol PI(3)P phosphoinositol-3-phosphate PI(4)P phosphoinositol-4-phosphate PI(4,5)P2 phosphoinositol-4,5-bisphosphate PS phosphatidylserine S.cerevisiae Saccharomyces cerevisiae SM sphingomyelin SNXs sorting nexins SPT single particle tracking TGN trans-Golgi network TORC2 target of rapamycin complex 2 5 | Page 4. Summary Structure and Architecture of Eisosomes 4 Summary The plasma membrane is a fundamental feature of life, since it constitutes the boundary between the cell and its environment. To coordinate the various different reactions occuring there, it is highly dynamic and its constituents are organized into domains of distinct protein and lipid composition.This lateral compartmentalization is at least partially mediated by huge protein complexes, termed eisosomes, which localize in a uniform punctuate pattern at the plasma membrane and are mainly composed of two proteins, Pil1 and Lsp1. Essential questions towards an understanding of eisosome-mediated plasma membane organization refer to their molecular architecture as well as to the mechanism mediating the lateral segregation of proteins and lipids into distinct compartments. In order to answer these questions, the work of my thesis characterizes the structure and architecture of eisosomes at several levels of resolution in vitro and in vivo. Using a combination of different biochemical and electron microscopy (EM) approaches, I show that eisosome proteins Pil1 and Lsp1 self-assemble into higher order structures, such as thin filaments and helices. Furthermore, both proteins Pil1 and Lsp1 directly bind and deform preferentially PI(4,5)P2-containing membranes into long tubules. By fitting of the crystal structure of a stable core domain of Lsp1 (named “Lsp1 ASIA”) into 3D models of eisosome proteins bound to membranes, I unveil the mechanism how these proteins self-assemble and bind to membranes and pinpoint amino acids that are essential in this process. Using these EM-derived 3D models of Pil1 and Lsp1, I demonstrate that these structures resemble eisosomes in vivo, From these data, I introduce a model, explaining how eisosomes are molecularly built and how they organize the plasma membrane by self-assembly into a protein scaffold that directly binds and deforms membranes with lipid-binding-specificity. Beyond the immediate gain of knowledge, characterization of eisosomes will most likely have an impact on our understanding of self assembly systems and how they organize cellular structure, and ultimately how that is used to regulate plasma membrane organization and endocytosis. 6 | Page 5. Introduction Structure and Architecture of Eisosomes 5 Introduction 5.1 The composition of the plasma membrane The plasma membrane constitutes the boundary between the cell and its environment. As such, it is a fundamental feature of life and crucial for a broad variety of functions, such as cell polarity establishment, cell motility or nutrient absorption. To achieve these many tasks, the plasma membrane is highly dynamic and its composition has to be constantly remodeled according to need. Consequently, the presence of receptors, transporters or signaling molecules achieves tight regulation, by a delicate interplay of proteins and lipids within the plasma membrane. 5.1.1 The fluid mosaic model In 1972 Singer and Nicolson proposed a concept for membrane organization called “fluid mosaic model”, which describes biological
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