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In Vitro Reconstitution of the Molecular Mechanisms of Vesicle Tethering and Membrane Fusion

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In Vitro Reconstitution of the Molecular Mechanisms of Vesicle Tethering and Membrane Fusion In vitro reconstitution of the molecular mechanisms of vesicle tethering and membrane fusion D I S S E R T A T I O N zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Enrico Daniele Perini Dottore in Biotecnologie Mediche Dottore Magistrale in Biotecnologie Mediche e Medicina Molecolare geboren am 9. Juli 1984 in Milano, Italien Gutachter: Prof. Dr. Bernard Hoflack, Biotechnologisches Zentrum, Dresden Prof. Dr. Marino Zerial, Max Planck Institut für Molekulare Zellbiologie und Genetik, Dresden Eingereicht am: 19. Oktober 2012 Tag der Verteidigung: 21. März 2013 Erklärung entsprechend §5.5 der Promotionsordnung Hiermit versichere ich, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigthabe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht. Die Arbeit wurde bisher weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt. Die Dissertation wurde im Zeitraum vom 3/11/2008 bis 19/10/2012 verfasst und von Prof. Dr. Marino Zerial , Max Planck Institut für Molekulare Zellbiologie und Genetik, Dresden betreut. Meine Person betreffend erkläre ich hiermit, dass keine früheren erfolglosen Promotionsverfahren stattgefunden haben. Ich erkenne die Promotionsordnung der Fakultät für Mathematik und Naturwissen- schaften, Technische Universität Dresden an. ______________________________ Date, Signature i Acknowledgements I would like to express my gratitude to Marino Zerial, with whom I share not only the passion for membrane trafficking, but also the one for bikes and football. He has been a fair boss, an excellent biker, an expert football opinionist. I am particularly grateful to him for the freedom he has given me in my work, and for the possibility of having a technician working on my side. This has been helpful not only to “free my hands from the bench” as he once said, but also to learn how to manage and organize the work of other people. I therefore sincerely acknowledge Ramona, who has been working with me for almost three years now. Her technical assistance has been invaluable for many aspects of this thesis. My thanks go to Marcus Jahnel, who has been and will be teaming with me on the optical tweezers. His motivation has been a great inspiration for me. A special mention goes to the members of my thesis advisory committee Bernard Hoflack and Stephen Grill, for their being supportive at all levels. I have to acknowledge Takeshi Ohya who, although only for a few months, introduced me to the harsh experimental work of the in vitro reconstitution. I am also enormously thankful to the people that have been teaching me or helping me on the experimental part: Uenal Coskun, Michal Grzybek, Julio Sampaio, and Christian Klose for the lipid work; David Drechsel, Barbara Borgonovo, and Regis Lemaitre for the protein production and purification; Anna Shevchenko for the Mass Spectrometry; Giovanni Marsico and Thierry Galvez for the image analysis. I am particularly grateful to Sunando Datta, Thierry Galvez, and Claudio Collinet, former members of the Zerial lab, for the scientific discussions that are fuel that spawns passion for this job. To be acknowledged are also all the members of the Zerial lab, but in particular Angelika Giner for her efficiency. I want to thank Marisa McShane and Alex Kalinka whom I asked to read this manuscript, for their critical comments. Finally I want to thank my wife Mara who has been always supporting, especially during the frustrating times of my work. She is like a ray of sunshine between clouds at the end of my day. Mara together with my friends Rashim, René, Alex, Iva, Labi, Paco, Giovanni, Jerome and many others made me have a wonderful time here in Dresden. ii Summary Eukaryotic cells are populated by membrane-enclosed organelles possessing discrete molecular and biochemical properties. Communication between organelles is established by shuttling vesicles that transport proteins and other molecules. Vesicles bud from a donor organelle, travel in the cytosol, and are delivered to a target organelle. All these steps are regulated to ensure that cargoes are transported in a specific and directed manner. The focus of this thesis is on the last part of the journey of a vesicle: the process of vesicle targeting. Two phases can be distinguished in this process: vesicle tethering, defined as the first interaction between the shuttling vesicle and the target membrane, and membrane fusion, which is the mixing of the lipid bilayers and of lumen content. Both phases are mediated by a minimal set of molecular components that include one member of the family of Rab GTPases, a vesicle tethering factor, a phosphoinositide lipid, and four SNAREs together with their regulatory proteins. While many studies have investigated the molecular details of how SNAREs mediate membrane fusion, the process of vesicle tethering is less well understood. The overall scope of my study is to describe the molecular details of vesicle tethering and how they can contribute to the general process of vesicle targeting. To address this question I developed an in vitro assay where I reconstitute in vitro the process of vesicle tethering. This bottom-up approach allows the molecular dissection of cellular processes outside of the complex context of the cell. With this assay I have characterized the vesicle tethering abilities of individual proteins involved in vesicle tethering on early endosomes. I show that a minimal vesicle tethering machinery can be formed by the concomitant interaction between one vesicle tethering factor and a phosphoinositide on the membrane of one vesicle, and by a vesicle tethering factor and a Rab GTPase on the membrane of another vesicle. These results provide an explanation for how vesicle tethering contributes to the specificity of vesicle targeting and to the directionality of cargo transport. In particular, specificity of vesicle targeting can arise from the specific interaction between a Rab and a vesicle tethering factor that is an effector of the Rab. I show that the asymmetric distribution of binding sites in the structure of a vesicle iii tethering factor can generate a heterotypic vesicle tethering reaction that can account for the directionality of cargo transport. The outcome of this thesis emphasizes the role that vesicle tethering factors have in the self-organized system of vesicle trafficking of eukaryotic cells. To identify novel Rab5 effectors implicated in vesicle tethering, I carried out a Rab5-chromatography on mouse liver. Amongst other novel Rab5 effectors, I identify a multi-subunit vesicle tethering complex that was not previously characterized in mammalian cells. The complex, named CORVET, is conserved from yeast to humans and plays a major role in cell physiology since its removal causes embryonic death in mice. I define its subunits composition, determine its subcellular localization, and elucidate its role in cargo transport. This finding reconciles a disharmony between findings in mammals and yeast regarding the molecular machinery responsible for the conversion from early to late endosomes. I also show that the newly identified subunit of the mammalian CORVET complex is the only Rab5 effector to localize to autophagosomes. I hypothesise that it is through the CORVET complex that Rab5 is involved in the formation and maturation of autophagosomes. iv Table of contents 1. Preface – Epistemology..................................................................... 1 2. Introduction ....................................................................................... 2 2.1 Part I – Membrane trafficking ............................................................................... 2 2.1.1 Origin of the eukaryotic endomembrane system..................................................2 2.1.2 Principles of membrane trafficking........................................................................4 2.1.3 Forces involved in membrane fusion.....................................................................6 2.2 Part II: Cellular machineries for vesicle targeting ............................................. 8 2.2.1 Rab GTPases specify compartment identity..........................................................8 2.2.2 SNARE proteins mediate vesicle fusion ..............................................................14 2.2.3 SM proteins facilitate SNARE complex assembly...............................................17 2.2.4 Phosphoinositides localize proteins to membranes............................................18 2.2.5 Vesicle tethering factors bind vesicles to the acceptor compartment................21 2.2.6 Current model of vesicle targeting.......................................................................23 2.3 Part III – Experimental methods and models to study vesicle targeting...... 25 2.3.1 In vitro approaches to study vesicle fusion.........................................................25 2.3.2 Early endosomes as a model for membrane fusion ............................................26 2.3.3 Machineries for early endosomes tethering in yeast ..........................................27 2.3.4 Machineries for early endosomes tethering in mammals ..................................30 2.4 Part IV - Molecular mechanics of the transition from vesicle tethering to membrane fusion...........................................................................................................
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