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Ricardaâ•Žs Doktorarbeit Insights into membrane binding of PROPPINs and Reconstitution of mammalian autophagic conjugation systems Dissertation for the award of the degree "Doctor rerum naturalium" (Dr. rer. nat.) of the Georg-August-Universität Göttingen in the GGNB program of Biomolecules: Structure - Function - Dynamics of the Georg-August University School of Science (GAUSS) submitted by Ricarda Angela Busse from Leinefelde, now Leinefelde-Worbis, Germany Göttingen, 2012 Members of the Thesis Committee: Dr. Karin Kühnel (1st Reviewer) Department of Neurobiology, Max Planck Institute for Biophysical Chemistry Prof. Dr. Michael Thumm (2nd Reviewer) Department of Biochemistry II, University of Göttingen Prof. Dr. Nils Brose Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine Date of the oral examination: January 08th, 2013 Declaration of Authorship Declaration of Authorship Hereby, I confirm that I have created this work Insights into membrane binding of PROPPINs and Reconstitution of mammalian autophagic conjugation systems entirely on my own and that I have only used the sources and materials cited. Göttingen, November 14th, 2012 Ricarda Angela Busse V VI VII For Patrick Acknowledgements First and foremost I would like to thank my advisor Dr. Karin Kühnel for giving me the opportunity to do the research in her lab. Without her supervision and training this thesis would not have been possible. Her door was always open for questions and discussions. Moreover, I would like to thank Prof. Dr. Reinhard Jahn for offering the generous financial support during my thesis and for the regular discussions we had about my work during the department seminars. Next, I would like to express my gratitude to my thesis committee formed by Prof. Dr. Nils Brose and Prof. Dr. Michael Thumm. A special thanks goes to my close collaborator in Thumm lab, Dr. Roswitha Krick. In addition, I would like to thank Prof. Dr. Reinhard Lührmann for access to his insect cell culture laboratory and crystallization facility. In terms of insect cell culture expertise, my gratitude goes to Karine dos Santos and her excellent training. Moreover, I would like to thank Dr. Vladimir Pena and his group, especially Dr. Jana Schmitzova for access and guidance to their infrastructure such as the Thermofluor experiment. Furthermore, I would like to mention the help of Monika Raabe and He-Hsuan Hsiao from the department of Prof. Dr. Henning Urlaub for the assistance with the mass spectrometry analysis for which I am grateful. I want to thank my colleagues and friends Andreea, Amanda and Caroline. They were all helpful with nice discussions about science and beyond. Especially, I would like to mention Andreea’s help during the last period of my thesis that made it possible to finalize all the repetitive experiments in a very short time. Besides having a great colleague, I also won a wonderful friend in you. Next I would like to express my appreciation of the Jahn department in which I have worked for three and a half years. It was a nice time both from the exciting scientific discussions and friendly atmosphere. First and foremost, I would like to thank Dr. Gottfried Mieskes for his assistance in everything ranging from machine maintenance to IT support. Heike, who has probably the most important but least appreciated tasks in the lab. Ursel and Matias for their assistance in the liposome related experiments: IX X DLS, MALLS, LUVs and phosphate determination. In addition, I am thankful to Esra and Angel for their helpful discussions related to ITC measurements. From Janina I had helpful insights into Cova PIP plates and the pH switch data analysis. I also want to say thank you to Michaela and Dagmar for their technical support. I want to thank all the people in the Jahn department that touched my life over the period of my PhD thesis and for becoming so good friends: Beyenech, Julia, Hale, Sina, Dieter, Sabrina, Saskia, Momchil, Elisa, Zohreh, John, Yongsoo, Seichii, Dominika, Dragomir, Partho, Brigitte, Peter and Barbara. Very important to me is my family and their love. First of course my husband Patrick, who is at my side since the time before I started to study. He supported me during my Diploma project and now we also managed the PhD thesis. Without him at my side I would not have been strong enough to succeed in all my tasks and plans. In addition, I thank my parents and parents-in-law, who provided all their possible support. I want to express my gratitude to my sister, my brothers and their partners for completing the supportive atmosphere of a family. Finally, I want to say that I am proud of being a member of GGNB. This institution provides so many resources for a young scientist, from which I was able to take advantage and helped me in developing as a scientist. There are many more people I have to thank! Abstract Autophagy is a degradation pathway conserved in eukaryotes. Upon induction of au- tophagy a double layered membrane is formed de novo and engulfs the cytosolic content. After fusion of the membrane, an autophagosome vesicle is formed, which then fuses with the vacuole (or lysosome) where its content is degraded. PROPPINs, β-propeller proteins that bind polyphosphoinositides, play a role in autophagy and phosphoinosi- tide binding depends on a conserved FRRG motif. The three yeast PROPPINs Atg18, Atg21 and Hsv2 are involved in different subtypes of autophagy. In this study, I purified different Atg18, Atg21 and Hsv2 PROPPIN homologs and showed that they bind specifically to PI3P and PI(3,5)P2 using protein-liposome co- flotation assays. Recently, we published the first structure of the PROPPIN Hsv2. Based on our structure I performed mutagenesis studies to probe phosphoinositide bind- ing of Hsv2. I analyzed phosphoinositide binding of the alaninine mutants with liposome flotation assays. I identified conserved residues essential for binding right and left of the FRRG motif, indicating the presence of two phosphoinositide binding sites, which was an unexpected finding. Using ITC measurements I then confirmed the binding sto- ichiometry of two phosphoinositides to one Hsv2 molecule and determined the binding affinities of PROPPINs to both PI3P and PI(3,5)P2 incorporated in small unilamellar vesicles. Phosphoinositide binding of S. cerevisiae Hsv2 is pH dependent. Acidic envi- ronment increases and basic environment decreases the affinity. In addition, I showed the involvement of loop 6CD in membrane binding. Mutagenesis analysis of loop 6CD residues revealed that membrane insertion is dependent on both ionic and hydrophobic interactions. Two ubiquitin-like conjugation systems modifying Atg8 (in mammals MAP1LC3) and Atg12 are essential for autophagy. Homologs of the canonical ubiquitin conjugation system, E1- and E2-like enzymes, are involved in the conjugation of Atg8 and Atg12 to their specific targets phosphatidylethanolamine and Atg5, respectively. XIII XIV A in vivo reconstitution system for the two human ubiquitin-like conjugation sys- tems Atg12 and MAP1LC3 was established using the MultiBac baculovirus expression system in insect cells. This allowed full length expression of the involved proteins and purification of the Atg5-Atg12 conjugate and lipidated MAP1LC3 in small yields. Contents 1 General introduction 1 1.1 Definitionofautophagy ........................... 1 1.2 Typesofautophagy ............................. 1 1.3 Molecularplayersinautophagy . 3 1.4 Structural characterization of autophagy proteins . ...... 3 1.5 Involvementofautophagyindisease . 4 2 Materials & Methods 7 2.1 Materials ................................... 7 2.1.1 Chemicals............................... 7 2.1.2 Enzymes................................ 8 2.1.3 Lipids . 8 2.1.4 Kits .................................. 9 2.1.5 Columnsforchromatography . 10 2.1.6 Antibodies .............................. 11 2.1.7 Buffersandmedia .......................... 11 2.1.8 Antibiotics . 13 2.1.9 Insect cell lines and bacterial strains . 13 2.1.10 DNAconstructs............................ 14 2.1.11 Oligonucleotides . 15 2.2 Methods.................................... 20 2.2.1 Molecular cloning . 20 2.2.2 Methodsforinsectcellculture . 22 2.2.2.1 Insect cell culture . 22 2.2.2.2 Bacmid extraction from E. coli DH10 .......... 22 2.2.2.3 Transfection of insect cells . 23 2.2.2.4 Pull down of infected insect cells . 23 2.2.2.5 Measuring YFP in infected insect cells . 24 XV XVI CONTENTS 2.2.3 Purification protocols of protein complexes from insect cells . 24 2.2.3.1 MAP1LC3 ......................... 24 2.2.3.2 Atg5-Atg12......................... 25 2.2.3.3 Increase of solubility of Atg5-Atg12 using a detergent screen............................ 26 2.2.4 Purification protocols for PROPPINs . 27 2.2.4.1 Batch purification for test expression . 27 2.2.4.2 Purification protocol for ScAtg18 . 27 2.2.4.3 Purification of PaAtg18, DmAtg18, CeAtg18, KlAtg21 andPaAtg21 ........................ 28 2.2.4.4 Purification protocol for SpHsv2 . 30 2.2.4.5 Purification protocol for ScHsv2 and mutants . 32 2.2.4.6 GST SpinTrap purification of ScHsv2 and mutants . 33 2.2.4.7 Purification protocol for KlHsv2 and mutants . 34 2.2.5 Purification protocol for PaAtg8 and KlAtg8 . 35 2.2.6 Biochemicalmethods......................... 36 2.2.6.1 SDS-PAGE and Western Blotting . 36 2.2.6.2 Protein stability assay . 37 2.2.6.3 Circular dichroism spectroscopy . 38 2.2.6.4 Limited proteolysis . 39 2.2.6.5 N-terminal protein sequencing . 39 2.2.6.6 Protein-lipid co-flotation assay and liposome preparation 40 2.2.6.7 Isothermal titration calorimetry . 42 2.2.6.8 Protein-lipid overlay assay (PIP strip, PIP array, mem- brane lipid strip) . 43 2.2.6.9 Cova PIP specificity plate . 43 2.2.6.10 Analytical gel filtration . 44 2.2.7 Crystallization and structure determination . 46 2.2.7.1 Crystallization screen setup . 46 2.2.7.2 Flash cooling of crystals . 47 3 Project I: Reconstitution of mammalian Atg12 and MAP1LC3 con- jugation pathways 49 3.1 Introduction.................................
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