A Role for Amphiphysin in AP-1/Clathrin Coat Formation Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Sonja Huser Studer aus Knonau (ZH) Basel, 2012 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Martin Spiess Prof. Anne Spang Basel, den 11. Dezember 2012 Prof. Dr. Jörg Schibler Acknowledgements I would like to thank Prof. Martin Spiess for giving me the opportunity to work on this project, for continuous support, and for many fruitful discussions. Special thanks go to Dr. Gregor Suri, who initially started this project, and to Dr. Pascal Crottet for his help and expertise during experiments and for being a walking encyclopedia. Many thanks go to Nicole Beuret for her professional technical advice and assistance and for her patience to answer the countless questions. I would also like to thank the past and present lab members for creating a great working atmosphere, for scientific and other discussions, and for their support at all times: Cristina Baschong, Dr. Julia Birk, Dominik Buser, Erhan Demirci, Dr. Michael Friberg, Franziska Hasler, Dr. David Hirschmann, Tina Junne, Simone Kälin, Dr. Lucyna Kocik, Dr. Deyan Mihov, and Dr. Barry Shortt. Summary Transport of cargo within the endocytic and secretory pathway is generally mediated by coated vesicles. Clathrin, in combination with different adaptor proteins, is the major coat protein for vesicle formation at the plasma membrane, endosomes, and the trans-Golgi network (TGN). Best characterized is the formation of clathrin coats for endocytosis at the plasma membrane involving the adaptor protein complex AP-2. Clathrin and AP-2 were shown to be at the centre of a complex interactome of proteins accessory to vesicle formation. Considerably less is known about the formation of clathrin coated carriers at the TGN and endosomes, where the adaptor protein complex AP-1 plays a major role. In vitro studies showed the minimal requirements for association of AP-1 to liposomal membranes to be activated ARF1, phosphoinositides, and either sorting signals or unknown cytosolic factors. We have used a liposome floatation assay to identify cytosolic proteins collaborating with AP-1 at the membrane. Separation of proteins from bovine brain cytosol with several chromatographic methods yielded an active fraction containing amphiphysin 1, amphiphysin 2, and endophilin A1. All three proteins are expressed in brain and known to be involved in AP-2/clathrin coat formation. They consist of an N- terminal N-BAR (Bin, amphiphysin, Rvs) domain for dimerization and membrane binding and a C-terminal SH3 (Src homology 3) domain for interaction with dynamin and synaptojanin. Amphiphysin 1 and 2 in addition contain a middle domain with binding sites for adaptors and clathrin. It was proposed that amphiphysins and endophilin are targeted to membranes with high curvature, such as the neck of a forming vesicle, where they recruit dynamin and synaptojanin in preparation for vesicle fission and uncoating. In this thesis, I bacterially expressed and purified all three proteins and tested them in the floatation assay for AP-1 membrane binding activity. Only amphiphysin 2 showed activity, both as a homodimer and as a heterodimer with amphiphysin 1. Activity depended on a motif that was shown to bind to AP-1, AP-2, and clathrin in GST pull-down experiments. Endogenous amphiphysins in primary neurons, as well as transiently expressed in neuronal or fibroblast cell lines, co-localized with AP-1 at the TGN. In addition, when expressed at high levels in neuronal cells, amphiphysins aggregated and interfered dominantly with the TGN localization of AP-1. Both phenomena depended on the presence of the clathrin and adaptor interaction sequence in the amphiphysins. Furthermore, both amphiphysins could be cross-linked to AP-1 in vivo. Our results indicate that amphiphysin 1 and 2 function not only in clathrin coated vesicle formation for endocytosis at the plasma membrane, but are also part of the machinery forming AP-1/clathrin coats at the TGN and endosomes. This suggests that the machineries for CCV formation with AP-1 and AP-2 at different locations in the cell share more components than previously anticipated. Table of contents 5 Table of Contents Abbreviations 6 Introduction 10 1. Intracellular transport 10 1.1 The secretory pathway 11 1.1.1 ER transport 11 1.1.2 ER to Golgi transport 12 1.1.3 Intra-Golgi transport 13 1.1.4 Post-Golgi transport at the TGN 14 1.2 The endocytic pathway 16 1.3 The recycling system 18 1.4 Retrograde transport from the endosomes to the TGN 19 2. Coated transport vesicles 21 2.1 Steps of vesicular transport 21 2.2 COPI 24 2.3 COPII 26 2.4 Clathrin coated vesicles 28 2.4.1 Clathrin 29 2.4.2 CCV formation 30 2.4.3 The clathrin adaptor interactome 31 2.4.4 Clathrin adaptors 32 2.4.5 Accessory factors 44 Aim of the Thesis 60 Material and Methods 61 Material 61 Methods 64 Results 72 Cytosolic factors contribute to membrane association of CCV-derived or cytosolic AP-1 to 73 peptide-free liposomes Purification and identification of the cytosolic activity 75 Amphiphysin 1, Amphiphysin 2, and Endophilin A1 77 Purification of Amphiphysin 1, Amphiphysin 2, and Endophilin A1 78 Purified Amphiphysins and Endophilin are present on floating liposomes 80 Amphiphysin 2 mediates AP-1 association with liposomal membranes 81 Amphiphysin 2-mediated liposome association of AP-1 depends on the Amphiphysin 2 82 WDLW motif in its middle domain Combinations of purified proteins did not enhance AP-1 membrane association 83 Amphiphysin 1 and 2 heterodimers mediate AP-1 membrane binding 84 Amphiphysin 2 also mediates binding of AP-2 to liposomal membranes 85 Amphiphysin 1 middle domain M1 is not functional 86 Table of contents 6 Amphiphysin 1 co-localizes with AP-1 at the TGN in primary neurons 87 Amphiphysin 1 and 2 co-transfect and co-localize with AP-1 at the TGN in transfected 88 neuronal cells Amphiphysin 1 and Amphiphysin 2 homodimers co-localize with AP-1 at the TGN region 90 TGN localization of amphiphysins depends on the CLAP domain 91 Amphiphysin co-localizes with clathrin and dynamin at the TGN 93 High expression levels of amphiphysin lead to its aggregation and interfere with TGN 94 localization of AP-1 Amphiphysin localizes to the TGN in an ARF1-dependent manner 96 Amphiphysin 1 and 2 can be cross-linked to AP-1 in vivo 98 Amphiphysin and AP-1 interact independently of clathrin 99 Amphiphysin is not essential for AP-1 localization to the TGN 101 Amphiphysins are not the only proteins stabilizing AP-1 on liposomal membranes 102 Discussion 103 Amphiphysins act at the plasma membrane in endocytosis 103 A role for amphiphysins in CCV formation at the TGN 104 Amphiphysin is not only involved in endocytosis 105 One motif in amphiphysin for adaptor and clathrin binding 106 Amphiphysin 1 middle domain is not functional 106 Endophilin is not in a stable complex with amphiphysin 107 Non-essential proteins function in tissues with high activity 107 References 111 Curriculum vitae 136 Abbreviations 7 Abbreviations AAK1 Adaptor associated kinase 1 AP-1, -2, -3, - 4 Adaptor protein complex 1, 2, 3, 4 ARF ADP-ribosylation factor ARP2/3 Actin-related protein 2/3 BAR Bin, amphiphysin, Rvs BDNF Brain-derived neurotrophic factor BFA Brefeldin A BIN1 Box-dependent myc-interacting protein-1 Bip Binding immunoglobulin protein CALM Clathrin assembly lymphoid myeloid leukemia CCP Clathrin coated pit CCV Clathrin coated vesicle CHC Clathrin heavy chain CLASP Clathrin-associated sorting protein CLC Clathrin light chain CME Clathrin mediated endocytosis COPI/II Coat protein I/II DAPI 4’,6-Diamidino-2-phenylindole EEA1 Early endosome antigen 1 EGF Epidermal growth factor EH Epsin homology EM Electron microscopy ENTH Epsin N-terminal homology Abbreviations 8 EPS15 Epidermal growth factor protein substrate 15 Epsin1 EPS15 interacting protein ER Endoplasmic reticulum ERAD ER-associated degradation ERC Endocytic recycling compartment ERES ER exit site ERGIC ER-Golgi intermediate compartment ESCRT Endosomal sorting complex required for transport FCHo Fer/Cip4 homology domain-only GAE γ-adaptin ear GAG Glycosaminoglycan GAK cyclin-G-associated kinase GAP GTPase-activating protein GAT GGA and TOM GBF1 Golgi-specific brefeldin A resistance factor 1 GEF Guanine nucleotide exchange factor GGA Golgi-localized, γ-ear containing, ARF-binding protein GMP-PNP Guanylyl imidodiphosphate GPCR G-protein coupled receptors GPI Glycophosphatidylinositol HSC70 Heat shock cognate 70 HSP Heat shock protein IF Immunofluorescence IPTG Isopropyl β-D-1-thiogalactopyranoside LAMP1 Lysosome-associated membrane protein 1 LDL Low-density lipoprotein LDLR Low-density-lipoprotein receptor Abbreviations 9 MMCC-DOPE (N-((4-maleimidylmethyl)cyclohexane-1-carbonyl)-1,2-dioleolyl-sn-glycero-3- phospho-ethanolamine MPR Mannose 6-phosphate receptor MTOC Microtubule organizing center MVB Multivesicular body NCS Newborn calf serum NGF Nerve growth factor NTID N-terminal insert domain N-WASP Neural Wiskott-Aldrich syndrome protein PA Phosphatic acid PC Phosphatidylcholine PCR Polymerase chain reaction PDI Protein disulfide isomerase PE Phosphatidylethanolamine PFA Paraformaldehyde PH Pleckstrin homology PMSF Phenylmethylsulfonyl fluoride PRD Proline rich domain PS Phosphatidylserine PtdIns(3)P Phosphatidylinositol-3-phosphate PtdIns(3,5)P2 Phosphatidylinositol (3,5)-bisphosphate PtdIns(4)P Phosphatidylinositol-4-phosphate