TECPR2 cooperates with LC3C to regulate COPII dependent ER export Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich 15 Biowissenschaften der Johann Wolfgang Goethe – Universität in Frankfurt am Main von Daniela Stadel aus Karlsruhe Frankfurt 2015 D30 vom Fachbereich 15 der Johann Wolfgang Goethe – Universität als Dissertation angenommen. Dekanin: Prof. Dr. Meike Piepenbrink Gutachter: Prof. Dr. Heinz D. Osiewacz Dr. Christian Behrends Datum der Disputation: __________________________________________________________________________ Content 1. Introduction ....................................................................................................................... 9 1.1 Hereditary spastic paraparesis (HSP) ....................................................................... 9 1.1.1 Types and clinical aspects of HSP ..................................................................... 9 1.1.2 Tectonin β-propeller containing protein 2 (TECPR2) ....................................... 11 1.2 The secretory pathway ............................................................................................ 12 1.2.1 COPII coat formation ........................................................................................ 13 1.2.2 ERES ............................................................................................................... 14 1.2.3 Role of SEC24D ............................................................................................... 15 1.2.4 Regulation and assisting factors of the secretory pathway .............................. 15 1.2.5 Role of ER architecture in HSP disease .......................................................... 16 1.3 The endocytic pathway ............................................................................................ 17 1.3.1 HOPS complex ................................................................................................. 18 1.3.2 BLOC-1 complex .............................................................................................. 20 1.4 Autophagy ............................................................................................................... 21 1.4.1 The ubiquitin like conjugation system .............................................................. 23 1.4.2 HOPS and BLOC-1 in autophagy .................................................................... 26 1.5 Aim of the present work ........................................................................................... 27 2. Material and Methods ..................................................................................................... 28 2.1 Materials .................................................................................................................. 28 2.1.1 Chemicals ........................................................................................................ 28 2.1.2 Buffers and media ............................................................................................ 30 2.1.3 Kits ................................................................................................................... 33 2.1.4 Vectors, oligonucleotides and siRNAs ............................................................. 34 2.1.5 Antibodies ........................................................................................................ 38 2.2 Methods ................................................................................................................... 39 2.2.1 Molecular biological methods ........................................................................... 39 2.2.2 Cell biological methods .................................................................................... 43 2.2.3 Protein biochemical methods ........................................................................... 45 2.2.4 Biophysical and structural methods ................................................................. 49 I __________________________________________________________________________ 2.2.5 Phenotypical assays ........................................................................................ 51 3. Results ............................................................................................................................ 53 3.1 TECPR2 associates and colocalizes with SEC24D, BLOC-1 and HOPS ............... 53 3.2 TECPR2 binds LC3 and GABARAP proteins in a LIR-dependent manner ............. 57 3.3 Association with SEC24D, BLOC-1 and HOPS requires LC3 binding of TECPR2 . 66 3.4 TECPR2 depletion destabilizes SEC24D, HOPS and BLOC-1 ............................... 69 3.5 Loss of TECPR2 compromises ERES .................................................................... 73 3.6 TECPR2 is required for ER export .......................................................................... 77 3.7 TECPR2 depletion affects ER morphology ............................................................. 81 3.8 TECPR2 contributes to autophagosome formation ................................................. 83 4. Discussion ...................................................................................................................... 90 4.1 Model: TECPR2 as scaffold for endomembrane signaling ...................................... 90 4.2 Assembly of the inner COPII coat ........................................................................... 92 4.3 HOPS ...................................................................................................................... 95 4.4 BLOC-1 ................................................................................................................... 96 4.5 LIR motif of TECPR2 and binding preference ......................................................... 97 4.6 ER export and autophagy ........................................................................................ 98 4.7 ER maintenance and neurodegeneration .............................................................. 101 4.8 Concluding remarks and future perspectives ........................................................ 103 5. References ................................................................................................................... 106 Zusammenfassung .............................................................................................................. 118 Danksagung ......................................................................................................................... 123 Curriculum vitae ................................................................................................................... 125 Publications ......................................................................................................................... 126 Presentations at conferences .............................................................................................. 127 Eidesstattliche Erklärung ..................................................................................................... 128 II __________________________________________________________________________ Abbreviations µ micro A Ampere Å Ångström aa aminoacid ABC ammonium bicarbonate ACN acetonitril ACTBL2 Beta-actine-like protein 2 AIM ATG interacting motif ALFY autophagy-linked FYVE protein α alpha, anti ANP32A Acidic leucine-rich nuclear phosphoprotein 32 family member A APS ammonium persulfate APSM average peptide spectral matches Asp aspartic acid ATG autophagy-related protein ATL1, SPG3A atlastin 1 BafA1 Bafilomycin A1 BCA bicinchoninic acid β beta BLOC-1 biogenesis of lysosome-related organelles complex 1 BLOS-1/2/3 biogenesis of lysosome-related organelle complex subunit-1/2/3 Btz Bortezomib °C degree celsius C.elegans Caenorabditis elegans CALR calreticulin CKAP4, CLIMP63 cytoskeleton-associated protein 4 CMA chaperone-mediated autophagy CMV cytomegalo virus CNO cappuccino COL10A1,COL1A1 collagen alpha-1 chain COPI/II coat protein complex I/II CORVET class c core vacuole/endosome tethering 1 __________________________________________________________________________ CPNC condensed perinuclear cluster CSP chemical shift perturbation C-terminal carboxy terminal D Aspartic acid DAPI 4,6-diamidin-2-phenylindol DAVID Database for Annotation, Visualization, and Integrated Discovery del deletion DFCP1 double FYVE-contining protein 2 DIC dynein intermediate chain DKFZ Deutsches Krebsforschungszentrum DMEM Dulbeccos modified eagle medium DMSO dimethylsulfoxide DNA deoxyribonucleic acid DTBP dimethyl-3,3-dithiobispropionimidate DTNBP1 dysbindin 1 DTT dithiotreitol DUB deubiquitinase E.coli Escherichia coli e.g. latin: exemplar gratia, english: for example E1/2/3 enzyme ECL Enhanced chemilumiscence EDTA ethylenediaminetetraacetic acid EGFR epidermal growth factor receptor EM Electron microscopy ER endoplasmic reticulum ERES ER exit sites ERGIC ER-to-Golgi intermediate compartment ESCRT escort complex FRAP fluorescence recovery after photobleaching FBS fetal bovine serum fig. figure FIP200, RB1CC1 RB1-inducible coiled-coil protein 1 FYCO1 FYVE and coiled-coil domain-containing protein 1 g gravity 2 __________________________________________________________________________ GABA gamma aminobutyric acid GABARAP gamma-aminobutyric acid receptor-associated protein GABARAPL1/2 gamma-aminobutyric acid receptor-associated protein-like1/2 GATE-16 gamma-aminobutyric acid receptor-associated protein-like 2 GAP guanosine triphosphatase activting protein GAT1