Sec16 as an integrator of signaling to the endoplasmic reticulum Dissertation submitted for the degree of Doctor of Natural Sciences (Dr. rer. nat.) Presented by Kerstin Tillmann at the Faculty of Sciences Department of Biology University of Konstanz Date of the oral examination: September 11th, 2015 First referee: Prof. Dr. Daniel Legler Second referee: PD Dr. Hesso Farhan Third referee: Prof. Dr. Sebastian Springer Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-305723 Table of Contents Summary ........................................................................................ 1 Zusammenfassung ........................................................................ 2 Introduction .................................................................................... 3 The Secretory Pathway ........................................................................................... 4 1 Structure of the Secretory Pathway ..............................................................................4 2 Endoplasmic reticulum ...................................................................................................9 2.1 Protein translocation into the ER ...............................................................................9 2.2 Protein maturation in the ER lumen ....................................................................... 10 2.3 Quality control and ERAD ...................................................................................... 11 3 ER exit sites .................................................................................................................. 13 3.1 Cargo selection ....................................................................................................... 13 3.2 COPII ...................................................................................................................... 14 a. Sec12/Sar1 ........................................................................................................ 16 b. Sec23/Sec24 ..................................................................................................... 18 c. Sec13/Sec31 .................................................................................................... 20 d. Structure of the COPII coat .............................................................................. 21 3.3 Sec16 ..................................................................................................................... 23 3.4 Regulation of ER Export ......................................................................................... 28 4 Post-ER trafficking in the early secretory pathway .................................................. 31 4.1 COPI coat ............................................................................................................... 31 4.2 ERGIC .................................................................................................................... 33 4.3 Golgi ....................................................................................................................... 35 5 References .................................................................................................................... 38 Response of the early secretory pathway to the environment ........................... 59 1 Response of the early secretory pathway to signaling ............................................ 59 1.1 The MAPK signaling pathways ............................................................................... 59 1.2 Growth factor signaling via the ERK1/2 cascade ................................................... 61 1.3 The non-ERK1/2 cascades .................................................................................... 62 1.4 Regulation of MAPK cascades ............................................................................... 67 1.5 The Egr transcription factor family .......................................................................... 69 Table of Contents I 2 Integration of extracellular signaling to the early secretory pathway .................... 72 2.1 ERES ...................................................................................................................... 73 2.2 ERGIC .................................................................................................................... 75 2.3 Golgi ....................................................................................................................... 77 2.4 Evidence from screens for kinase/phosphatase regulation of the early secretory pathway ......................................................................................................................... 78 3 Response of the endomembrane system to nutrients and starvation ................... 80 4 References .................................................................................................................... 85 The secretory pathway in cell growth and cancer ............................................. 101 1 Signaling pathways controlling proliferation and cell growth .............................. 101 2 The role of the secretory pathway in proliferation ................................................. 104 3 The role of the ER-to-Golgi trafficking machinery in cancer ................................. 106 3.1 The ER stress response ....................................................................................... 107 3.2 The response of the early secretory pathway to increased cargo load ................ 110 4 References .................................................................................................................. 111 Aim of the thesis .................................................................................................. 120 Materials and Methods .............................................................. 121 1 Cell culture and transfection ..................................................................................... 122 2 Immunofluorescence staining .................................................................................. 122 3 ERES quantification ................................................................................................... 122 4 In vitro recruitment assay ......................................................................................... 122 5 Retention Using Selective Hooks (RUSH) assay .................................................... 123 6 Fluorescence Recovery After Photobleaching (FRAP) .......................................... 123 7 Fluorescence Correlation Spectroscopy (FCS) ...................................................... 124 8 Modeling ...................................................................................................................... 125 9 Regulatory sequence analysis ................................................................................. 125 10 Cell lysis and Western Blotting .............................................................................. 125 11 Co-immunoprecipitation .......................................................................................... 126 12 Subcellular fractionation assay .............................................................................. 126 13 References ................................................................................................................ 127 Table of Contents II Results ........................................................................................ 128 Regulation of Sec16A at the transcriptional and posttranslational level links proliferation and secretion .................................................................................. 129 1 Sec16A integrates growth factor signaling at the level of ERES .......................... 129 2 Absence of growth factor signaling decreases Sec16A synthesis ...................... 133 3 Sec16A expression might be controlled by Egr1+3 transcription factors ........... 138 4 Sec16A as part of a coherent feed-forward loop (CFFL) ....................................... 146 5 Growth factor treatment increases ERES number and alters Sec16A dynamics 146 6 Interaction with COPII modulates the turnover of Sec16A on ERES .................... 155 7 Interaction with COPII is required for Sec16A to generate more ERES ............... 158 8 Cell proliferation is dependent on Sec16A .............................................................. 161 9 Summary ..................................................................................................................... 166 10 References ................................................................................................................ 167 Characterization of the role of TECPR2 in the early secretory pathway .......... 168 1 Background information ........................................................................................... 168 2 Results ........................................................................................................................ 169 3 Summary ..................................................................................................................... 175 4 References .................................................................................................................. 176 Discussion .................................................................................. 177 1 Sec16A as an integrator of signaling and nutritional stimuli ......................