Steps to reconstitute in vitro a complete round of COPI vesicle budding, uncoating and fusion [Elektronische Ressource] / presented by Hatim Jawhari

Dissertation Submitted to the Combined Faculties for The Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by Hatim Jawhari, MS. Rabat, Morocco Oral examination: Steps to Reconstitute in vitro a Complete Round of COPI vesicle Budding, Uncoating and Fusion Referees: Prof. Dr. Felix T. Wieland Prof. Dr. Michael Brunner A Julia, pour son soutien de tous les instants iContents Abstract 1 Introduction 2 1 Protein sorting within the eukaryotic cell 3 1.1 Protein sorting to mitochondria and chloroplasts 5 1.2 Protein sorting to peroxisomes 5 1.3 The Classical secretory pathway 2 Golgi vesicular transport, a focus on COPI coated vesicles 9 2.1 Recruitment of COPI coat components to the membrane 9 2.2 COPI coat formation 11 2.3 In vitro generation of COPI coated vesicles from liposomes 11 2.4 SNAREs in the in vitro budding assay 12 Results 13 1 Cloning, expression and purification of proteins 15 1.1 GST-fusion SNAREs 15 1.2 The p24 family members 18 1.3 Native yeast ARF1p and human ARF1 19 1.3.1 Yeast ARF1p 20 1.3.2 Human ARF1 25 1.4 Expression, purification and refolding of Glo3p, an ARF GTPase activating protein (GAP) in E. coli 27 1.