Identification and characterization of Dop1p as an essential component of the Neo1p-Ysl2p-Arl1p membrane remodeling complex [Elektronische Ressource] / vorgelegt von Sónia Cristina de Oliveira Barbosa

Sonia C. De Oliveira Barbosa

Sujets

• Biologie

Informations

• Publié par: universitat_stuttgart
• Publié le: 01 janvier 2011
• Nombre de lectures: 133
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Identification and characterization of Dop1p as
an essential component of the Neo1p-Ysl2p-Arl1p
membrane remodeling complex






Von der Fakultät Energie-, Verfahrens- und und Biotechnik der Universität Stuttgart zur
Erlangung der Würde eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte
Abhandlung




Vorgelegt von Dipl.-Bioch.
Sónia Cristina de Oliveira Barbosa
geboren in Paris (Frankreich)



Hauptberichter: Prof. Dr. Dieter H. Wolf
Mitberichter: Privatdozentin Dr. Birgit Singer-Krüger






Tag der mündlichen Prüfung: 14.01.2011




Institut für Biochemie der Universität Stuttgart
2011


































Hiermit versichere ich, dass ich die Arbeit selbst verfasst und dabei keine enderen als
die angegebenen Quellen und Hilfsmittel verwendet habe.
Stuttgart, den 23.06.2010



Sónia Cristina de Oliveira Barbosa
Table of contents

Abbreviation...................................................................................................................vi
Abstract ........................................................................................................................viii
Zusammenfassung .......................................................................................................... x
1 Introduction .............................................................................................................. 1
1.1 The secretory and endocytic pathways are interconnected at the TGN/endosomes 1
1.2 Vesicular transport................................................................................................... 3
1.2.1 Vesicle formation ................................................................................................. 3
1.2.2 Vesicle targeting and fusion................................................................................. 7
1.2.3 The role of phosphoinositides as regulators of vesicular transport ...................... 7
1.3 Cellular mechanisms of membrane remodelling and membrane curvature
generation.......................................................................................................................... 9
1.4 P -ATPases: their role in maintaining lipid asymmetry and in vesicle-mediated 4
transport 11
1.4.1 Transbilayer lipid asymmetry in biological membranes .................................... 11
1.4.2 P -ATPases are prime candidates for being aminophospholipid translocases ... 13 4
1.4.3 The Cdc50 family members are β-subunits of the P -ATPases ......................... 16 4
1.4.4 Role of P -ATPases in vesicle-mediated transport............................................. 17 4
1.5 Scope of this work................................................................................................. 20
2 Materials and Methods .......................................................................................... 22
2.1 Materials................................................................................................................ 22
2.1.1 Saccharomyces cerevisiae strains ...................................................................... 22
2.1.2 Escherichia coli strain ........................................................................................ 24
2.1.3 Plasmids.............................................................................................................. 25
2.1.4 Kits and enzymes used for molecular biology ................................................... 26
2.1.5 Chemicals ........................................................................................................... 26
2.1.6 Media.................................................................................................................. 27
2.1.6.1 Yeast media ........................................................................................................ 27
2.1.6.2 E. coli media....................................................................................................... 28
2.2 Methods ................................................................................................................. 28
2.2.1 S. cerevisiae and E. coli growth ......................................................................... 28
2.2.1.1 Yeast cell cultures .............................................................................................. 28
2.2.1.2 Yeast growth test................................................................................................ 28
ii 2.2.1.3 E. coli cell cultures ............................................................................................. 29
2.2.2 Molecular biology methods................................................................................ 29
2.2.2.1 Generation of DNA constructions...................................................................... 29
2.2.2.1.1 Mutagenesis of the PPSY motif present in the C-terminal tail of Neo1p ....... 29
2.2.2.1.2 Cloning the DOP1 gene................................................................................... 30
2.2.2.1.3 Generation of pRS315-P -GFP-DOP1 and pRS315-P -GFP-dop1-3.. 31 ADH1 ADH1
2.2.2.1.4 Generation of GFP fused domains of Dop1p .................................................. 32
2.2.2.2 Generation of strains by homologous recombination......................................... 33
2.2.2.3 Mating, sporulation and dissection of S. cerevisiae cells................................... 36
2.2.2.4 Transformation of S. cerevisiae cells ................................................................. 36
2.2.3 Protein biochemistry methods ............................................................................ 37
2.2.3.1 Preparation of yeast cell extracts........................................................................ 37
2.2.3.2 Western blotting ................................................................................................. 37
2.2.3.2.1 SDS-PAGE...................................................................................................... 37
2.2.3.2.2 Protein transfer to nitrocellulose membranes.................................................. 38
2.2.3.2.3 Immunodetection............................................................................................. 39
2.2.3.3 Immunoprecipitation experiments...................................................................... 41
2.2.3.3.1 Preparation of cell aliquots.............................................................................. 41
2.2.3.3.2 General procedure for immunoprecipitation................................................... 41
2.2.3.3.3 Immunoprecipitation of Cdc50p-13-Myc and 3-HA-Neo1p .......................... 42
2.2.3.3.3.1 Immunoprecipitation from total membranes................................................ 42
2.2.3.3.3.2 Immunoprecipitation from total cell extracts............................................... 43
2.2.3.3.4 Determination of the extent of protein solubilisation...................................... 43
2.2.3.3.5 Co-immunoprecipitation experiments using GFP tagged Dop1p, Nterm,
internal and Cterm domains of Dop1p............................................................................ 44
2.2.3.3.6 Immunoprecipitation of HA-Neo1p to detect ubiquitination.......................... 44
2.2.3.4 Co-isolations with Dop1p-TAP.......................................................................... 45
2.2.3.5 Pulse-chase analysis ........................................................................................... 47
2.2.4 Cell biology methods.......................................................................................... 48
2.2.4.1 Fluorescence microscopy ................................................................................... 48
2.2.4.1.1 Indirect immunofluorescence 48
2.2.4.1.2 GFP fluorescence ............................................................................................ 49
2.2.4.1.3 Alexa598-conjugated alpha-factor uptake and visualisation .......................... 49
2.2.4.1.4 Nuclei staining with Hoechst 33342 ............................................................... 50
iii 2.2.4.1.5 Actin staining with Rodamine phaloidin......................................................... 50
2.2.4.2 CPY missorting test............................................................................................ 51
3 Results...................................................................................................................... 52
3.1 The P4-ATPase Neo1p does not interact with Cdc50p......................................... 52
3.2 Dop1p is physically associated with the Neo1p-Ysl2p-Arl1p network ................ 54
3.2.1 Dop1p interacts with Neo1p and Ysl2p ............................................................. 54
3.2.2 The interaction between Dop1p and Neo1p is independent from Ysl2p............ 57
3.3 Dop1p, Neo1p and Ysl2p are interdependent........................................................ 58