Molecular mechanisms of COPI vesicle biogenesis [Elektronische Ressource] / presented by Rainer Beck

Rainer Bauböck

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 13 Mo

Extrait

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
Rainer Beck, Diplom-Biologe
born in Berchtesgaden
Oral examination:...................





Molecular Mechanisms of COPI Vesicle
Biogenesis






Referees: Prof. Dr. Felix Wieland
Prof. Dr. Walter Nickel
2




Für Katharina
3
Table of Contents
Abstract ............................................................................................................................................ 9
Abstract (German version) ............................................................................................................. 11
Abbreviations ................................................................................................................................. 13
Introduction .................................................................................................................................... 14
The secretory pathway ........................................................................................................ 14
COPI- mediated Transport ................................................................................................. 15
Formation of COPI-coated vesicles.................................................................................... 16
ArfGAP1 activity and COPI vesicle formation .................................................................. 17
A novel Role for Arf1 during COPI vesicle Biogenesis .................................................... 17
Functions of coatomer isotypes .......................................................................................... 19
Results Part I Roles of ArfGAP-activity in COPI vesicle biogenesis and function ...................... 21
Generation and characterization of an ArfGAP1 antibody ................................................ 21
Cloning of full length ArfGAP1-wt and mutants into a Pichia pastoris expression vector24
Expression of full length ArfGAP1 in Pichia pastoris ...................................................... 24
Purification of full-length ArfGAP1-HIS from Pichia pastoris ....................................... 25 6
Purification of full length myristoylated Arf1 .................................................................... 25
Measuring ArfGAP1 activity ............................................................................................. 27
GTP-hydrolysis prevents the accumulation of COPI coated vesicles in vitro ................... 29
The COPI-budding assay .................................................................................................... 31
ArfGAP1 activity and vesicle formation ............................................................................ 35
ArfGAP1-activity allows uptake of anterograde cargo into COPI vesicles ....................... 38
A two-step incubation system for COPI-budding challenges the prevalent model of
ArfGAP1 in COPI-vesicle biogenesis ................................................................................ 39
The stoichiometry of Arf1 to coatomer on COPI vesicles ................................................. 45
4
Results Part II A novel role for Arf1-GTP in COPI vesicle biogenesis ......................................... 49
Arf1-GTP generates membrane curvature .......................................................................... 49
Arf1-GTP dimerizes on membranes ................................................................................... 54
The Arf1-dimer interface .................................................................................................... 56
Characterization of an Arf1 variant that cannot dimerize .................................................. 58
Lack of function of monomeric Arf1 analyzed in vivo and in vitro ................................... 67
Dissecting contributions to membrane deformation by Arf1 and coatomer ...................... 71
Results Part III Coatomer Isoforms ................................................................................................ 77
Purification of recombinant coatomer isoforms ................................................................. 78
Coatomer isoforms and their ability to mediate COPI-vesicle biogenesis ......................... 80
Preferences in cargo selection of homogeneously coated COPI-vesicles .......................... 81
Structural studies on coatomer ........................................................................................... 82
Discussion ...................................................................................................................................... 86
Discussion ...................................................................................................................................... 86
Part I ................................................................................................................................... 86
Part II .................................................................................................................................. 89
Part III ................................................................................................................................. 91
Materials ......................................................................................................................................... 93
Chemicals ........................................................................................................................... 93
Peptides............................................................................................................................... 93
Beads .................................................................................................................................. 93
Molecular weight standards for SDS – PAGE ................................................................... 94
Protease – Inhibitors ........................................................................................................... 95
Antibodies........................................................................................................................... 95
(a) Primary antibodies ....................................................................................................... 95
5
(b) Secondary antibodies ................................................................................................... 96
Restriction enzymes and DNA polymerases ...................................................................... 96
Bacterial strains .................................................................................................................. 96
Growth media ..................................................................................................................... 97
Buffers ................................................................................................................................ 97
PBS: ........................................................................................................................................ 97
Pichia dialysis buffer .............................................................................................................. 97
Coatomer isoforms purification buffers ................................................................................. 98
5x DNA loading buffer .......................................................................................................... 98
50x TAE ................................................................................................................................. 98
Endogenous coatomer purification buffers ............................................................................ 98
3x SDS loading buffer ............................................................................................................ 99
Coomassie staining solution ................................................................................................... 99
Destaining solution ................................................................................................................. 99
Ponceau-S solution ................................................................................................................. 99
Semi-dry transfer buffers ..................................................................................................... 100
SDS PAGE stock solutions .................................................................................................. 100
Methods ........................................................................................................................................ 101
Agarose gel electrophoresis .............................................................................................. 101
DNA concentration determination ................................................................................... 101
Polymerase chain reactions .............................................................................................. 101
PCR products and plasmid fragments purification ........................................................... 102