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

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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....................................................................................

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Published 01 January 2009
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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
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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
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(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
Restriction and ligation..................................................................................................... 102
Transformation ................................................................................................................. 103
Site directed mutagenesis ................................................................................................. 103
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Protein expression ............................................................................................................ 104
Protein purification ........................................................................................................... 105
Peptide coupling to thiopropyl-sepharose beads .............................................................. 106
SDS PAGE ....................................................................................................................... 107
Western blot analysis ........................................................................................................ 109
Immunochemical detection of proteins on PVDF-membranes ........................................ 110
Bradford assay .................................................................................................................. 110
Tri-Chloro-acetic acid (TCA) – precipitation................................................................... 111
Immunoprecipitation (IP) ................................................................................................. 111
Synthesis of p23 lipopeptide ............................................................................................ 111
Isolation of coatomer from rabbit liver ............................................................................ 112
Isolation of rat liver Golgi ................................................................................................ 115
Recombinant Coatomer isoforms purification ................................................................. 116
Purification of ArfGAP1 from Pichia pastoris ................................................................ 116
Purification of myrArf1-wt from E.coli ........................................................................... 117
Purification of Arf1-Y35A from E.coli ............................................................................ 117
Preparation of liposomes .................................................................................................. 117
Golgi binding assay .......................................................................................................... 118
Chemical crosslinking ...................................................................................................... 118
Dimerization assay with protein-free liposomes .............................................................. 118
Nucleotide exchange activity on Golgi membranes of Arf1-wt and Arf1-Y35A ............ 119
Recruitment to Golgi membranes of coatomer and adaptin-1 by Arf1-wt and Arf1-Y35A120
Arf1 nucleotide exchange measurements by trpytophane fluorescence according to
(Bigay, et al., 2003) .......................................................................................................... 120
Monitoring Arf1-mediated coat recruitment and release in real time by static light
scattering according to (Bigay and Antonny, 2005) ......................................................... 121
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Flotation experiments according to (Bigay, et al., 2003) ................................................. 122
Arf1-wt and mutant localization to the Golgi................................................................... 122
Complementation assays in yeast ..................................................................................... 123
GAL1-overexpression of Arf1-Y35A in yeast ................................................................. 124
Formation of COPI vesicles in vitro ................................................................................. 124
Complementation assays in yeast ..................................................................................... 125
Arf1 induced tubulation of membrane sheets ................................................................... 125
References .................................................................................................................................... 126
Acknowledgements ...................................................................................................................... 134


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Abstract
COPI vesicles are defined by a coat consisting of (i) the small GTPase Arf1 and (ii) coatomer.
Arf1 in its GTP-loaded form recruits coatomer to the membrane to form a COPI vesicle. GTP
hydrolysis leads to uncoating, and is catalyzed by (iii) ArfGAP-activity. In this work we studied
the molecular contributions of the named three key players in order to further elucidate the
mechanisms underlying COPI vesicle biogenesis and functions:
(i) We show that Arf1-GTP induces positive membrane curvature, and find that the
small GTPase can dimerize on the membrane. Investigating a possible link between
Arf1 dimerization and curvature formation, we isolated an Arf1-mutant (Arf1-Y35A)
that cannot dimerize. Although it was capable to exert the classical role of Arf1 as a
coat receptor, it could not mediate the formation of COPI vesicles and is lethal in
yeast. Strikingly, this mutant was not able to deform membranes, suggesting that
GTP-specific dimerization of Arf1 is a critical step which induces membrane
curvature during the formation of coated vesicles. We observed that, while the
curvature in the budding zone of a COPI vesicle is mediated by coatomer, Arf1
contributes to membrane tension in such a way that fission can occur.
(ii) The heptameric coat complex coatomer exists in four isoforms, defined by their γ1/γ2
and ζ1/ζ2 subunits, which are distributed differentially over the Golgi. Here we show
a biochemical characterization of the four recombinant isoforms of the heptameric
complex. We demonstrate that the recombinant protein complexes yield COPI-
vesicles in vitro with an efficiency comparable to coatomer isolated from tissue.
(iii) To investigate roles of ArfGAP activity during in vitro budding, we find that catalytic
amounts of full length ArfGAP1 reduce the yield of COPI-coated vesicles
significantly, in line with the proposed function of the enzyme to mediate the
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uncoating reaction. We also experimentally addressed discrepancies in the literature
about the role of ArfGAP1 during COPI-vesicle formation.
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