Regulation of membrane traffic at the Golgi apparatus by Rab GTPases and their GAPs [Elektronische Ressource] / vorgelegt von Evelyn Fuchs

Evelyn Fuchs

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 21 Mo

Extrait

Regulation of Membrane Traffic
at the Golgi Apparatus by
Rab GTPases and their GAPs



Dissertation
zur Erlangung des
Doktorgrades der Naturwissenschaften
(Dr. rer. nat.)
der Fakultät für Biologie der
Ludwigs-Maximilians-Universität München



vorgelegt von

Evelyn Fuchs

München 2007



Dissertation eingereicht am: 30. August 2007
Tag der mündlichen Prüfung: 22. Februar 2008

Erstgutachter: Prof. Dr. Erich Nigg
Zweitgutachterin: PD Dr. Angelika Böttger
Ehrenwörtliche Versicherung und Erklärung über frühere
Promotionsversuche


Hiermit versichere ich, Evelyn Fuchs, ehrenwörtlich, dass die vorgelegte
Dissertation von mir selbständig und ohne unerlaubte Hilfe angefertigt ist.
Desweiteren erkläre ich, dass die vorgelegte Dissertation weder ganz noch in
wesentlichen Teilen einer anderen Prüfungskomission vorgelegt wurde. Ich habe
mich anderweitig keiner Doktorprüfung unterzogen.



München, 19. August 2007



Evelyn Fuchs
Table of contents

Table of contents
Summary ..................................................................................5
1 Introduction ........................................................................6
1.1 The Golgi apparatus: Processing and sorting station along
intracellular transport routes.................................................................6
1.1.1 The Golgi apparatus in a cell biological view....................................... 6
1.1.2 Structure and function of the Golgi apparatus ..................................... 6
1.1.3 Molecular mechanisms of intracellular transport ................................. 8
1.1.4 Anterograde transport and Exocytosis .............................................. 10
1.2 Endocytosis and retrograde transport .....................................11
1.2.1 Endocytic vesicle formation at the plasma membrane....................... 11
1.2.2 Endocytic sorting pathways............................................................... 13
1.2.3 Retrograde transport of Shiga toxin .................................................. 15
1.3 Rab GTPases: Molecular switches regulating membrane
traffic.......................................................................................................18
1.3.1 The Rab family of small GTPases..................................................... 18
1.3.2 The Rab GTPase cycle..................................................................... 20
1.3.3 Catalytic mechanism and members of the human Rab GAP family... 22
1.4 Rab6: Role of a Golgi localised Rab..........................................25
1.4.1 Isoforms and features of Rab6.......................................................... 25
1.4.2 Rab6 function in membrane transport ............................................... 26
1.4.3 Rab6 effector proteins....................................................................... 27
2 Identification and Characterisation of PIST as a novel
Rab6 effector protein ............................................................29
2.1 Aim of the work............................................................................29
2.2 Results ..........................................................................................29
2.2.1 Identification of PIST as a putative Rab6 effector.............................. 29
2.2.2 Generation and validation of a PIST specific antibody ...................... 31
2.2.3 PIST binds Rab6 in a GTP-dependent fashion.................................. 32
2.2.4 PIST is a specific binding partner of Rab6 and not of TC10 .............. 33
2.2.5 PIST is a Golgi localised protein ....................................................... 36
I Table of contents

2.2.6 Domain specific Rab6 binding and Golgi localisation of PIST ........... 37
2.2.7 Interfering with Rab6 levels does not affect PIST at the Golgi........... 41
2.2.8 Golgin160 is a binding partner of PIST.............................................. 45
2.2.9 PIST is unaltered in Golgin160 depleted cells................................... 47
2.2.10 Depletion of PIST does not have an effect on Golgi structure ........... 49
2.3 Conclusions .................................................................................51
3 Rab GTPase-activating proteins defining Shiga toxin
and epidermal growth factor uptake pathways..................52
3.1 Aim of the work............................................................................52
3.2 Results ..........................................................................................52
3.2.1 Establishment of the Shiga toxin and EGF retrograde transport assay .
......................................................................................................... 52
3.2.2 Shiga toxin transport but not EGF uptake is Rab6 dependent........... 56
3.2.3 TBC1D11/GAPCenA acts on Rab4 and does not block Shiga toxin
transport ......................................................................................................... 59
3.2.4 A subset of human RabGAPs influences Shiga toxin transport ......... 62
3.2.5 Only RabGAP-5 is able to block EGF uptake.................................... 69
3.2.6 Different RabGAPs define Shiga toxin and EGF uptake pathways.... 71
3.2.7 Biochemical identification of target Rabs........................................... 74
3.2.8 Functional verification of target Rabs for RN-tre and RabGAP-5....... 76
3.3 Conclusion....................................................................................78
4 Discussion ........................................................................80
4.1 The novel Rab6 binding protein PIST .......................................80
4.1.1 PIST binds Rab6 with high affinity and specificity ............................. 80
4.1.2 PIST does not bind the Rho GTPase TC10 ...................................... 81
4.1.3 How is PIST localised to Golgi membranes?..................................... 82
4.1.4 What is the function of PIST in complex with Rab6? ......................... 84
4.2 Rab GTPase activating proteins defining Shiga toxin and
epidermal growth factor uptake ..........................................................86
4.2.1 A subset of human Rab GAPs influences Shiga toxin transport ........ 86
4.2.2 The role of Rab6 in the Shiga toxin trafficking pathway..................... 89
4.2.3 Only RabGAP5 is able to block EGF uptake ..................................... 90
4.2.4 What are the target Rabs for the GAPs blocking Shiga toxin transport
or EGF endocytosis?....................................................................................... 91
II Table of contents

5 Material and Methods ......................................................93
5.1 Material..........................................................................................93
5.1.1 Reagents .......................................................................................... 93
5.1.2 Equipment ........................................................................................ 93
5.1.3 Solutions........................................................................................... 94
5.1.4 Plasmids ........................................................................................... 95
5.1.5 siRNA oligonucleotides ................................................................... 101
5.1.6 Antibodies....................................................................................... 102
5.2 Bacterial Methods......................................................................103
5.2.1 Growth and maintenance of E. coli ................................................. 103
5.2.2 Bacterial strains .............................................................................. 103
5.2.3 Preparation and transformation of chemically competent bacteria .. 104
5.2.4 Preparation and transformation of electrocompetent bacteria ......... 105
5.2.5 Plasmid DNA preparation from bacteria.......................................... 105
5.2.6 Purification of 6xHis- and 6xHisGST-tagged proteins from bacteria 105
5.2.7 Purification of MBP-tagged proteins from bacteria .......................... 106
5.3 DNA methods .............................................................................107
5.3.1 "Shortway" cloning strategy ............................................................ 107
5.3.2 Restriction digests and agarose gel electrophoresis of DNA........... 107
5.3.3 Cloning digested DNA fragments.................................................... 108
5.3.4 mRNA isolation and cDNA synthesis .............................................. 108
5.3.5 PCR and cloning of PCR products .................................................. 108
5.3.6 Site-directed mutagenesis............................................................... 109
5.3.7 DNA sequencing............................................................................. 109
5.4 Protein methods.........................................................................109