The small GTPase Arf1p from Saccaromyces cerevisiae goes new ways [Elektronische Ressource] : novel roles in mRNA transport and in the formation of specialized vesicles from the Golgi = Die kleine GTPase Arf1p aus Saccharomyces cerevisiae geht neue Wege / vorgelegt von Mark Trautwein

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Sujets

• Gesundheit

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2004
• Nombre de lectures: 41
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

The small GTPase Arf1p from Saccaromyces cerevisiae goes new ways
- Novel roles in mRNA transport
and in the formation of specialized vesicles from the Golgi -



Die kleine GTPase Arf1p aus Saccharomyces cerevisiae geht neue Wege
- Neue Funktionen im mRNA Transport
und bei der Bildung von spezialisierten Golgi-Vesikeln -


D I S S E R T A T I O N


der Fakultät für Chemie und Pharmazie
der Eberhard-Karls-Universität Tübingen

zur Erlangung des Grades eines Doktors
der Naturwissenschaften



2004

vorgelegt von

Mark Trautwein


I


























Tag der mündlichen Prüfung: 16. November 2004


Dekan: Prof. Dr. Stefan Laufer
Erster Berichterstatter: Prof. Dr. Hans-Georg Rammensee
Zweiter Berichterstatter: Prof. Dr. Frank Madeo

II III
























Die vorliegende Arbeit wurde angefertigt unter der Anleitung von Dr. Anne Spang in der
Arbeitsgruppe „Hefen und Würmer: Modellsysteme für intrazellulären Transport“ am
Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Tübingen.
Prof. Dr. Hans-Georg Rammensee vom Interfakultären Institut für Zellbiologie, Abteilung
Immunologie, Universität Tübingen, übernahm die Vertretung der Arbeit vor der Fakultät.
IV V















“Previous generations have been absolutely convinced that their scientific theories were
well-nigh perfect, only for it to turn out that they had missed the point entirely. Why
should it be any different for our generation? Beware of scientific fundamentalists who try
to tell you everything is pretty much worked out, and only a few routine details are left to
do. It is just when the majority of scientists believe such things that the next revolution in
our world-view creeps into being, its feeble birth-squeaks all but drowned by the
earsplitting roar of orthodoxy.” (Pratchett et al., 2002)



VI VII
Table of Contents

1 Introduction ........................................................................... 1

1.1 Intracellular protein sorting: Vesicular traffic, secretion and endocytosis........1

1.2 Molecular mechanism of vesicular traffic .............................................................4

1.3 Early stages of the secretory pathway....................................................................5

1.3.1 COPII-coated vesicles....................................................................................5
1.3.2 COPI-coated vesicles.....................................................................................7

1.4 Later stages of the secretory pathway, endocytosis and sorting of internalized
proteins ...................................................................................................................10

1.4.1 Clathrin-coated and related vesicles ............................................................10
1.4.2 Endocytosis and endosomal sorting.............................................................12

1.5 The Arf GTPases: Structure and function ..........................................................15

1.6 Interactors of Arf1.................................................................................................18

1.6.1 Arf1-regulators: Arf1-GEFs and Arf1-GAPs ..............................................18
1.6.2 Vesicle coat proteins and adaptors...............................................................19
1.6.3 Proteins interacting with Arf1-GDP ............................................................19
1.6.4 Phospholipid-metabolizing enzymes ...........................................................19
1.6.5 Other effectors .............................................................................................20

1.7 Aim of this study ....................................................................................................21 VIII
2 Materials and Methods........................................................22

2.1 Instrumentation......................................................................................................22
2.2 Chemicals................................................................................................................22
2.3 Materials .................................................................................................................23
2.4 Kits ..........................................................................................................................24
2.5 Media.......................................................................................................................24
2.6 Commonly used solutions and buffers .................................................................26
2.7 Strains, plasmids, antibodies, oligonucleotide primers ......................................29
2.8 Biochemical Methods.............................................................................................41
2.8.1 Determination of yeast cell density..............................................................41
2.8.2 Preparation of yeast total cell extract...........................................................41
2.8.3 Preparation of yeast cytosol .........................................................................41
2.8.4 Preparation of enriched Golgi membranes from yeast.................................42
2.8.5 Protein determination...................................................................................42
2.8.6 Trichloro acetic acid precipitation ...............................................................43
2.8.7 SDS-PAGE ..................................................................................................43
2.8.8 Coomassie-Blue staining of polyacrylamide gels........................................44
2.8.9 Colloidal Cooomassie-Blue staining of polyacrylamide gels according to
Fairbanks......................................................................................................44
2.8.10 Silver staining of polyacrylamide gels.........................................................45
2.8.11 Blue Native PAGE.......................................................................................45
2.8.12 Immunoblots ................................................................................................47
2.8.13 Purification of recombinant Arf1p proteins with His -tag from E. coli.......48 6
2.8.14 Purification of other recombinant His -tagged proteins from E. coli ..........49 6
2.8.15 binant wild-type Arf1p protein from E. coli..............49
2.8.16 Preparation of affinity material....................................................................50
2.8.17 Differential Arf1p-affinity chromatography ................................................50
2.8.18 Affinity purification of immunoglobulins....................................................51
2.8.19 In-gel digestion and mass spectrometric identification of proteins .............52