Characterization of a myosin transport complex from yeast [Elektronische Ressource] / Alexander Heuck

Alexander Heuck

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 35
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und
Pharmazie der Ludwig-Maximilians-Universität München












Characterization of a myosin transport complex from yeast









Alexander Heuck
aus Rodewisch i.V.
2009 Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordung vom 29. Januar 1998
von Herrn Prof. Dr. Ralf-Peter Jansen betreut.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.



München, den 23. März 2009


Alexander Heuck






























Dissertation eingereicht am 24. März 2009
1. Gutachter: Prof. Dr. Ralf-Peter Jansen
2. Gutachter: Prof. Dr. Karl-Peter Hopfner
Mündliche Prüfung am 20. Mai 2009 TABLE OF CONTENTS
ABBREVIATIONS ......................................................................................................IV
1. INTRODUCTION..... 1
1.1 The cytoskeleton.......................1
1.2 The actin cytoskeleton in yeast ................................................................................................. 2
1.3 Dynein and kinesin motor proteins............................3
1.4 Myosin motor proteins............................................................................................................... 5
1.5 Type-V myosins.........................6
1.5.1 The motor domain and lever arm ...................................................................................................7
1.5.2 The tail domain - the coiled-coil rich region....................8
1.5.3 The tail domain - cargo complexes on type-V myosins..8
1.6 Regulation of myosin-adapter interactions..............................................................................10
1.7 The yeast type-V myosin Myo4p.............................................................................................10
1.7.1 mRNA transport in yeast..............11
1.7.2 Inheritance of the cortical endoplasmic reticulum in yeast...........................13
1.8 Structure of the Myo2p globular-tail domain ........................................................................... 14
1.9 Objectives................................................................16
2. RESULTS.............................................. 17
2.1 Expression and purification of the Myo4p-tail and She3p-N...................17
2.2 Myo4p and She3p form stable complexes ..............................................17
2.3 The Myo4p-tail can be divided into three parts....................................... 18
2.4 The Myo4p-tail contains two distinct binding sites for She3p..................19
2.5 The coiled-coil region stabilizes Myo4p-She3p complexes.....................................................20
2.6 The residues 1056 and 1057 of Myo4p are required for She3p binding ................................. 21
2.7 The Myo4p-tail is strictly monomeric.......................................................23
2.8 The Myo4p-tail forms homodimers when linked to artificial dimerization domains .................25
2.9 Artificially dimerized Myo4p fragments bind to She3p and form stable complexes ................26
2.10 Disruption of Myo4p dimerization results in disassembly of complexes with She3p............. 27
2.11 The globular-tail domain of Myo4p is not required for ER inheritance .................................. 28
2.13 The Myo4p globular-tail domain is required for localized ASH1-mRNA translation ..............29
2.14 The globular tail is required to localize Myo4p at the bud tip ................................................30
2.15 Crystallization of the Myo4p globular tail...............................................32
2.16 Structure determination and refinement of the Myo4p globular tail....... 33
2.17 Crystal structure of the Myo4p globular tail........................................... 34
2.18 Structural comparison of the Myo4p and Myo2p globular-tail domain.. 36
2.19 The globular-tail domain of Myo4p interacts directly with membranes. 39
2.20 Identification of a membrane interacting region within the Myo4p globular tail.....................40
2.22 Quantification of the Myo4p globular-tail-vesicle interaction .................................................42
2.23 Crystallization of the Myo4p-She3p complex ........................................ 43
i TABLE OF CONTENTS
3. DISCUSSION ........................................................................................................ 45
3.1 The oligomerization state of Myo4p........................45
3.2 Structure of theMyo4p-She3p complex – an experimental outlook......... 49
3.3 Two binding regions of Myo4p interact with She3p................................................................. 49
3.4 Importance of complex stability for directional transport.........................51
3.5 The Myo4p globular-tail domain is conserved in terms of fold but not function ......................51
3.6 The globular-tail domain as a peripheral membrane-binding domain ..................................... 53
3.7 The function of the globular-tail domain ..................................................57
3.7.1 On cortical ER inheritance ...........................................57
3.7.2 On mRNA transport......................................................58
3.7.3 Anchoring at the bud tip...............59
3.8 Summary................................................................................................. 61
4. MATERIALS AND METHODS .............................................. 62
4.1 Consumables ..........................................................................................62
4.2 Plasmid DNA...........................62
4.2.1 Purchased plasmids.....................................................................................62
4.2.2 Plasmids for E. coli expression (biochemical characterization) ...................................................62
4.2.3 Plasmids for E. coli expression (crystallization)...........63
4.3 E. coli strains...........................................................................................64
4.4 S. cerevisiae strains................................................64
4.5 Oligonucleotides......................64
4.6 Antibodies................................................................................................................................65
4.7 Molecular biology....................66
4.7.1 Standard cloning methods...........66
4.7.2 Transformation of E. coli and isolation of plasmid DNA ...............................................................66
4.7.3 Transformation of yeast cells .......................................................................66
4.7.4 Isolation of yeast genomic DNA...66
4.8 Protein analysis.......................................................67
4.8.1 Protein separation by SDS-PAGE................................................................................................67
4.8.2 Western blot.67
4.9 Protein expression, purification and crystallization. 67
4.9.1 Recombinant protein expression in E. coli ...................................................................................67
4.9.2 Selenomethionine labeling ...........................................................................68
4.9.3 Purification of Myo4p fragments...................................68
4.9.4 Purification of She3p fragments...69
4.9.5 Crystallization and structure determination of the Myo4p-GT ......................................................69
4.9.6 Crystallization of the Myo4p-She3p complex ...............................................70
4.10 In vitro characterization of the Myo4p-tail function................................70
4.10.1 Ni-pull down................................................................................................70
4.10.2 Surface Plasmon-Resonance....70
4.10.2.1 Myo4p-She3p-N interaction........................70
4.10.2.2 Myo4p-GT vesicle interaction.....................................................................................71
4.10.3 Floatation assay with ER-like protein-free liposomes................................71
4.10.3.1. Preparation of protein free Liposomes ......................................71
4.10.3.2 In vitro binding and floatation of liposomes71
4.10.4 Reflectometric Interference Spectroscopy.................................................71
ii TABLE OF CONTENTS
4.11. Fluorescence microscopy..................................................................................................... 72
4.11.1 Preparation of cells for Immunofluorescense microscopy..........................72
4.11.2 Preparation of cells for fluorescence microscopy.......72
4.11.3 Fluorescence microscopy..........72
4.12. Bioinformatics.......................................................................................................................73
4.12.1 Homology searches and alignme