Coordination of endoplasmic reticulum and mRNA localization in Saccharomyces cerevisiae [Elektronische Ressource] / Maria Schmid

Maria Schmid

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

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





Coordination of endoplasmic reticulum
and mRNA localization in
Saccharomyces cerevisiae





Maria Schmid
aus Oberviechtach

München
2008



Erklärung

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



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.


München, am………………………….


…………………………………………..
Maria Schmid




Dissertation eingereicht am 24.06.08
1. Gutachter: Prof. Dr. Ralf-Peter Jansen
2. Gutachter: Prof. Dr. Klaus Förstemann
Mündliche Prüfung am 28.07.08








Table of contents


Table of contents
1. Introduction ..................................................................................................................1
1.1. mRNA localization, a conserved process essential for somatic cell polarity and
embryonic development.............................................................................................1
1.1.1. mRNA localization in polarized somatic cells.......................................................2
1.1.1.1. Migrating fibroblasts....................................................................................2
1.1.1.2. Neurons......................................................................................................2
1.1.2. mRNA localization in oocytes and developing embryos.......................................4
1.1.2.1. Drosophila melanogaster ............................................................................4
1.1.2.2. Xenopus laevis ...........................................................................................4
1.2. Mechanisms of mRNA localization.............................................................................6
1.3. mRNA localization in S. cerevisiae7
1.3.1. ASH1 mRNA - the most prominent localized mRNA in yeast regulates mating
type switching8
1.3.2. Trans-acting factors: the mRNA localization machinery.....................................11
1.3.2.1. The core locasome ...................................................................................11
1.3.2.1.1. Myo4 (She1p), a motor protein of the myosin V family .......................11
1.3.2.1.2. She3p, the adaptor protein.................................................................12
1.3.2.1.3. She2p, an unconventional RNA-binding protein.................................12
1.3.2.2. Other trans-acting and accessory factors for mRNA localization...............15
1.3.3. Additional localized mRNAs in S. cerevisiae......................................................16
1.4. Inheritance of cortical endoplasmic reticulum in S. cerevisiae..................................18
1.4.1. Structure and function of the ER........................................................................18
1.4.2. Inheritance of the ER.........................................................................................20
1.5. First indications for a link between mRNA localization and ER inheritance in
S. cerevisiae ............................................................................................................23
1.6. Aim of this work........................................................................................................27
2. Results........................................................................................................................28
2.1. Loss of ASH1-MS2 RNP localization in cells defective for ER inheritance................28
2.2. IST2-MS2 does not localize as efficiently as ASH1-MS2 mRNA ..............................30
2.3. WSC2 mRNA can be used as a model mRNA which is expressed earlier in cell cycle
than ASH1 mRNA....................................................................................................31
2.4. Biochemical analysis: co-migration of ER and the ASH1 mRNA binding protein She2p
during subcellular fractionation.................................................................................33
2.4.1. She2p co-migrates with ER markers in a discontinuous velocity sucrose gradient
2.4.2. She2p is present in the fraction of purified ER microsomes...............................34
2.4.3. Flotation of ER membranes by equilibrium density centrifugation: She2p floats
along.................................................................................................................35
2.5. Intact polysomes are not required for She2p-ER association ...................................36
2.6. The She2p-ER interaction is not dependent on mRNA.............................................38
2.6.1. RNase treatment of whole cell extracts does not disrupt She2-ER association .38
2.6.2. The mRNA binding mutant She2p-N36S,R63K accumulates in the nucleus......40
2.7. In vitro assay: recombinant She2p co-migrates with ER on a velocity sucrose gradient
2.7.1. Purification of recombinant She2p.....................................................................41
2.7.2. Recombinant She2p behaves like endogenous She2p......................................43
2.8. The RNA binding mutant She2p-N36S,R63K is not impaired in ER association.......45
2.9. None of the best characterized mutations in She2p shows an effect on ER-
association...............................................................................................................46
2.10. Search for a protein factor acting as mediator for the She2p – ER interaction..........48
2.11. She2 pellets with flotation purified ER membranes in an in vitro binding assay........51
2.11.1. In vitro binding assay with flotation purified ER membranes..............................51
2.11.2. Protease treatment of ER membranes ..............................................................54
2.12. She2p directly binds to synthetic liposomes.............................................................56



Table of contents


2.12.1. She2p floats along with ER-like protein-free liposomes.....................................56
2.12.2. She2p behaves like a bona fide peripheral membrane protein ..........................57
2.12.3. Phosphatidylserine and phosphatidylinositol are not essential for She2p-
liposome interaction ..........................................................................................58
2.12.4. She2p interacts with liposomes in the presence of its RNA ligand.....................59
3. Discussion..................................................................................................................62
3.1. Functional linkage between mRNA localization and cortical ER inheritance.............62
3.2. A connection between mRNAs and membranes: lessons from other organisms......64
3.3. The RNA binding protein She2p associates with ER membranes ............................66
3.4. She2p has the ability to directly interact with lipid membranes.................................69
3.5. Benefits of coordinated mRNA and ER transport and implications for a possible
model.......................................................................................................................77
4. Materials .....................................................................................................................81
4.1. Consumables and Chemicals...................................................................................81
4.2. Equipment................................................................................................................81
4.3. Commercially available kits......................................................................................82
4.4. Enzymes..................................................................................................................82
4.5. Oligonucleotides ......................................................................................................83
4.6. Plasmids87
4.7. E. coli strains............................................................................................................89
4.8. S. cerevisiae strains.................................................................................................89
4.9. Antibodies................................................................................................................91
5. Methods ......................................................................................................................92
5.1. E. coli-specific techniques........................................................................................92
5.1.1. Preparation of competent E. coli cells ...............................................................92
5.1.2. Transformation of competent E. coli cells..........................................................92
5.1.3. Preparation of Plasmid-DNA .............................................................................93
5.2. S. cerevisiae-specific techniques93
5.2.1. Cell densi