Analysis of the recruitment of the class I myosin Myo5p to endocytic sites in Saccharomyces cerevisiae [Elektronische Ressource] / vorgelegt von Helga Grötsch

. . Seeboorapha

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

INAUGURAL-DISSERTATION


zur Erlangung der Doktorwürde
der Naturwissenschaftlich-Mathematischen Gesamtfakultät
der Ruprecht-Karls-Universität Heidelberg







vorgelegt von
Diplom-Biologin Helga Grötsch,
geboren in Lüneburg



Tag der mündlichen Prüfung:






Title:
Analysis of the recruitment of the class I myosin Myo5p to
endocytic sites in Saccharomyces cerevisiae










Referees: Prof. Dr. Felix Wieland
Prof. Dr. Christine Clayton

Acknowledgments

First of all, I would like to thank Maribel Geli for giving me the opportunity to do my PhD in
her lab and for all her support, not only concerning the work, but also personally.
I am very grateful to Prof. Dr. Felix Wieland and Prof. Dr. Christine Clayton for reviewing
this thesis.
Also, I would like to thank Prof. Dr. Wieland and Barbara Schröter for pushing me a little
bit to finish this thesis.
I am more than grateful to all the people of my group: Isabel Fernandez, Fatima Idrissi
and Henrique Girao. “You are all fantastic!” It helped me a lot also in very hard days to
enjoy your company!!! Gracias!!!! Thank you also for critically reading this thesis, this
helped a lot. Un abrazo fuerte por todo!
Thank you also to Jonatan Perez, who made the work really easier when he was in our
lab and who was a very pleasant person to have around. Also very nice company was
Monica Pons, who helped a lot with the microscope.
I would also like to thank all the nice people who worked next to me at the BZH and at the
IBMB, because all together they were creating a nice atmosphere at the working place.
VIELEN DANK and MUCHAS GRACIAS to all my friends!!! Although most of you were far
away, it helped me a lot already to talk to you on the phone and when we saw each other
it always gave me “good energy” for a longer time! Here in Barcelona, “the Cristinas” were
really a big support always, muchas gracias para esto!!!
Vielen Dank auch an meine Eltern, meine Schwester Birgit und Florian, die mich immer
unterstützt haben. Jetzt beginnt eine neue Zeit! Birgit danke ich auch besonders für die
vielen tollen Pakete, die sie mir immer geschickt hat!
Paul, como esposo y como amigo me has ayudado muchisimo. Espero que un dia puedo
aprender mas de ti como no estresarme siempre tanto.


Index
Index

1. Zusammenfassung 1
2. Summary 2
3. Abbreviations 3
4. Introduction 4
4.1. Class I myosins 4
4.1.1. Class I myosins function in membrane dynamics 5
4.1.2. The functional domains of the class I myosins 6
4.1.2.1. The conserved domains characterizing myosins I: the head, the neck and
the TH1 domain 6
4.1.2.2. The C-terminal extension of the long-tailed class I myosins 9
4.1.2.1.1. The C-terminal extension of the long-tailed class I myosins forms a
linkage to the Arp2/3 complex 11
4.2. Endocytosis in S. cerevisiae: the assembly of a highly dynamic endocytic
patch 13
4.3. The S. cerevisiae class I myosins in the endocytic uptake step 18
4.3.1. Mechanisms of Myo5p regulation 20

5. Objectives 22

6. Results 23

6.1. Analysis of Myo5p recruitment to endocytic patches 23
6.1.1. An interaction between different Myo5p tail domains regulates the
recruitment of Myo5p to cortical patches 23
6.1.1.1. The cellular localization of GFP-Myo5p constructs bearing different
truncations suggests that an interaction between the TH1 domain and
the Myo5p C-terminus prevents Myo5p recruitment to cortical patches 23
6.1.1.2. The Myo5p neck and TH1 domains directly interact with a C-terminal
Myo5p fragment containing the GPA domain, the SH3 domain
and the acidic peptide in in vitro binding assays 25
6.1.1.3. The TH1 domain blocks the interaction of the Myo5p C-terminus with
Verprolin in cis and in trans 26
6.1.2. Calmodulin regulates the recruitment of Myo5p to endocytic patches at
the plasma membrane 28
6.1.2.1. Calmodulin dissociation promotes efficient lipid binding of Myo5p 28
6.1.2.2. Cmd1p dissociation from the Myo5p neck releases the interaction of
the neck and TH1 domains with the Myo5p C-terminus in vitro 30
6.1.2.3. Cmd1p dissociation from the Myo5p neck promotes Myo5p binding
to Vrp1p 31
6.1.2.4. At the plasma membrane Myo5p releases Cmd1p and binds to Vrp1p 33
6.1.2.5. No evidence for homo-oligomerization of the cytosolic or plasma
membrane associated Myo5p 35
6.1.2.6. Cmd1p binding to Myo5p influences the average lifespan of Myo5p at
cortical patches in vivo 36
I Index
6.2. Screening for factors in trans involved in Myo5p localization 37
6.2.1. Visual screening for factors in trans required for Myo5p localization 37
6.2.1.1. Las17p might exhibit redundant function with Vrp1p in Myo5p patch
Recruitment 37
6.2.1.2. PI(4,5)P2 might be involved in Myo5p recruitment to the plasma
Membrane 40
6.2.2. The Plasma Membrane Recruitment System (PRS): a reporter system to
investigate the close association of proteins with the plasma membrane
in vivo 43
6.2.2.1 The PRS can monitor the plasma membrane localization of Myo5p 44
6.2.2.2. The PRS monitors close plasma membrane association, but not cortical
patch localization of proteins 45
6.2.2.3. The Myo5p domains mediating lipid binding (the neck and TH1 domains)
allow growth of the 5’Sos fusion construct in the PRS 46
6.2.2.4. A genetic screen to search for factors in trans required for neck and TH1
domain mediated Myo5p recruitment to the plasma membrane 48
6.2.2.4.1. Screening for mpr (Myo5p plasma membrane recruitment) mutants 49
6.2.2.4.2. Identification of genes bearing the mpr mutations 51
6.2.2.4.3. Mpr mutants bear mutations in Class C VPS genes 52
6.2.2.4.4. The vps mutants identified in the screening show no delocalization of
Myo5p 53
6.2.2.4.5. The vps mutantthe screening exhibit a slight defect in the
uptake step of endocytosis 54
7. Supplementary data 56
8. Discussion 57
8.1. Cmd1p regulates the Myo5p-Vrp1p interaction and the association of Myo5p
to the endocytic patch 57
8.2. Phospholipid binding of Myo5p 60
8.3. A model for Cmd1p-regulated recruitment of Myo5p to endocytic patches 64
8.4. The PRS screening 66
8.5. Outlook 67
9. Materials and methods 70
9.1. Cell culture 70
9.1.1. E. coli cell culture 70
9.1.2. S. cerevisiae cell culture 70
9.2. Genetic techniques 70
9.2.1. Generation of yeast strains 70
9.2.1.1. Generation of double mutants by diploid construction, sporulation and
tetrad dissection 70
9.2.1.2. Plate assay for the detection of bar1 mutants 71
9.2.2. Yeast strains 71
9.2.3. Methods of the PRS screening 73
II Index
9.2.3.1. Chemical mutagenesis with ethymethane sulfonate 73
9.2.3.2. Selection of mpr (myosin plasma membrane recruitment) mutants 73
9.2.3.3. Outcrossing of mpr mutants 74
9.2.3.4. Identification of mutants by complementation with a genetic library 74
9.2.3.4.1. The yeast genomic library 74
9.3. DNA techniques and plasmid construction 75
9.3.1. Introduction of DNA into cells 75
9.3.2. Extraction and purification of plasmid DNA from S. cerevisiae 75
9.3.3. Extraction and purification of genomic DNA from S. cerevisiae 75
9.3.4. Plasmids 76
9.3.4.1. Plasmids for expression of 5’Sos constructs 79
9.3.4.2. Plasmids for expression of GFP constructs 80
9.3.4.3. Plasmids for expression of ProtA-Myo5p constructs 82
9.3.5. Primers 83
9.4. Purification of yeast plasma membrane and cytosol 85
9.5. Protein techniques 86
9.5.1. SDS-PAGE, immunoblots and antibodies 86
9.5.2. Purification of recombinant GST fusion proteins 88
9.5.2.1. Purification of GST 88
9.5.2.2. Purification of GST-C 88
9.5.2.3. Purification of GST-Cmd1p and Cmd1p 89
9.5.3. Purification of Protein A-tagged proteins from yeast 89
9.5.4. In vitro protein binding assay 89
9.5.5. Protein overlay assay 90
9.5.6. Binding of purified proteins to lipid strips 90
9.5.7. IgG pull-down experiments 91
9.5.7.1. IgG pull-downs from yeast extract 91
9.5.7.2. IgG pull-down from plasma membrane and cytosolic fractions 91
9.5.8. Immunoprecipitations from plasma membrane and cytosolic fractions 92
9.6. The α-factor internalization assay 92
9.7. Fluorescence microscopy 93
9.7.1. Fluorescence microscopy of living cells expressing GFP-Myo5p constructs 93
9.7.2. Time-lapse fluorescence microscopy of cortical patches in living cells 93
9.7.3. Plasma membrane staining with FM4-64 and confocal fluorescence
microscopy 93
10. Literature 94
11. Publications 104


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