Characterization of the yeast proteins Neo1p and Sjl2p, two highly conserved regulators of phospholipid composition within endosomal membranes [Elektronische Ressource] / vorgelegt von Sidonie Wicky John

Sujets

• Biologie

Informations

• Publié par: universitat_stuttgart
• Publié le: 01 janvier 2005
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Characterization of the yeast proteins Neo1p and Sjl2p,
two highly conserved regulators
of phospholipid composition within endosomal membranes
Von der Fakultät Geo- und Biowissenschaften der Universität Stuttgart
zur Erlangung der Würde eines Doktors der
Naturwissenschaften (Dr. rer. Nat.) genehmigte Abhandlung
vorgelegt von
Sidonie Wicky John
aus Schelten (Schweiz)
Hauptberichter: Priv.-Doz. Dr. Birgit Singer-Krüger
Mitberichter: Prof. Dr. Dieter H. Wolf
Tag der mündlichen Prüfung: 15.12.2004
Institut für Biochemie der Universität Stuttgart
2004
12Table of contents
Abbreviations............................................................................................................................5
Zusammenfassung....................................................................................................................6
Abstract...................................................................................................................................14
1 Introduction .........................................................................................................................16
1.1 Endocytosis in the budding yeast Saccharomyces cerevisiae................................................. 16
1.1.1 Yeast as a model for studying endocytosis......................................................................... 16
1.1.2 The endocytic compartments.............................................................................................. 16
1.1.3 The transport pathways within the endomembrane system ................................................ 18
1.2 Structural and regulatory proteins involved in endocytic vesicle formation ...................... 19
1.2.1 Role of the clathrin coat in vesicle formation..................................................................... 19
1.2.2 Function of the actin cytoskeleton in early stages of endocytosis....................................... 21
1.2.3 Arf GTPases and their role as regulators of vesicle formation ........................................... 22
1.3 Phospholipids and membrane deformation events ............................................................... 23
1.3.1 Role of phospholipid asymmetry in membrane budding and regulation by the Drs2 family
of P-type ATPases....................................................................................................................... 23
1.3.1.1 Glycerophospholipid asymmetry and transport across the membrane bilayer ........................... 23
1.3.1.2 Relevance of the lipid asymmetry in cellular processes............................................................ 24
1.3.1.3 The Drs2 family of P-type ATPases and APL translocation..................................................... 26
1.3.1.4 Localization and function of the Drs2 family members in Saccharomyces cerevisiae............... 27
1.3.1.5 Neo1p is functionally connected to the endosomal proteins Ysl2p and Arl1p........................... 28
1.3.2 Role of phosphoinositides in membrane trafficking and their regulation by synaptojanin
family members .......................................................................................................................... 28
1.3.2.1 Phosphoinositide isoforms and their subcellular distribution.................................................... 28
1.3.2.2 The synaptojanin family .......................................................................................................... 30
1.3.2.3 Synaptojanin proteins in Saccharomyces cerevisiae................................................................. 31
1.4 Goal of this project .................................................................................................................. 32
2 Materials and Methods .......................................................................................................33
2.1 Materials .................................................................................................................................. 33
2.1.1 Saccharomyces cerevisiae strains....................................................................................... 33
2.1.2 Escherichia coli strains ...................................................................................................... 34
2.1.3 Plasmids ............................................................................................................................. 35
2.1.4 Antibodies.......................................................................................................................... 35
2.1.4.1 Antibodies used for immunoblotting........................................................................................ 35
2.1.4.2 Antibodies used for immunoprecipitations............................................................................... 36
2.1.4.3 Primary antibodies used for indirect immunofluorescence ....................................................... 36
2.1.4.4 Secondary antibodies used for indirect immunofluorescence ................................................... 36
2.1.5 Enzymes and kits used for molecular biology .................................................................... 37
2.1.6 Chemicals........................................................................................................................... 37
2.1.7 Media ................................................................................................................................. 37
2.2 Methods.................................................................................................................................... 38
2.2.1 Generation of DNA constructs ........................................................................................... 38
2.2.2 Mating, sporulation, transformation of yeast cells, CPY missorting, and two-hybrid assays
.................................................................................................................................................... 39
2.2.3 Biochemical methods ......................................................................................................... 40
2.2.3.1 Cell extracts, immunoblotting of proteins, and quantitative analysis of soluble Ysl2p and Neo1p
........................................................................................................................................................... 40
2.2.3.2 Co-immunoprecipitation experiments using TAP-tagged Ysl2p and HA-tagged Neo1p ........... 41
2.2.3.3 Fluorescence microscopy......................................................................................................... 43
2.2.3.4 Sucrose density gradient centrifugations.................................................................................. 45
2.2.3.5 Pulse-chase labeling and immunoprecipitation of HA-Neo1 proteins, HA-Ysl2p and CPY...... 45
32.2.3.6 Analysis of invertase glycosylation.......................................................................................... 46
2.2.3.7 ATPase activity assay of Neo1p .............................................................................................. 47
2.2.3.8 Liposome floatation assay ....................................................................................................... 49
3 Results ..................................................................................................................................50
3.1 Characterization of wild-type and mutant Neo1 proteins and their interactions with Ysl2p
and Arl1p ....................................................................................................................................... 50
3.1.1 Neo1p interacts with Ysl2p in vivo..................................................................................... 50
3.1.2 Neo1p localizes to endosomes and the Golgi complex....................................................... 52
3.1.3 The temperature-sensitive neo1-37 and neo1-69 mutants are defective in vacuolar protein
sorting, but not in normal secretion............................................................................................. 54
3.1.4 Localization and stability of the temperature-sensitive Neo1p mutants.............................. 57
3.1.5 Neo1-69p affects the subcellular distribution of HA-Arl1p................................................ 60
3.1.6 HA-Ysl2p localization and stability are affected in the neo1 mutants ................................ 61
3.1.7 Modifications of the Neo1p C-terminal tail affect the localization and stability of Neo1p. 63
3.1.8 The Neo1p ATPase activity is essential for Neo1p function in vivo, but is not impaired in
the neo1 mutants ......................................................................................................................... 65
3.2 Characterization of the localization of Sjl2p and of its newly-identified interacting partner
Bsp1p.............................................................................................................................................. 69
3.2.1 Sjl2p localizes to punctate structures by indirect immunofluorescence.............................. 69
3.2.2 The staining pattern of HA-Sjl2p is affected in cells lacking the actin-regulating kinases
Ark1p and Prk1p ......................................................................................................................... 71
3.2.3 Bsp1p, a binding partner of Sjl2p, localizes to cortical actin patches and p