Dynamics and architecture of the HOPS tethering complex in yeast vacuole fusion [Elektronische Ressource] / presented by Clemens Werner Ostrowicz

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DISSERTATION SUBMITTED TO THE COMBINED FACULTIES FOR THE NATURAL SCIENCES AND FOR MATHEMATICS OF THE RUPERTO-CAROLA UNIVERSITY OF HEIDELBERG, GERMANY FOR THE DEGREE OF DOCTOR OF NATURAL SCIENCES Dynamics and Architecture of the HOPS Tethering Complex in Yeast Vacuole Fusion PRESENTED BY DIPLOM-BIOCHEMIKER CLEMENS WERNER OSTROWICZ, BORN IN KIEL, GERMANY DISSERTATION SUBMITTED TO THE COMBINED FACULTIES FOR THE NATURAL SCIENCES AND FOR MATHEMATICS OF THE RUPERTO-CAROLA UNIVERSITY OF HEIDELBERG, GERMANY FOR THE DEGREE OF DOCTOR OF NATURAL SCIENCES PRESENTED BY DIPLOM-BIOCHEMIKER CLEMENS WERNER OSTROWICZ, BORN IN KIEL, GERMANY ORAL EXAMINATION: Dynamics and Architecture of the HOPS Tethering Complex in Yeast Vacuole Fusion Referees: Prof. Dr. Eduard Hurt Prof. Dr. Christian Ungermann Declaration I herewith declare that I wrote this thesis independently and used no other sources and aids than those indicated. April 28,2009 __________________________________ (Clemns W.Ostrowicz) Table of Contents Publications and Manuscripts ____________________________________________I Summary ____________________________________________________________ II Zusammenfassung____________________________________________________ IV 1 Introduction _______________________________________________________ 1 1.

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DISSERTATION
SUBMITTED TO THE
COMBINED FACULTIES FOR THE
NATURAL SCIENCES AND FOR MATHEMATICS OF THE
RUPERTO-CAROLA UNIVERSITY OF HEIDELBERG, GERMANY
FOR THE DEGREE OF
DOCTOR OF NATURAL SCIENCES




Dynamics and Architecture of the HOPS
Tethering Complex in Yeast Vacuole Fusion











PRESENTED BY
DIPLOM-BIOCHEMIKER CLEMENS WERNER OSTROWICZ,
BORN IN KIEL, GERMANY DISSERTATION
SUBMITTED TO THE
COMBINED FACULTIES FOR THE
NATURAL SCIENCES AND FOR MATHEMATICS OF THE
RUPERTO-CAROLA UNIVERSITY OF HEIDELBERG, GERMANY
FOR THE DEGREE OF
DOCTOR OF NATURAL SCIENCES





















PRESENTED BY
DIPLOM-BIOCHEMIKER CLEMENS WERNER OSTROWICZ,
BORN IN KIEL, GERMANY
ORAL EXAMINATION:








Dynamics and Architecture of the HOPS
Tethering Complex in Yeast Vacuole Fusion













Referees: Prof. Dr. Eduard Hurt
Prof. Dr. Christian Ungermann Declaration
I herewith declare that I wrote this thesis independently and used no other sources and aids than
those indicated.



April 28,2009 __________________________________
(Clemns W.Ostrowicz) Table of Contents
Publications and Manuscripts ____________________________________________I
Summary ____________________________________________________________ II
Zusammenfassung____________________________________________________ IV
1 Introduction _______________________________________________________ 1
1.1 The Eukaryotic Endomembrane System __________________________________ 1
1.1.1 Intracellular Organelles_____________________________________________________ 2
1.1.1.1 Transport Pathways involving the vacuole __________________________________ 6
1.2 Membrane Trafficking ________________________________________________ 9
1.2.1 Vesicle Formation________________________ 10
1.2.1.1 Clathrin mediated vesicle formation______ 11
1.2.1.2 AP-3 dependent vesicle formation _______________________________________ 12
1.2.2 Vesicle targeting _________________________________________________________ 14
1.2.3 Membrane Tethering_____________________ 15
1.2.4 Membrane Fusion________________________ 16
1.3 The Tethering machinery _____________________________________________ 17
1.3.1 Rab GTPases____________________________ 17
1.3.2 Tethering Factors ________________________________ 20
1.3.2.1 The TRAPP complexes________________ 22
1.3.2.2 The COG complex____________________ 24
1.3.2.3 The GARP complex__________________ 25
1.3.2.4 The exocyst complex 27
1.3.2.5 The HOPS tethering complex ___________________________________________ 29
1.4 The yeast vacuole as a model system for intracellular membrane fusion _______ 33
1.4.1 Stages of vacuole fusion___________________ 34
1.4.1.1 Priming ____________________________________________________________ 35
1.4.1.2 Tethering___________________________ 36
1.4.1.3 Docking and fusion___________________ 36
1.5 SNARE function in vacuole fusion ______________________________________ 37
1.5.1 SNAREs during priming________________________________ 39
1.5.2 SNAREs during tethering, docking and fusion__________________________________ 40
2 Rationale ________________________________________________________ 41
3 Results___________________________________________________________ 43
3.1 Regulation of the HOPS tethering complex by Yck3- mediated phosphorylation of
its subunit Vps41 _________________________________________________________ 43
3.1.1 Identification of phosphorylation sites in Vps41_________________________________ 44
3.1.2 The vacuolar fusion factor Mon1 is a substrate of Yck3___________________________ 49
3.2 Purification of HOPS complex and its subunits for crystallization and electron
microscopy analysis _______________________________________________________ 50
3.2.1 Recombinant expression of HOPS subunits____ 51
3.2.1.1 Bicistronic expression _________________________________________________ 51
3.2.2 Purification of HOPS proteins from yeast______ 52
3.2.2.1 Purification of Vps41 for crystallographic trials _____________________________ 54
3.2.2.2 Screening for optimal purification conditions for Vps41_______________________ 55
3.2.2.3 Purification of HOPS complex for electron microscopy 58
3.3 Identification and characterization of the novel CORVET tethering complex and
intermediate complexes ____________________________________________________ 61
3.3.1 Vps3 interacts with the class C Vps proteins ___________________________________ 61
3.3.2 Vps3 is a subunit of a novel HOPS homologous complex _________________________ 62
3.3.3 Identification of intermediate complexes______ 64
3.4 Identification of stable HOPS subcomplexes ______________________________ 65
3.4.1 Deletion of single HOPS subunits reveals the existence of stable subcomplexes________ 65
3.4.2 Overexpression of apposite subunits allows formation of stable subcomplexes_________ 67
3.4.3 HOPS related subcomplexes are naturally occurring in vivo _______________________ 71
3.4.4 In vivo localization of overexpressed HOPS subunits ____________________________ 72
3.4.5 In vitro reconstitution of HOPS complex assembly ______________________________ 74
3.5 Functional characterization of HOPS subunits and subcomplexes ____________ 76
3.5.1 Vps41 and Vam6 differentially interact with the vacuolar Rab GTPase Ypt7 __________ 76
3.5.2 The HOPS complex interacts with Ypt7 via Vps41 alone _________________________ 79
4 Discussion________________________________________________________ 82
4.1 Regulation of the HOPS tethering complex by Vps41 phosphorylation ________ 82
4.2 Structural analysis of the HOPS complex ________________________________ 84
4.3 Identification of the novel CORVET complex and intermediate complexes_____ 85
4.4 Identification of stable subcomplexes and functional characterization of HOPS
subunits _________________________________________________________________ 87
4.5 Conclusion __________________________________________________________ 89