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Identifying Potential Binding Partners of VPS16B and VPS33B in Mammalian Cells

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Identifying Potential Binding Partners of VPS16B and VPS33B in Mammalian Cells Identifying Potential Binding Partners of VPS16B and VPS33B in Mammalian Cells by Shao Zun Chen A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Biochemistry University of Toronto © Copyright by Shao Zun Chen 2013 Identifying Potential Binding Partners of VPS16B and VPS33B in Mammalian Cells Shao Zun Chen Master of Science Department of Biochemistry University of Toronto 2013 Abstract Platelets contain specialized granules that are required for platelet function in hemostasis. These granules are formed in the megakaryocyte precursor, and VPS33B and VPS16B are essential for this process as mutations in them cause arthrogryposis, renal dysfunction and cholestasis syndrome, where platelets lack α-granules. Here, it is shown that VPS33B and VPS16B exist as part of two complexes (480 kDa and 720 kDa). My project entailed the identification of the components of these complexes that could be required for platelet granule biogenesis. It was found that these complexes are distinct from the VPS33A HOPS complex as they are not associated with VPS11 or VPS18, but are formed through self- oligomerization. Yeast three hybrid and co-immunoprecipitation experiments suggest that VPS52, COG5 and ATP6AP2 may interact with VPS33B and VPS16B. In addition, VPS33B and VPS16B likely interact with Rab5 and/or Rab7, but not with Rab11A, based on both GST-pulldown assays and co-immunoprecipitation experiments. ii Acknowledgments I would like to express my deepest appreciation to my supervisor, Dr. Walter Kahr, for providing guidance and inspiration during my studies; to my supervisory committee Dr. Allen Volchuk and Dr. David Williams for their helpful inputs and guidance; to all members of the Kahr lab: Dr. Ling Li, Denisa Urban, Dr. Fred Pluthero and Michael Puhacz for helpful advice and support; to Dr. Carol Froese for technical assistance with the SF21 insect cell experiments; to Dr. John Rubinstein for his help in studying the structure of VPS33B- VPS16B complex by electron microscopy; and to Dr. John Brumell for providing various constructs, and all other members of the Trimble lab for helpful advice and sharing of reagents. iii Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Abbreviations ..................................................................................................................... ix Chapter 1 Introduction .................................................................................................................... 1 1.1 Platelets and megakaryocytes ............................................................................................. 1 1.1.1 Platelet structure and function ................................................................................ 1 1.1.2 Megakaryocyte maturation and platelet formation ................................................. 2 1.2 Granule Biogenesis ............................................................................................................. 4 1.3 Arthrogryposis renal dysfunction and cholestasis (ARC) syndrome and VPS33B ............ 7 1.4 The yeast HOPS/CORVET complex .................................................................................. 9 1.5 VPS33B complex in mammalian cells ............................................................................. 12 1.6 VPS16B and ARC syndrome ............................................................................................ 13 1.7 Rab GTPases ..................................................................................................................... 15 1.8 Rationale and hypothesis .................................................................................................. 18 Chapter 2 Materials and Methods ................................................................................................. 19 2.1 Antibodies and plasmids .................................................................................................... 19 2.2 Cloning ............................................................................................................................... 19 2.3 Cell cultures and transfections/infections .......................................................................... 20 2.4 Baculovirus generation ....................................................................................................... 21 2.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting ................................................................................................................ 21 2.6 Yeast hybrid screens .......................................................................................................... 22 2.7 Immunoprecipitation .......................................................................................................... 24 2.8 Blue native PAGE (BN-PAGE) and sample preparation ................................................... 24 2.9 Mass spectrometry ............................................................................................................. 25 2.10 His-tagged protein purification ........................................................................................ 26 2.11 Electron microscopy ......................................................................................................... 26 2.12 GST-protein bead preparation and pull-down assay ........................................................ 26 2.13 Size exclusion chromatography of the VPS33B-VPS16B complex ................................ 27 2.14 Tandem affinity purification (TAP) ................................................................................. 27 Chapter 3 Results .......................................................................................................................... 29 3.1 Yeast-two-hybrid (Y2H) screen using VPS16B as bait against a bone marrow library ................................................................................................................................ 29 3.2 Stable cell line immunoprecipitation (VPS16B-FLAG and VPS33B-FLAG) followed by mass-spectrometry ........................................................................................ 30 3.3 Identification of transient interacting proteins .................................................................. 33 3.3.1 Yeast-three hybrid (Y3H) assay with VPS16B as the bait and VPS33B as the bridge .............................................................................................................. 34 3.3.2 Confirmation of yeast three hybrid assay interactions by co- immunoprecipitation ............................................................................................. 35 3.4 Interaction with Rab proteins ............................................................................................ 37 3.4.1 GST-fusion protein pulldown assay ...................................................................... 37 3.4.2 Rab Co-immunoprecipitation experiments in mammalian cells ........................... 38 iv 3.5 Hetero-oligomer interaction analysis ................................................................................ 39 3.5.1 Verifying multiple copies of VPS33B/16B by co-immunoprecipitation experiments ........................................................................................................... 40 3.5.2 VPS16B oligomerization is not phosphorylation dependent ................................ 42 3.5.3 VPS33B-VPS16B protein purification from SF21 insect cells ............................ 43 3.5.4 Ultrastructure analysis using electron microscopy ............................................... 47 Chapter 4 Discussion .................................................................................................................... 49 4.1 The VPS33B-VPS16B complex does not include other HOPS components .................... 49 4.2 VPS52 as a potential interacting partner of VPS16B and VPS33B ................................... 50 4.3 COG1/COG5 as potential interacting partners of VPS16B and VPS33B ......................... 51 4.4 ATP6AP2 as a potential interacting partner of VPS16B and VPS33B ............................. 51 4.5 Rab5 and Rab7 as potential binding partners of VPS33B and VPS16B, but not Rab11A. ............................................................................................................................ 53 4.6 VPS33B and VPS16B can associate to form multimeric complexes ................................ 55 Chapter 5 Conclusion and Future Directions ................................................................................ 57 References ..................................................................................................................................... 62 Appendices .................................................................................................................................... 71 A.1 Complete
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