Identifying Potential Binding Partners of VPS16B and VPS33B in Mammalian Cells

Shao Zun Chen

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Biochemistry University of Toronto

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.

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.

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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 mass-spectrometry results from individual complexes ...... 71 A.2 Gel Filtration Profile of SF21 purified VPS33B-VPS16B complex ...... 74 A.3 His-VPS16B and VPS33B purification from SF21 insect cells ...... 75

List of Tables

Table 1. Summary of results from Y2H using human bone marrow library with VPS16B as bait………………………………………………………………...……………….29

Table 2. Summary of results from Y3H using human universal normalized tissue library with VPS16B as bait and VPS33B as bridge..…...... 34

List of Figures

Figure 1. Electron micrographs of platelets...... 1

Figure 2. Formation of platelets from megakaryocytes in bone marrow...... 3

Figure 3. Alpha granule biogenesis in megakaryocytes...... 6

Figure 4. Organelles including alpha granules travel along proplatelet shafts toward the tip in distinct populations...... 6

Figure 5. Abnormal platelets from patients with ARC syndrome...... 8

Figure 6. Predicted domains of VPS33B based on conserved domain search using entire protein sequence...... 9

Figure 7. Yeast Class C VPS complexes and their role in vesicle trafficking...... 10

Figure 8. VPS33B exists as a 480 kDa and 720 kDa complex in Dami cells...... 12

Figure 9. Predicted domains of VPS16B based on conserved domain search using entire protein sequence...... 13

Figure 10. VPS16B is required for α-granule formation...... 15

Figure 11. Rab GTPases and their function in vesicular trafficking...... 17

Figure 12. VPS16B forms a large complex regardless of addition of a tag...... 31

Figure 13. VPS16B and VPS33B do not elute well from the calmodulin beads...... 31

Figure 14. VPS33B and VPS16B form complexes in stable HEK293 cell lines...... 33

Figure 15. High molecular weight complexes isolated from VPS16B-FLAG and VPS33B- FLAG stable HEK293 cells contained mainly VPS33B and VPS16B...... 33

Figure 16. VPS52 as a potential binding partner of VPS33B or VPS16B...... 36

Figure 17. COG5 and ATP6AP2 as potential binding partners of VPS33B or VPS16B...... 37

Figure 18. GST-pulldown assay using Rab5, 7 and 11 with stable cell line lysates expressing VPS33B-FLAG or VPS16B-FLAG...... 38

Figure 19. Co-immunoprecipitations of GFP-Rab5A, - Rab7, and - Rab11A with VPS33B and VPS16B...... 39

Figure 20. VPS16B interacts with VPS16B and VPS33B interacts with VPS33B...... 41

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Figure 21. Phosphorylation mutation Y11F and ARC mutation C341R in VPS16B does not affect its interaction with itself or the assembly of the 480 kDa complex...... 42

Figure 22. VPS16B-FLAG co-purifies with His-VPS33B in SF21 insect cells...... 44

Figure 23. VPS16B-FLAG co-purifies with His-VPS33B in SF21 insect cells in a similar 480 kDa and the 720 kDa complex as in mammalian cells...... 45

Figure 24. VPS33B-VPS16B complexes purified from SF21 insect cells contained only VPS33B and VPS16B...... 47

Figure 25. Electron micrographs of purified VPS33B-VPS16B complexes from SF21 cells.47

Figure 26. ATP6AP2 is part of the V-ATPase complex...... 53

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List of Abbreviations

ATP6AP2: V-ATPase lysosomal accessory protein 2 ARC: Arthrogryposis, renal dysfunction, and cholestasis AP3: Adaptor protein complex 3 BLOC: Biogenesis of lysosome related organelle complex BN-PAGE: Blue Native polyacrylamide gel electrophoresis CIP: Calf-intestinal phosphatase COG: Conserved oligomeric Golgi complex CORVET: Class C core vacuole/endosome tethering DMEM: Dulbecco’s modification Eagle’s medium DMSO: Dimethyl sulfoxide ECL: Enhanced chemiluminescence EDTA: Ethylenediaminetetraacetic acid EM: Electron microscopy FBS: Fetal bovine serum GAP: GTPase activating protein GARP: Golgi associated retrograde proteins GDF: GDI displacement factors GDI: GDP dissociation inhibitors GEF: Guanosine exchange factor GFP: Green fluorescent protein GST: Glutathione S-transferase HA: Haemagglutinin HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HOPS: Homotypic fusion and protein sorting HRP: Horseradish peroxidase IP: Immunoprecipitate LB: Lysogeny broth MVB: Multivesicular body PBS: Phosphate-buffered saline PCR: Polymerase chain reaction PEG: Polyethylene glycol PVDF: Polyvinylidene fluoride SPE39: Spermatogenesis family member 39 SDS-PAGE: Sodium dodecyl sulphate polyacrylamide gel electrophoresis SM: Sec1/Munc18 SNARE: Soluble N-ethylmaleimide-sensitive factor attachment protein receptor TAP: Tandem affinity purification TBS: Tris-buffered saline TGN: Trans-Golgi network VIPAR: VPS33B-interacting protein involved in polarity and apical protein restriction VPS: Vacuolar protein sorting Y2H: Yeast two hybrid Y3H: Yeast three hybrid YPDA: Yeast extract, peptone, dextrose, adenine

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Chapter 1 Introduction

1.1 Platelets and megakaryocytes

Platelets are small (2-5 µm in diameter) and anucleate blood cells that are derived from megakaryocytes, which are rare myeloid cells that are primarily found in the bone marrow. Platelets circulate in the blood with a life span of 7 to 10 days, and they are required for hemostasis and are involved in many other functions including angiogenesis and innate immunity (Patel, Hartwig et al., 2005).

1.1.1 Platelet structure and function

Platelets contain a number of distinguishable structural elements (Figure 1): an invagination of the plasma membrane that forms the open canalicular system; a spectrin based membrane skeleton; an actin based cytoskeleton; a peripheral microtubule band; and four types of vesicles: lysosomes, peroxisomes, alpha (α)-granules and dense granules (Thon and Italiano, 2012).

Figure 1. Electron micrographs of platelets. Platelets contain distinguishable structural elements including the open canalicular system and spectrin based membrane skeleton. They contain secretory vesicles storing protein factors that are essential for platelet function. Image taken from Thon et al, 2012.

Platelets are best known for their involvement in forming blood clots at sites of vascular injury. In the case of vessel wall injury, platelets will first adhere and aggregate at the site of injury, and become activated by factors such as collagen and thrombin (Nurden, 2011). Platelet activation is characterized by rearrangement of cytoskeletons, formation of filopodial extensions, and secretion of granular proteins (Zucker and Nachmias, 1985). Release of protein factors and adherence of more platelets will lead to formation of a platelet plug, and the start of coagulation events by conversion of fibrinogen into fibrin (Nurden, 2011). Disorders that cause reduced platelet counts (Thrombocytopenia, <150,000/μL) or dysfunctional platelets can cause a bleeding phenotype in patients (Cox, Price et al., 2011). In addition to their essential role in hemostasis, platelets have been found to be involved in many other functions. Platelets influence inflammation by secretion of pro-inflammatory factors such as chemokines that induce neutrophil adhesion, monocyte activation and monocyte differentiation (Blair and Flaumenhaft, 2009; Semple, Italiano et al., 2011; Rondina, Weyrich et al., 2013). Platelets also facilitate antimicrobial host defense by recognition of microbial colonization in damaged endothelium, secretion of α-granule microbicidal proteins, and activation of the complement pathway (Semple, Italiano et al., 2011). The ability of platelets to modulate immune responses is also thought to contribute to the development of atherosclerosis and ischemic stroke (Blair and Flaumenhaft, 2009). There are well established studies supporting the platelet’s role in angiogenesis, where platelet granules may contain distinct populations of proteins with pro- and antiangiogenic properties (Italiano, Richardson et al., 2008). Platelets are known to promote wound healing through stimulation of tissue regeneration and the production of growth factors (Nurden, Nurden et al., 2008). There is a diverse range of physiological functions affected by platelets, and there are many unanswered questions about how platelets fulfill each of these roles.

1.1.2 Megakaryocyte maturation and platelet formation

Platelets are formed from megakaryocytes via the process of proplatelet formation. Megakaryocytes are large and rare myeloid cells that are primarily found in the bone marrow, but they are also found in the lung and the peripheral blood (Patel, Hartwig et al., 2005). Megakaryocytes are highly specialized for platelet biogenesis with their unique cytoplasm and membrane systems. Before megakaryocytes release platelets, they first

enlarge in diameter and increase the production of ribosomes to facilitate the synthesis of platelet specific proteins (Italiano, Patel-Hett et al., 2007). This cell growth is mediated by multiple rounds of endomitosis, which is a series of DNA replication events without cell division (up to 64-fold). This step is promoted by binding of thrombopoietin, a cytokine required for megakaryocyte maturation, to the c-Mpl receptor on megakaryocytes (Kaushansky and Drachman, 2002). In addition to becoming polyploid and multilobed (4N to 128N), megakaryocytes also reconstruct their membrane system and organelle organization to prepare for proplatelet formation (Patel, Hartwig et al., 2005). Proplatelet structures are long branched membrane structures (100-500 μm) that commence from a single site on the megakaryocyte. Organelles such as granules and mitochondria are transported along microtubules via motor proteins kinesin and dynein to the tip of the proplatelet (Richardson, Shivdasani et al., 2005). The proplatelets extend through the endothelium and into the vasculature, where they are fragmented by the sheer flow of the blood and are released into the blood stream as functional platelets (Figure 2). A single megakaryocyte can release from 100 to 1000 platelets through this process (Geddis and Kaushansky, 2007; Junt, Schulze et al., 2007).

Figure 2. Formation of platelets from megakaryocytes in bone marrow. Megakaryocytes adhere to the endothelial wall and form proplatelet structures that extend into the blood vessel. The sheer flow of blood allow the proplatelets to break down into functional platelets. Figure taken from Geddis and Kaushansky (2007).

1.2 Granule Biogenesis

Platelets contain two distinct types of granules: α-granules and dense (-) granules. Upon activation, the granules fuse with the plasma membrane to release their cargo and to increase platelet surface area (Blair and Flaumenhaft, 2009). Dense granules average about 3 to 8 per cell with a diameter of 200 to 300 nm. They are electron dense vesicles that contain mostly small molecules such as serotonin, adenosine diphosphate (ADP), adenosine triphosphate (ATP), polyphosphates, calcium ions and magnesium ions (Thon and Italiano, 2012). Alpha granules are the most abundant secretory granule in platelets as there are approximately 50 to 80 α-granules per platelet, varying in size from 200 to 500 nm. They are a source of membrane (10%) in addition to the open canalicular system that contributes to the increase in platelet surface area during activation (White and Clawson, 1980). Alpha granules are membrane bound vesicles where proteomic studies have identified over 300 soluble cargo proteins. These releasable proteins include adhesion molecules, chemokines, cytokines, immunologic modulators, fibrinolytic regulators, and also an array of coagulation factors, growth factors, complement factors, and angiogenic factors (Harrison and Cramer, 1993; Coppinger, Cagney et al., 2004; Reed, 2004). These cargoes can originate from both endogenous expression and endocytic events, and they play important roles in blood clotting, angiogenesis, inflammation, and development of atherosclerotic diseases (Blair and Flaumenhaft, 2009). There are studies suggesting sorting events within the α-granules to package distinct α-granule subpopulations for differential secretion, as α-granules contain both anti- and pro-angiogenic factors (Sehgal and Storrie, 2007; Italiano, Richardson et al., 2008). However, other observations suggest that there is differential packaging within α- granules (van Nispen tot Pannerden, de Haas et al., 2010) and that secretion is heterogeneous (Jonnalagadda, Izu et al., 2012).

Both α-granule and dense granule biogenesis occur in the megakaryocytes (Figure 3). Alpha and dense granules are partly derived from direct budding of small vesicles containing granule cargoes from the trans-Golgi network (Cramer, Harrison et al., 1990; Hegyi, Heilbrun et al., 1990). These vesicles are subsequently directed to multivesicular bodies (MVBs), which are typically transient endosomal structures involved in protein sorting. Vesicles containing endocytosed cargoes and newly synthesized proteins are sorted at this

site (Woodman and Futter, 2008). MVBs serve as an intermediate stage for both α and dense granule production in megakaryocytes (Youssefian and Cramer, 2000). Within megakaryocytes, there are two distinct populations of MVBs. Immature MVB (MVB I) contain internal vesicles alone, and they sort endocytosed proteins from endosomes and also from trans-Golgi network cargo vesicles. Sorted vesicles are then transported to mature MVBs (MVB II) which contain both internal vesicles and an electron dense matrix, which give rise to α-granules and dense granules (Heijnen, Debili et al., 1998). It is unclear whether MVBs are the only intermediate through which vesicle trafficking to α-granules occurs, but they represent a stage from which α-granules mature. Maturation of α-granules continues in circulating platelets where plasma proteins are incorporated into platelet α-granules via endocytic mechanisms (Behnke, 1992). Fibrinogen is first bound to a membrane receptor on platelets and internalized by clathrin-dependent endocytosis (Handagama, George et al., 1987). Other proteins such as albumin or immunoglobulin are taken up by pinocytosis (George and Saucerman, 1988).

Figure 3. Alpha granule biogenesis in megakaryocytes. Alpha granules are formed from vesicles that either bud off directly from the trans-Golgi network (TGN) or through sorted vesicles that passed through distinct populations of the multivesicular bodies (MVBs). Cargoes can also be endocytosed at the plasma membrane and delivered to α-granules. Figure adapted from Flaumenhaft (2012).

Newly synthesized organelles including the α- and dense granules are transported to the platelets in the final stages of megakaryocyte development, where proplatelet structures are formed (Richardson, Shivdasani et al., 2005). The proplatelet extensions are filled with microtubule bundles; and organelles and granules travel bi-directionally at the rate of 0.2 μm/sec along these microtubules. Although there are bidirectional movements at the same rate, some of the organelles are captured at the proplatelet tip by the microtubule coil structures. There appear to be two types of transport mechanism. First, organelles including granules are transported by motor proteins; and secondly, the microtubules can slide against each other to move the bound organelles in a “piggyback” fashion. The transport process along the proplatelet shaft and the capturing of organelles at the tip are both actin- independent processes (Richardson, Shivdasani et al., 2005). In addition, the organelles do not move as aggregates but as single particles along the proplatelets, and distinct α-granule populations have been visualized to be transported separately (Figure 4) (Italiano, Richardson et al., 2008).

Figure 4. Organelles including alpha granules travel along proplatelet shafts toward the tip in distinct populations. Alpha granules are transported from the megakaryocyte to the

proplatelet tip along the proplatelet structure. Immunofluorescence shows a limited colocalization between α-granule proteins fibrinogen (green) and von Willebrand Factor (red). This suggests that α-granules exist in distinct sub-populations or that cargo is packaged in distinct regions within granules. Figure adapted from Italiano, Richardson et al. (2008) and Blair and Flaumenhaft (2009).

Maturation of dense granules and α-granules appears to follow distinct biogenesis pathways after sorting at the MVB. This is based on studying platelet disorders that affect these two types of granules separately. Dense granule defects are observed in patients with Hermansky Pudlak syndrome and Chediak-Higashi syndrome. These patients completely lack dense granules and have abnormal bleeding, but have functional α-granules (Gunay-Aygun, Huizing et al., 2004). Mutations identified in these patients are in genes that encode proteins involved in vesicle trafficking, such as members of the biogenesis of lysosome-related organelle complexes (BLOC1-3), and adaptor protein complex 3 (AP3) (Dell'Angelica, Shotelersuk et al., 1999; Huizing, Parkes et al., 2007). In addition, a murine model of Hermansky Pudlak syndrome shows defects in a subunit of the Class C Vps complex, VPS33A (Suzuki, Oiso et al., 2003). Similarly, disorders that affect the formation of α- granules such as gray platelet syndrome and arthrogryposis renal dysfunction and cholestasis (ARC) syndrome, do not impair the formation of dense granules (Lo, Li et al., 2005).

1.3 Arthrogryposis renal dysfunction and cholestasis (ARC) syndrome and VPS33B

Arthrogryposis multiplex congenital, renal dysfunction and cholestasis (ARC) syndrome is an autosomal recessive disorder. Patients fail to thrive and they rarely survive their first year of life (Di Rocco, Callea et al., 1995). It is a multi-system disorder with many clinical manifestations including congenital heart disease, cerebral malformations, deafness, nephrogenic diabetes insipudus, ichthyosis, dysmorphic features, recurrent fevers, diarrhea and increased bleeding susceptibility. The bleeding problem in these patients is not due to decreased platelet number as they have normal platelet counts (Kim, Chang et al., 2010). Observations of blood films and thin-section transmission electron microscopy of platelets from ARC patients reveal large platelets that are completely depleted of α-granules (Figure 5). In contrast to gray platelet syndromes where α-granule membrane structures are intact and P-selectin, an α-granule membrane protein, is present, ARC platelets lack α-granule

membrane structures and have no detectable levels of P-selectin (Lo, Li et al., 2005). One of the genes identified to be mutated in patients with ARC syndrome is VPS33B (Gissen, Johnson et al., 2004; Lo, Li et al., 2005).

Figure 5. Abnormal platelets from patients with ARC syndrome. Patients with mutations in VPS33B (A) have platelets that appear large and pale compared to controls [black arrows] (B). When examined under electron microscopy, ARC platelets (C) also lack electron dense α-granules that are abundant in normal platelets [white arrows] (D). Figure adapted from Lo, Li et al. (2005).

VPS33B is a member of the Sec1/Munc18 (SM) protein family (Toonen and Verhage, 2003). SM proteins are highly conserved among species and are essential factors in vesicle trafficking. SM proteins have been implicated in binding to soluble N-ethylmaleimide- sensitive factor attachment protein receptors (SNAREs), which are membrane-bound proteins that function in docking the vesicle to their target membrane, and in catalyzing the fusion of the membrane of the donor vesicle with the target compartment. SM proteins bind to and affect the formation of the SNARE complexes, which provide the mechanical energy for membrane fusion to occur. The mammalian genome encodes 7 SM proteins: Munc18-1,

Munc18-2, Munc18-3, VPS45, SLY1, VPS33A and VPS33B. Each protein functions to regulate fusion events at different intracellular membrane compartments (Toonen and Verhage, 2003).

Of all the SM genes identified, VPS33B is among one of the least characterized. It is a protein of 617 amino acids, with an estimated molecular weight of 71.6 kDa. Similar to other SM proteins, it is largely hydrophilic. It shares 31% sequence identity and 51% sequence similarity with its mammalian homolog VPS33A, and it has a unique stretch of 31 amino acids (residues 450 to 480) that is not present in VPS33A (Figure 6). In addition, it also has three predicted syntaxin binding domains based on sequence alignment with neuronal Sec1 (residues 35-60, 260-275 and 310-340) (Gissen, Johnson et al., 2005).

Figure 6. Predicted domains of VPS33B based on conserved domain search using entire protein sequence. VPS33B contains the Sec1 domain that is common to the Sec1/Munc18 family (residues 36-612 in red). In addition, it also contains three putative syntaxin binding sites (blue) at residues 35-60, 260-75, and 310-40. Residues highlighted in yellow represent a unique stretch of amino acids (450-80) that is absent in VPS33A. Image modified from http://www.ncbi.nlm.nih.gov/Structure/cdd.

1.4 The yeast HOPS/CORVET complex

VPS33A and VPS33B are both homologues of the yeast protein vacuolar protein sorting (Vps) 33p (Gissen, Johnson et al., 2005). Vps33p was first discovered in Sacchromyces cerevisiae through a genetic screen that identified 41 VPS genes (Raymond, Howald- Stevenson et al., 1992). These VPS genes were organized into different classes based on distinct phenotypes. Vps33p is one of the 4 proteins that caused the Class C phenotype, in which mutant cells lacked identifiable vacuolar lysosomes. Other proteins that cause Class C phenotypes include Vps11p, Vps16p, and Vps18p (Raymond, Howald-Stevenson et al., 1992). Interestingly, Vps33p, Vps11p, Vps16p and Vps18p were later found to form the stable Vps-C core complex (Sato, Rehling et al., 2000; Seals, Eitzen et al., 2000; Wurmser, Sato et al., 2000; Peterson and Emr, 2001; Subramanian, Woolford et al., 2004; Collins,

Thorngren et al., 2005). This core complex can associate with two accessory subunits, Vps39p and Vps41p to form a 633 kDa complex called homotypic fusion and protein sorting (HOPS) complex (Figure 7) (Nickerson, Brett et al., 2009). Vps39p is a yeast guanosine exchange factor (GEF) that activates vacuolar protein Ypt7, which is the yeast Rab7 homolog. Rab proteins are effectors that facilitate membrane tethering with a GTPase domain. Vps41p binds to Ypt7 and physically links it to the HOPS complex, and it also acts as a direct effector of Ypt7 (Wurmser, Sato et al., 2000; Brett, Plemel et al., 2008). The HOPS complex controls all trafficking events from the late endosomes, autophagosomes and the Golgi into vacuolar lysosomes in yeast. The Vps-C core complex also associates with alternative accessory subunits Vps3p and Vps8p to form the Class C core vacuole/endosome tethering (CORVET) complex (659 kDa) (Nickerson, Brett et al., 2009). Vps3p and Vps8p share homology with the HOPS complex accessory subunits Vps39p and Vps41p. The CORVET complex interacts with the Rab5 ortholog Vps21p at early endosomes through Vps8p, and controls traffic into late endosomes (Markgraf, Peplowska et al., 2007; Peplowska, Markgraf et al., 2007). The two complexes interconvert by exchanging the two accessory subunits, forming chimeric complexes to provide specificity in vesicular trafficking (Ostrowicz, Brocker et al., 2010).

Figure 7. Yeast Class C VPS complexes and their role in vesicle trafficking. There are two VPS complexes in yeast that are distinguished by either VPS3, 8 (CORVET) or VPS39, 41 (HOPS). Each complex share the VPS11, 16, 18, 33 core and they alternate by forming chimeric structures. They regulate distinct trafficking events: The CORVET complex

regulates the pathways that are colored blue; and the HOPS complex regulates ones colored in red. Figure adapted from Nickerson, Brett et al. (2009).

In metazoan cells, there exist equivalents of the HOPS complex components: VPS11, VPS16A, VPS18 and VPS33A. Similar to the HOPS complex in yeast, the metazoan version of these proteins also forms a large complex. In Drosophila, dVPS33A is the product of the carnation (car) gene, where mutations in the gene lead to a carnation eye pigmentation defect (Sevrioukov, He et al., 1999; Akbar, Ray et al., 2009); dVPS33A has also been found as part of the 370 kDa HOPS complex. The Drosophila HOPS complex is required in lysosomal delivery of different cargoes and fusion of lysosomes with autophagosomes in Drosophila (Sriram, Krishnan et al., 2003; Pulipparacharuvil, Akbar et al., 2005; Simonsen, Cumming et al., 2007). In mouse, the mVPS11, mVPS16, mVPS18, mVPS39 and mVPS41 genes have been proposed to participate in early endosomal fusion (Kim, Kramer et al., 2001; Richardson, Winistorfer et al., 2004). Mice with mutations in mVPS33A is a model (buff) for Hermansky Pudlak syndrome, where they display hypopigmentation in their coat color and retina, and a platelet dense granule deficiency (Suzuki, Oiso et al., 2003).

VPS33A is one of the 16 genes proposed to be involved in regulation of vesicular trafficking to platelet dense granules and melanosomes (Suzuki, Oiso et al., 2003; Li, Rusiniak et al., 2004). Less is known about VPS33B, but loss of function of either VPS33A or VPS33B will result in distinct phenotypes, suggesting that they act at specific stages of vesicular trafficking. In Drosophila, dVPS33A and dVPS33B are part of distinct complexes with non- redundant functions. dVPS33A is required for late endosome to lysosome fusion, and loss of function does not affect early endosomal fusion (Akbar, Ray et al., 2009). On the other hand, dVPS33B is found to be present in a distinct complex from HOPS that interacts with Av1, a protein implicated in early endosomal fusion (Akbar, Tracy et al., 2011). In addition, loss of dVPS33A function that causes the carnation eye, was not rescued by over- expression of dVPS33B, confirming that these two proteins have distinct roles during vesicular trafficking (Pulipparacharuvil, Akbar et al., 2005; Akbar, Ray et al., 2009).

1.5 VPS33B complex in mammalian cells

Human VPS33B, like Drosophila VPS33B, is also found in a large complex. This is based on unpublished data from our lab using Blue native gel analysis (Figure 8). Blue native PAGE (BN-PAGE) differs from the traditional SDS-PAGE in that it uses Coomassie blue as the charge shift molecule (Wittig, Braun et al., 2006). Compared to the Laemmli system where SDS denatures protein and disrupts complexes, Coomassie G-250 selectively binds basic residues and hydrophobic residues, allowing positively charged proteins to migrate toward the anode while maintaining protein complex structure at native pH. Lysates from megakaryocytic Dami cells that expresses VPS33B endogenously were fractionated into cytosolic and membrane fractions; and protein complexes were visualized by BN-PAGE after immunoblotting. Using an antibody against endogenous VPS33B, distinct complexes containing VPS33B with approximate molecular weights of 480 kDa and 720 kDa were observed. Both the cytosolic and the membrane compartment contained the 480 kDa complex, whereas only the membrane compartment contained the 720 kDa complex.

Figure 8. VPS33B exists as a 480 kDa and 720 kDa complex in Dami cells. Blue native gel electrophoresis resolves cellular complexes on gels without denaturing proteins or destruction of complexes by using Coomassie G-250 as charge shift molecule. Running lysates from Dami cells, which are a megakaryocytic cell line, on Blue native gel and probing for VPS33B, a complex of 480 kDa was observed in both the cytosolic fraction and

the membrane fraction. A complex of 720 kDa was detected only in the membrane fraction (estimated size). Diagram adapted from Invitrogen Japan.

1.6 VPS16B and ARC syndrome

The objective of my thesis project was to identify all members of the large VPS33B complex. VPS16B is a previously identified protein that interacts with VPS33B. It was detected in a yeast two hybrid library screen using VPS33B as bait, and it was first identified as the uncharacterized gene product of chromosome 14 open reading frame 133 (C14orf133) (Urban, Li et al., 2012). Other groups have also identified similar interactions and named their protein human SPE-39 (SPErmatogenesis family member 39) (Zhu, Salazar et al., 2009) or VIPAR (VPS33B interacting protein involved in polarity and apical protein restriction) (Cullinane, Straatman-Iwanowska et al., 2010).

Figure 9. Predicted domains of VPS16B based on conserved domain search using entire protein sequence. VPS16B contains a Golgin A5 domain that spans most of the sequence (residues 26-471 in grey bar), and a Vps16_C domain (residues 178-416 in pink) found in yeast Vps16. (Image modified from http://www.ncbi.nlm.nih.gov/Structure/cdd)

In parallel to the presence of two VPS33 homologues, there are also two homologues of VPS16 in metazoans. VPS16B (57 kDa, 493 amino acids) only shares 20% sequence identity with VPS16A, however they have well-conserved secondary structures based on structure predictions that identify positions of alpha-helices (Urban, Li et al., 2012). A peptide BLAST search of VPS16B also revealed a Golgin A5 domain occupying most of the protein (residues 12 to 493) that is absent from VPS16A (Figure 9) (Cullinane, Straatman-Iwanowska et al., 2010; Urban, Li et al., 2012).

In Drosophila, dVPS16A is required for trafficking to lysosomes and biogenesis of pigment granules (Pulipparacharuvil, Akbar et al., 2005), while dVPS16B is encoded by the full-of- bacteria (fob) gene (Akbar, Tracy et al., 2011). dVPS16B is found to be required for

14 phagosome maturation, and mutations in the gene leads to increased sensitivity to infections. dVPS16A and dVPS16B are found to be part of two distinct VPS-C complexes in Drosophila. dVPS16A binds to only dVPS33A, while dVPS16B interacts specifically with dVPS33B (Pulipparacharuvil, Akbar et al., 2005). Similar to Drosophila, the human version of VPS16B also forms a distinct complex from VPS16A. Based on immunoprecipitation experiments, it was found that VPS16B specifically interacts with VPS33B and not VPS33A, and VPS16A specifically interacts with VPS33A and not VPS33B (Urban, Li et al., 2012). In addition, similar to Drosophila, VPS33A and VPS33B complexes in humans also have non- redundant functions in platelet granule biogenesis (Lo, Li et al., 2005; Urban, Li et al., 2012). Mutations in VPS33A cause defects in dense granule biogenesis (Suzuki, Oiso et al., 2003), while mutations in VPS33B causes α-granule defects (Gissen, Johnson et al., 2004; Lo, Li et al., 2005). Deficiency of VPS16B in zebrafish and mice also causes biliary secretion and tight junction defects, which is in agreement with the data that suggest the involvement of VPS16B in regulating apical-basolateral polarity in the liver and kidney (Cullinane, Straatman-Iwanowska et al., 2010).

It was recently confirmed in our laboratory that VPS16B, similar to its binding partner VPS33B, is also required in megakaryocyte and platelet α-granule biogenesis (Urban, Li et al., 2012). Loss of VPS16B due to a nonsense mutation in VPS16B has been identified in a patient with ARC syndrome. Ultrastructural studies done using platelets from this patient show a complete absence of α-granules and increased dense granules (Figure 10). This observation is similar to those seen in ARC platelets with mutations in VPS33B (Figure 5). Alpha granule cargo proteins are also absent in VPS16B null platelets, including the membrane protein P-selectin, suggesting an absence of α-granule membrane structures (Urban, Li et al., 2012).

Based on colocalization studies performed using Dami cells stably expressing GFP-VPS16B, it was found that VPS16B localized to the trans-Golgi network, late endosomes, lysosomes and α-granules. VPS16B did not co-localize with markers of dense granules, cis-Golgi, or early endosomes (Urban, Li et al., 2012). This is similar to a previous observation that showed VPS33B association with markers of late endosomes and α-granules, but not with dense granules or cis-Golgi complexes (Lo, Li et al., 2005). These results suggest that

VPS16B with VPS33B functions along the trans-Golgi network to late endosome, to α- granule vesicular trafficking pathway in megakaryocyte α-granule biogenesis.

Figure 10. VPS16B is required for α-granule formation. Platelets from an ARC patient with a mutation in VPS16B (B) show a complete absence of α-granules as compared to platelets from controls [white arrows] (A). Figure adapted from Urban, Li et al. (2012).

1.7 Rab GTPases

Rab (‘Ras-related in brain’) proteins are part of the Ras superfamily of small GTPases. They are central regulators of cycles of vesicle budding, transport, tethering and fusion (Stenmark, 2009). Similar to other regulatory GTPases, Rabs function as molecular switches that alternate between the GTP-bound state and the GDP-bound state, with the help of a guanine nucleotide exchange factor (GEF) and a GTPase-activating protein (GAP) (Figure 11A). The GTP-bound conformation is regarded as the active state that interacts with downstream effector proteins (Stenmark and Olkkonen, 2001). There are more than 60 members of the Rab family in the human genome, and they localize to distinct intracellular membrane compartments. Rab proteins can be dissociated from or attached to membranes via GDIs (GDP dissociation inhibitors) or GDFs (GDI-displacement factors) respectively. Recruitment to membranes via GDFs is followed by activation by GEFs, and active Rabs continue to interact with their effectors until they are reverted to GDP-bound forms by GAPs (Stenmark, 2009).

Different Rab GTPases are localized to distinct organelles, or distinct membrane microdomains on the same organelle (Figure 11B). In particular, Rab5 localizes to early endosomes, phagosomes, caveosomes and the plasma membrane; and it mediates endocytosis and endosome fusion of clathrin coated vesicles (Kitano, Nakaya et al., 2008; Stenmark, 2009). Rab7, on the other hand associates with late endosomes and mediates maturation of late endosomes and phagosomes, and their fusion with lysosomes (Rojas, van Vlijmen et al., 2008). Rab11 is found at the recycling endosome and the trans-Golgi network, and they regulate traffic from early endosomes to the trans-Golgi network and secretion from recycling endosomes (Wilcke, Johannes et al., 2000; Savina, Fader et al., 2005; Ducharme, Williams et al., 2007).

The HOPS and CORVET complexes in yeast interact with the Rab7 orthologue Ypt7 and Rab5 orthologue Vps21 respectively (Nickerson, Brett et al., 2009). Although it is still unclear which Rab protein interacts with the VPS33B-VPS16B complexes in humans, some data suggest that Rab5, Rab7 and Rab11 are involved, as VPS16B functions in the endosome-lysosome trafficking pathway (Zhu, Salazar et al., 2009). In addition, VPS16B possesses a Golgin A5 domain, which is known to interact with members of the Rab GTPases family via their coiled-coil motif; and VPS16B has been proposed to interact with Rab11A through co-immunoprecipitation experiments by another group (Cullinane, Straatman-Iwanowska et al., 2010). This in agreement with the observation that VPS16B co- localizes with Rab5, Rab7 and Rab11 compartments based on immunofluorescence microscopy in HEK293 cells (Zhu, Salazar et al., 2009).

Figure 11. Rab GTPases and their function in vesicular trafficking. Rab proteins alternate between a GTP and GDP bound state mediated by Guanine nucleotide exchange factors (GEF) and GTPase activating proteins (GAP), and are dissociated from or attached to membranes by GDP dissociation inhibitors (GDI) and GDI-displacement factors (GDF) (A). They are often localized to distinct organelles (B) and they function in all stages of vesicular trafficking including budding, tethering, docking and fusion by interaction with multisubunit tethering complexes, SNARE proteins and other factors (C). There are three modes of vesicle interaction with the multisubunit tethering complex: via 1) Rab-GTP and SNAREs, 2) coat proteins and SNAREs or 3) multiple Rab protein recognition along coiled-coil tethers. Figure A and B are adapted from Stenmark (2009), and C is adapted from Brocker, Engelbrecht- Vandre et al. (2010).

1.8 Rationale and hypothesis

Identification of VPS16B was only the first step towards identifying all the binding partners of the VPS33B complexes. The combination of VPS16B (57 kDa) and VPS33B (71 kDa) is only approximately 128 kDa, which falls short of the 480 kDa and 720 kDa complexes that were observed on the Blue native gel. Potential interacting partners could include Rab proteins that are known to interact with the HOPS/CORVET complex in yeast and SNARE proteins that are known to interact with the Sec/Munc protein family to facilitate membrane fusion events. Other candidates include the human equivalents of the yeast HOPS/CORVET complex components (VPS11, VPS18, VPS39, VPS41, VPS3, and VPS8).

My project was focused on identifying and characterizing the VPS33B-VPS16B complexes. Since VPS33B and VPS16B have been established to be essential in α-granule biogenesis in megakaryocytes, I hypothesize that other proteins that interact with the VPS33B-VPS16B complex could also be specifically required for this process. Determining and characterizing the precise role of each of these proteins would help clarify the mechanism of platelet granule development, and contribute to our understanding of platelet granule functions.

Chapter 2 Materials and Methods

2.1 Antibodies and plasmids

Antibodies used for immunoprecipitation and immunoblotting experiments include: α-HA (Rabbit polyclonal, Bethyl); α-GFP (Rabbit polyclonal; mouse monoclonal 3E6, Invitrogen Life Technologies); α-myc (mouse monoclonal 9E10, Covance); α-VPS33B (rabbit polyclonal, produced in-house); α-VPS16B (mouse polyclonal, produced in-house; rabbit polyclonal, Abcam); α-FLAG (mouse monoclonal M2, Sigma); Anti-VPS52 (rabbit polyclonal, Aviva Systems Biology); Anti-His (mouse monoclonal 6-His, Covance).

Plasmids used for yeast hybrid experiments were pGBKT7 and pBridge (Clontech Laboratories). Plasmids used for co-immunoprecipitation experiments were pCMV-Myc and pCMV-HA (BD biosciences), pEGFP-C2 and pEGFP-C3 (BD biosciences), and p3xFLAG- CMV-14 (Sigma). pFastBacHT-A and pFastBacHT-Δ-His(Δ4050-4119) (Invitrogen Life Technologies).

2.2 Cloning

All cloning was performed using the same protocol, with varying buffers and restriction enzymes depending on the reactions. All required inserts were acquired via polymerase chain reaction (PCR) using: primers designed, and ordered from IDT Integrated DNA Technology or Sigma Aldrich, cDNAs (ordered from Thermos) or constructs previously established in the lab as templates, and Pfx DNA polymerase from Invitrogen Life Technologies. PCR products were purified with either PCR Purification Kits or Gel Extraction Kits from Qiagen after resolving them on 10% agarose gels. Purified PCR products were digested with the appropriate restriction enzymes (New England Biolabs Inc.). The vectors were digested with restriction enzymes and were treated with Calf Intestine Phosphatase (CIP) (New England Biolabs Inc.). Ligation of the digested PCR product and vector was done using T4 DNA Ligase (New England Biolabs Inc.) in its supplied buffer for 1 hour at room temperature. Ligation products were transformed into competent DH5α E.coli, and plated on Lysogeny

Broth (LB) plates containing the required antibiotic for selection. Colonies were picked and grown in liquid LB culture containing antibiotics overnight. DNA was then extracted using a QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer’s instructions.

For experiments done using DH10Bac cells, cells were plated on Luria Agar plates supplemented with 50 μg/ml Kanamycin, 7 μg/ml Gentamycin, 10 μg/ml Tetracyclin, 40 μg/ml IPTG and 200 μg/ml Bluo-Gal. BacMid DNA from DH10Bac cells were then extracted using ethanol precipitation.

FLAG-VPS16B inserts were cloned into pFastBacHT- Δ-His (Δ4050-4119) by PCR cloning from the P3xFLAG-CMV-14 vector. The Rab11A insert was cloned into pGEX-6p-1(GE healthcare life sciences) from the pEGFP-C2 vector by subcloning. VPS16B was cloned into the TAP-plasmid (done by Denisa Urban).

2.3 Cell cultures and transfections/infections

HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Wisent Bioproducts) supplemented with 10% v/v fetal bovine serum (Hyclone). All transient transfections with HEK293 cells were performed using the Lipofectamine 2000 reagent (Invitrogen Life Technologies) or the jetPRIME reagent (Polyplus transfection) according to the manufacturer’s instructions. For the immunoprecipitation experiments, HEK293 cells were grown to 90% confluency on 6 well plates, and transfected using 2 μg total DNA in the presence of 12 μL jetPRIME reagent in 200 μL of jetPRIME buffer. Transfection mixtures were added to the cells growing in 2 mL DMEM with 10% FBS. Cells were harvested and lysed 24h after transfection.

Adherent SF21 insect cells were grown in Grace’s media (Gibco) supplemented with 10% v/v fetal bovine serum (Hyclone). Suspended SF21 cells were grown in Sf-900 II serum free medium and Grace’s Media (50:50 v/v) with 10% (v/v) fetal bovine serum (Hyclone). Suspended cells were incubated at room temperature with shaking at no more than 105 rpm. Transfection of SF21 insect cells were performed using Cellfectin. SF21 cells were grown to 90% confluency on 6 well plates, and transfected using 3 μg total DNA in the presence of 8 μL Cellfectin reagent in 100 μL of Grace’s media without FBS. Transfection mixtures were

incubated for 30 minute before addition to cells growing in 1 ml Grace’s media with no FBS. Media was replaced 24 hours later with Grace’s media supplemented with 10% FBS (v/v).

Stable HEK293 cells expressing VPS33B-FLAG or VPS16B-FLAG fusion protein were made as follows. HEK293 cells were first transfected with p3xFLAG-VPS33B or p3xFLAG- VPS16B as described previously. Stably transfected cells expressing VPS33B or VPS16B- 3xFLAG fusion proteins were then generated by selection for neomycin resistance using 2.5 mg/mL G418. G418 resistant colonies were isolated and selected based on VPS33B or VPS16B-3xFLAG expression levels.

2.4 Baculovirus generation

For generation of the baculovirus used for protein expression in SF21 cells, transfection was first done using pFastBac-HT-A containing gene of interest (VPS33B or VPS16B) on adherent SF21 cells in 6 well plates grown to 90% confluency. Transfection was done as described above, and cells were left to grow in Grace’s media supplemented with 10% FBS (v/v) for 7 days at or until most of the cells have detached from the surface at room temperature. Cells were pelleted and the supernatant were collected (Passage 1). The entire Passage 1 viruses were added to SF21 cells grown in a T25 flask to 90% confluency. The cells were left to grow again for 7 days or until most of the cells had detached at room temperature. The supernatant was collected and marked as Passage 2 (10 mL), and about 2-3 mL of the Passage 2 viruses were added to a 50 mL culture of suspension SF21 cells. Cells were left to grow on shaker at 100 rpm for 7 days at room temperature or until all cells appeared to be infected. Supernatant from these cells were collected and marked as Passage 3, which was used for all subsequent protein expression infections.

2.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) and Western blotting

All SDS-PAGE and Western blotting was performed using the same protocol, varying the antibodies used depending on the experiment. Samples were treated with SDS-loading buffer (20% v/v glycerol, 1% v/v β-mercaptoethanol, 2% w/v SDS, 65 mM Tris-HCl, pH 6.8, 0.001% Bromophenol Blue) and boiled for 10 min. They were then loaded onto polyacrylamide gels (4% stacking, 10% resolving), and run at 200V for ~45 minutes, and

subsequently transferred onto nitrocellulose membranes at 100V for 1 hour or 30V overnight. Membranes were blocked in Tris-buffered saline-Tween (TBS-T) (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% v/v Tween 20) containing 5% skim milk powder for 1 hour, then incubated with primary antibody in blocking solution for 1 hour. This was followed by incubation with horse radish peroxidise (HRP)-conjugated secondary antibody in blocking solution for 1 hour. The membranes were washed 3 times for 10 minutes in TBS-T, and treated with enhanced chemiluminescence (ECL) reagent for 1 minute and exposed to film.

2.6 Yeast hybrid screens

The yeast hybrid library screen against a pre-made human bone marrow library (Clontech, Mountain View, CA Cat: 630477) or a normalized mixed tissue library (Clontech, Mountain View, CA Cat: 630480) with VPS16B as bait was performed as per the manufacturer’s specifications. Briefly, AH109 cells were first transformed with the pGBKT7-VPS16B or pBridge-VPS16B-VPS33B using the following protocol. Initially, the AH109 strain were grown from glycerol stock onto plates containing the rich media YPDA (20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose, 0.1 g/L NaOH, 20 g/L agar, 0.003% adenine hemisulfate in distilled water). Single colonies were picked and grown in 5 mL YPDA overnight at 30°C. The overnight cultures were then used to inoculate 300 mL YPDA to reach an OD600 of 0.1. The cultures were shaken vigorously at 30°C until OD600 has reached 0.5-0.7, and the yeast cells were harvested by centrifugation at 1,500x g for 5 minutes and the supernatant was removed. The cells were resuspended in 20 mL distilled water and centrifuged again at 1,500xg for 5 minutes, and the supernatant was again removed. The cells were then resuspended in 1.5 mL 1x TE/LiOAc buffer (0.01 M Tris, pH 7.5, 0.001 M EDTA, 0.1 M lithium acetate, in distilled water) and 100 μL aliquots were used for each transformation. Salmon sperm DNA was denatured by boiling for 5 minutes, and 50 μg was added, along with 100 ng of the plasmid DNA to the cells. 300 μL of 1x TE/LiOAc/PEG buffer (0.01 M Tris, pH 7.5, 0.001 M EDTA, 0.1 M lithium acetate, in 50% PEG-3350) were added to each reaction and mixed by inversion, and the mixtures were incubated at 30°C for 30 minutes. Next, 70 μL DMSO was added to each reaction, and the cells were heat shocked at 42 to 45°C for 15 minutes. 200 μL was removed from the mixture and plated onto selective growth media SD/-Trp (6.7 g/L yeast nitrogen base with ammonium sulphate, without amino

acids, 0.6 g/L –Trp dropout mix, 20 g/L glucose, 20 g/L agar in distilled water). The plates were placed at 30°C incubator for 2-3 days until colonies were observed.

Mating to yeast pre-transformed with cDNA libraries from human bone marrow or a mixed

normalized tissue was done as follows. A concentrated overnight culture (OD600 > 0.8) of the bait strain (AH109 cells transformed with pGBKT7 or pBridge) was prepared by inoculating 50mL of SD/-Trp for incubation at 30°C overnight shaking at 250-270rpm. Cells were centrifuged at 1,000xg for 5 minutes and resuspended in 5 mL of residual liquid by vortexing. One frozen aliquot (~1.0 mL) of the pre-transformed library culture (Human bone marrow library for the yeast two hybrid screen, and universal normalized human cDNA library from mixed tissue for the yeast three hybrid screen) was thawed at room temperature. The pre-transformed library cultures and the overnight AH109 cultures were added to a 2 L sterile flask, and topped with 50 mL of 2x YPDA. The mating cultures were then incubated at 30 °C overnight with gentle swirling (30-50 rpm). After incubation, the mating mixtures were pelleted by centrifugation at 1,000xg for 10 minutes, and resuspended in 10 ml of 0.5X YPDA/Kanamycin. The mixtures were plated onto medium stringency plates (SD/-His/-Leu/- Trp, with X-α-gal) at 200 μL per plate, and incubated at 30°C until colonies appeared (1 to 2 weeks).

Positive colonies were restreaked on SD/-Leu/-Trp/X-α-gal plates 2 to 3 times to ensure the correct phenotype. Plasmids from these colonies were isolated by first preparing 5 ml overnight cultures that were resuspended in 150 μL of plasmid isolation buffer (2% Triton X- 100, 1% SDS, 100 mM NaCl, 10 mM Tris, 1 mM EDTA). To the resuspended pellets, 0.3g glass beads and 400 μL of phenol:chloroform:isoamyl solution were added. The mixtures were vortexed for 2 minutes and centrifuged at maximum speed for 5 minutes. The aqueous phases were extracted and an additional 150 μL of the plasmid isolation buffer was added to the beads, and they were centrifuged again for 5 minutes. The combined aqueous phases were applied onto mini-prep columns where DNA purifications were done as per manufacturer’s recommendations (Qiagen). Plasmid DNAs were amplified through re- transformation and sequenced at the ACGT Corporation sequencing facility.

2.7 Immunoprecipitation

Hela or HEK293 cells were grown in 10cm dishes or 6 well plates until 90% confluency. If transfections were required, they were done 24 hours prior to the immunoprecipitation protocol. Cells were first washed three times in ice-cold PBS, and then scraped off into 1 ml lysis buffer (50 mM Tris HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA and 1% TRITON X-100, supplemented with 1x Protease inhibitor cocktail (Roche) and 1x phosphatase inhibitor cocktail (Roche)) and incubated on ice for 30 minutes. The lysates were then centrifuged at 16, 100 xg for 10 minutes at 4°C. The lysates were then added to washed mouse IgG beads (Sigma A0919) for preclearance at 4°C for an hour. The pre-cleared lysates were then added to washed anti-FLAG beads (Sigma A2220), and incubated at 4°C for 2 hours or overnight. The beads were then collected by centrifugation at 1000xg for 1 minute and washed 3 times with lysis buffer. Proteins bound to the beads were eluted either by addition of 35μL 2x sample buffer followed by 10 minutes boiling, or by addition of lysis buffer containing FLAG-peptide (Sigma).

For immunoprecipitation experiments done using antibodies other than anti-FLAG, protein G or protein A Sepharose beads were used. Lysates were prepared as described above, and antibody was added to the desired concentration and incubated at 4°C for 1 hour. Antibody bound complexes were then precipitated by addition of 40 μL of Sepharose beads followed by incubation at 4°C for 1 hour. Beads were then washed three times and eluted as described above with 2x sample buffer.

2.8 Blue native PAGE (BN-PAGE) and sample preparation

BN-PAGE was performed as per the manufacture’s specifications. Samples were prepared in or dialyzed against NativePAGE Sample Buffer (50 mM BisTris, 6 N HCl, 50mM NaCl, 10 % w/v Glycerol, 0.001% Ponceau S, pH 7.2). Gels used in the electrophoresis were 4-16% Bis-Tris Gel (NativePAGE Novex, Invitrogen Life Technology). Electrophoresis was performed at room temperature, at constant 150V for 105-120 minutes in NativePAGE running buffer (50 mM BisTris, 50 mM Tricine, 0.002% Coomassie G-250, pH 6.8). Transfer onto PVDF membrane was done at 100V in transfer buffer (25 mM Bicine, 25 mM

Bis-Tris, 1 mM EDTA, 50 μM chlorobutanol, pH 7.2) for 1 hour. Native ladder (NativeMark Unstained Protein Standard, Invitrogen) was used to estimate the protein complex size.

For preparing cytosolic and membrane fractions for BN-PAGE, HEK293 cells or Dami cells were grown in 3 T-75 flasks until 90% confluency. Each were washed 3x with 10 mL cold PBS, before collection in 2.5mL of cold PBS using a cell scraper. Cells were then resuspended using a cut pipette tip, and transferred into a 15mL conical tubes. Cells were centrifuged at 1000xg for 5 minutes at 4 °C, and resuspended in 1.5mL of homogenization buffer (25 mM HEPES pH 7.4, 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 1x protease inhibitor cocktail) with a cut pipette. The resuspended cells were then collected by centrifugation at 300xg for 10 minutes at 4 °C, and resuspended again in 300 μL of homogenization buffer. The resuspended cells were lysed mechanically by passing the 300 μL of resuspended cells through a 1mL syringe with 25 gauge needle. The needle was pressed against the side the conical vial near the bottom and cells were expelled aggressively. This step was repeated 10 times, and the extent of cell lysis was examined under the microscope with 5 μL of homogenization buffer and 1 drop of lysate. If lysis was incomplete (>80% free nuclei), the mechanical lysis was repeated for 2-3 more times. The lysates were then centrifuged at 3000xg for 15 minutes at 4°C. The pellets included unbroken cells, nuclei, and cytoskeleton. The post-nuclear supernatants were transferred to small plastic ultracentrifuge tubes (Beckman Cat. 347357, 11x34mm) and centrifuged at 42,000 rpm(~120,000xg) in a TLX Beckman centrifuge in a TLS55 rotor for 2 hours at 4 °C. The supernatant was the cytosolic fraction, and the pellet was the membrane fraction. Membrane fractions were resuspended in homogenization buffer containing 1% Triton-X-100. Both the cytosolic fractions and the membrane fractions were dialyzed against 4L of 1x BN-Sample buffer (50 mM BisTris, 6 N HCl, 50 mM NaCl, 10% w/v Glycerol, 0.001% Ponceau S, pH 7.2) overnight at 4 °C before they were loaded onto the BN gel.

2.9 Mass spectrometry

Mass spectrometry analysis was performed at the Hospital for Sick Children mass- spectrometry facility. Results were visualized using Scaffold software (version 3.6, Proteome Software Inc.).

2.10 His-tagged protein purification

His-tagged protein purifications from SF21 insect cells were performed by first resuspending the infected cells in ice cold lysis buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM

MgCl2,2% TRITON-X 100, 0.05% Tween-20, 25 mM imidazole and 1x protease inhibitor). Resuspended cells were incubated on ice for 10 minutes and centrifuged at 12,000 rpm (JA17 rotor) for 30 minutes. Lysates were then added to 300 μL of washed Ni-NTA resins and incubated at 4°C for 2 hours or overnight. The Ni-NTA resin were then washed 3x with lysis buffer and 2X with PBS. Bound proteins were eluted with 500 mM imidazole in PBS.

2.11 Electron microscopy

Carbon grids used for negative staining preparations were glow-discharged for 15 seconds (30mA, negative polarity, 10-1 mBar). Proteins were blotted directly onto the carbon grid and incubated for 1 minute. Excess samples were then washed from the grid by rinsing three times with water (50μL) for ten seconds each. Between rinsing, excess water was removed by touching the edge of the grid to a piece of Whatman filter paper. Grids were then stained with freshly prepared 2% uranyl acetate (2% w/v) by placing the grid directly onto it. Excess stain solution was removed using filter paper. EM microscopy was then performed using a FEI Co. (Eindhoven, The Netherlands) Tecnai F20 electron microscope equipped with a field emission gun, operating at 200 kV, with a charge coupled device camera (CCD) for image acquisition.

2.12 GST-protein bead preparation and pull-down assay

Starter cultures of BL21 Magic cells expressing the GST-tagged protein of interest were induced with 0.2mM IPTG for 2 hours at 25°C. Cells were then pelleted at 6000g for 10 minutes. The pellets were then resuspended in the resuspension buffer (25 mM HEPES

KOH, pH 7.8, 100 mM NaCl, 5 mM MgCl2, 0.05% Tween-20, 1 mM DTT and 1x protease inhibitors). The resuspended solution was then subjected to a French press twice at 1000 psi, and centrifuged at 10,000xg for 10 minutes. Lysates were then added to 300 μL of washed Glutathione Sepharose beads and incubated at 4°C for 2 hours. Supernatants were then removed by centrifugation at 1,000xg for 1 minute, and the remaining beads were then washed 3x with resuspension buffer. Washed beads were then resuspended in 0.3mL of

resuspension buffer to make a 50% slurry mixture, and the protein concentrations were measured via a Bradford assay (Pierce).

Lysates from mammalian cells were prepared as described in the immunoprecipitation experiments, and incubated with 40 μg of GST fusion protein and glutathione beads for 2-4 hours at 4°C. Beads were then washed 3x with lysis buffer. Proteins bound to the beads were eluted by addition of 30 μL of 2x sample buffer and boiled for 5 minutes.

2.13 Size exclusion chromatography of the VPS33B-VPS16B complex

Size exclusion chromatography was performed to separate the VPS33B-VPS16B complexes using Superose 6 columns. The column were first washed with water and then equilibrated with the buffer of choice, before 100 μL of purified protein from SF21 cells were loaded onto the column. Elutions were collected in 1 ml fractions after 5 ml of void volume had passed, and the elution process was monitored by UV absorbance at 280 nm.

2.14 Tandem affinity purification (TAP)

TAP-tagged purification of VPS16B was performed as per Anne-Claude Gingras –TAP- tagging protocols (http://www.proteomecenter.org/protocols/ ). HEK293 cells stably expressing TAP-tagged VPS16B were grown on 10cm dishes until 90% confluency. Cells were washed 3x in ice cold PBS and lysed for 30 minutes in 1 ml lysis buffer (10% glycerol, 50mM HEPES-KOH, pH 8.0, 100mM KCl, 2mM EDTA, 0.1% NP-40, 2mM DTT, 1x protease inhibitor, 1x phosphatase inhibitor). Cell debris was cleared by centrifugation for 10 minutes at maximum speed in a 4°C microcentrifuge. Cleared lysate was then added to 30 μL of washed IgG sepharose beads (Amersham) and incubated at 4°C for 2-4 hours. After incubation, IgG beads were centrifuged and washed 3x with lysis buffer and 3x with TEV buffer (10 mM HEPES-KOH, pH 8.0, 150 mM NaCl, 0.1% NP-40, 0.5 mM EDTA and 1mM DTT). Washed beads were then resuspended in 500 μL TEV buffer containing 4 μL TEV protease (Invitrogen Life Technologies), and incubated at 4°C overnight. Post-cleavage IgG beads were centrifuged and the supernatant containing the protein of interest was transferred to a fresh tube containing 1 volume of calmodulin binding buffer (10 mM β-mercaptoethanol, 10 mM Hepes-KOH pH8.0, 150 mM NaCl, 1 mM MgOAc, 1 mM imidazole, 0.1% NP-40, 2

mM CaCl2). 1M CaCl2 was added to the mixture at 1:250 (v/v) dilution, and the mixture was added to 30 μL of washed calmodulin Sepharose beads (Amersham), and incubated at 4°C for 90 minutes with gentle agitation. Post incubation calmodulin beads were washed 3x with 10-20 volumes of calmodulin binding buffer, and 2x with 10-20 volumes of calmodulin rinsing buffer (50mM Ammonium bicarbonate, pH 8.0, 75 mM NaCl, 1 mM MgOAc, 1 mM imidazole, 2 mM CaCl2). Proteins were eluted off the beads by resuspending the beads in calmodulin elution buffer (50 mM ammonium bicarbonate, pH 8.0, and 25 mM EGTA).

Chapter 3 Results

3.1 Yeast-two-hybrid (Y2H) screen using VPS16B as bait against a bone marrow library

VPS33B and VPS16B form distinct complexes from VPS33A and VPS16A in mammalian cells, and the Vps33p ortholog in yeast is part of complexes termed CORVET and HOPS complex. By analogy, additional proteins could be part of the mammalian VPS33B-VPS16B complex, such as HOPS complex homologues or other novel proteins. The proteins that interact with VPS33B likely also play an important role in α-granule biogenesis. To identify other binding proteins, I performed a Y2H screen using VPS16B as the bait against a human bone marrow cDNA library. Since VPS16B was detected in a Y2H screen where VPS33B was the bait against a bone marrow library, additional interacting partners could potentially be detected using the same library using VPS16B as the bait.

VPS16B was cloned into a pGBKT7 vector and transformed into AH109 cells that were mated to yeast pre-transformed with cDNA from a human bone marrow library. 23 positive interaction colonies were detected using the x-α-galactosidase assay, and identified using sequencing and BLAST. The results from this Y2H screen are shown in Table 1.

Table 1. Summary of results from Y2H using human bone marrow library with VPS16B as bait Name Hit VPS33B 2 Hemoglobin (alpha) 2 Hemoglobin (alpha1/2) 1 Hemoglobin (beta) 1 Hemoglobin (zeta) 1 Immunoglobulin (kappa) 2 ATP synthase F1 complex subunit alpha 1 Spermine/Spermidine N1-acetyltransferase (SAT) 1 Human defense alpha (DEFA1) 1 Erythrocyte membrane band 4.9 1 Ribosomal protein S2 1 Thyroid hormone receptor interacting protein 6 (TRIP6) 2 Others 7

As seen from Table 1, the majority of the hits in this Y2H screen were likely non-specific interactions that include hemoglobin isoforms, immunoglobulin, erythrocyte membrane band 4.9 and ribosomal proteins. The 7 proteins in the Others category included ones that tested positive for X-α-Gal assay, but were not able to be sequenced. VPS33B was found among the detected interactions, and acted as a positive control for this screen. 3.2 Immunoprecipitation of VPS16B-FLAG and VPS33B-FLAG from stable cell lines followed by mass-spectrometry

Since the Y2H screen was not successful, I attempted to purify the VPS33B-VPS16B complex and potential binding proteins using tagged VPS33B and VPS16B in mammalian cells. To do this, I established HEK293 cells stably expressing C-FLAG-tagged VPS16B fusion protein, or N-TAP-tagged VPS16B fusion protein at near endogenous levels, with a plan to purify the VPS16B associated complex by affinity purification. Interacting proteins that co-purified with VPS16B were then identified by mass spectrometry.

To exclude the possibility that the tag interfered with complex formation, I tested the effect of tag size and location on the ability of VPS16B and VPS33B to form the 480 kDa and the 720 kDa complexes. I purified the cytosolic fraction and membrane fraction and ran the samples on Blue native PAGE. The results showed that the position and the size of the tag did not affect the formation of the 480 kDa complex (Figure 12).

After confirming that the fusion proteins can form the same 480 kDa complex as observed in vivo, I proceeded to purify the VPS16B-FLAG and N-TAP-VPS16B fusion protein and its potential interacting partners. While purifying N-TAP tagged VPS16B fusion protein from stable HEK293 cells, I was unable to elute any protein due to low EGTA elution efficiency. The first purification step using IgG beads and TEV protease was successful, but both N- TAP-VPS16B and endogenous VPS33B were found to be stuck on the calmodulin beads (Figure 13). There were also numerous washes required in performing the TAP tag purification, which could have led to loss of transient interactions. For this reason, I focused on using HEK293 cells stably expressing C-terminal tagged VPS16B-FLAG for the rest of the affinity purification experiments.

Figure 12. VPS16B forms a large complex regardless of addition of a tag. Lysates from HEK293 cell lines stably expressing N-terminally TAP tagged VPS16B and C-terminally FLAG tagged VPS16B were resolved on BN-PAGE. Western blotting was performed using antibody against VPS16B. The 480 kDa complex was not disrupted by the presence of the tag.

Figure 13. VPS16B and VPS33B do not elute well from the calmodulin beads. 20 μL fractions were taken throughout the purification process and examined by immunoblotting. TAP-VPS16B and VPS33B bind to IgG beads as shown in IgG beads pre-TEV treatment (lane 3). TEV protease eluted VPS16B and VPS33B successfully (lane 4), although some

32 were left on IgG beads (lane 5 and lane 9). Elution from calmodulin beads was not successful (lane 7 and 8), VPS16B and VPS33B were still bound to calmodulin beads as seen in lane 10.

Affinity purifications were performed using anti-FLAG agarose beads using lysates from VPS16B-FLAG HEK293 stable cell lines and VPS33B-FLAG HEK293 stable cell lines. HEK293 cells served as negative controls. Bound proteins were eluted with 3xFLAG peptides and sent for mass spectrometry analysis. Most of the hits were ribosomal proteins, nuclear transcription factors and heat shock proteins (results not shown). It was unclear which of the hits were true interactions or non-specific interactions. To resolve this, I eluted the bound proteins from the anti-FLAG beads using 3xFLAG peptides suspended in 1x Blue native PAGE sample buffer and ran them on the Blue native gel. As seen from the western blot probing with anti-FLAG antibody in Figure 14, the complexes of 480 kDa and 720 kDa were maintained through the IP process. Two new complexes of 240 kDa and 140 kDa were observed in VPS33B-FLAG stable cells, but not in the VPS16B-FLAG stable cells. Another Blue native gel loaded with the same sample was stained with Coomassie, where individual bands were excised and sent for mass-spectrometry to analyze the components of each complex.

IP: Anti-FLAG

IB: Anti-FLAG

Figure 14. VPS33B and VPS16B form complexes in stable HEK293 cell lines. VPS33B- FLAG and VPS16B-FLAG stable HEK293 cells and control HEK293 cells were used to perform anti-FLAG IP’s. The elutions were resolved on BN-PAGE. VPS16B forms the same 480 kDa and 720 kDa complexes as VPS33B in Dami cells. VPS33B-FLAG fusion protein forms complexes of 146 kDa, 242 kDa and 480 kDa (estimated size).

Mass spectrometry results from individual complex bands are shown in Figure 15. The top two hits from the analysis were VPS33B and VPS16B.

Figure 15. High molecular weight complexes isolated from VPS16B-FLAG and VPS33B-FLAG stable HEK293 cells contained mainly VPS33B and VPS16B. The individual complexes were sent for MS analysis. The 480 kDa and 720 kDa complexes are composed of mainly VPS33B and VPS16B. The 242 kDa complex from the VPS33B-FLAG stable cell line contained only VPS33B. The total numbers of peptides detected are shown.

3.3 Identification of transient interacting proteins

Since the Y2H screen and mass spectrometry failed to identify any new targets that could function in the same pathway as the VPS33B-VPS16B complex, I hypothesized that interacting proteins could require the presence of both VPS33B and VPS16B. To test this possibility, I performed a yeast three hybrid assay, using VPS16B as the bait and VPS33B as the bridge protein.

3.3.1 Yeast-three hybrid (Y3H) assay with VPS16B as the bait and VPS33B as the bridge

I removed NotI and BglII cut sites in both VPS16B and VPS33B in order to clone them into the pBridge vector. I transformed AH109 cells with this vector and mated them with yeast pre-transformed with a normalized cDNA library from human mixed tissues. The human mixed tissue cDNA library was used in this assay because VPS33B and VPS16B are widely expressed in human tissues, and specific interactions could occur in other cell types, which could be important for the complex function of these two proteins.

The results show that many more interactions were observed compared to the Y2H assay, as tabulated in Table 2, organized by gene function. Table 2. Summary of results from Y3H using human universal normalized tissue library with VPS16B as bait and VPS33B as bridge. Genomic Contigs/Unknowns Hits Homo sapiens chromosome 1 genomic contig 2 Homo sapiens chromosome 4 genomic contig 1 Homo sapiens chromosome 10 genomic contig 1 Homo sapiens chromosome 16 genomic contig 1 Homo sapiens chromosome 17 genomic contig 1 Homo sapiens hypothetical LOC100506621 partial miscRNA 1 Homo sapiens chromosome 8 open reading frame 71 (C8orf71), non-coding RNA 1 DNA/RNA Binding/Nuclear Proteins Homo sapiens poly(A) polymerase alpha 1 Homo sapiens TBP-like 1 (TBPL1) (TATA BP) 1 Homo sapiens transcription factor Dp-1 (TFDP1) 1 Homo sapiens cell growth regulator with ring finger domain 1 (CGRRF1) 1 Adenosine deaminase, RNA-specific, B1 (ADARB1) 1 Homo sapiens heterogeneous nuclear ribonucleoprotein H1 1 Homo sapiens alpha thalassemia/mental retardation syndrome X-linked (ATRX) 1 Synaptonemal complex protein 1 (SYCP1) 1 Homo sapiens budding uninhibited by benzimidazoles 1 homolog (yeast) (BUB1) 1 Homo sapiens unc-50 homolog (C. elegans) (UNC50) 3 Enzymes/Transporters Homo sapiens fucosyltransferase 9 1 Retinol saturase (all-trans-retinol 13,14-reductase) 1 Solute carrier family 39 (zinc transporter), member 8 (SLC39A8) 1 Homo sapiens solute carrier family 25 (mitochondrial carrier; adenine nucleotide 1 translocator), member 31 (SLC25A31) Homo sapiens abhydrolase domain containing 15 (ABHD15) 1 Microsomal glutathione S-transferase 1 (MGST1) 1

Homo sapiens phosphofructokinase 1 TRIM41 1 Homo sapiens phosphatidic acid phosphatase type 2A (PPAP2A) 1 Homo sapiens mitogen-activated protein kinase kinase kinase 1 1 Homo sapiens aldehyde dehydrogenase 3 family, member A2 (ALDH3A2) 3 Homo sapiens ATPase, H+ transporting, lysosomal accessory protein 2 (ATP6AP2) 3 Homo sapiens spermatogenesis associated 9 (SPATA9) 2 Homo sapiens RAD50 interactor 1 (RINT1) 1 Homo sapiens decorin (DCN) 1 Homo sapiens kinesin family member 5B (KIF5B) 1 Homo sapiens vacuolar protein sorting 13 homolog A (S. cerevisiae) (VPS13A) 1 IQ motif containing GTPase activating protein 2 (IQGAP2) 1 Homo sapiens component of oligomeric golgi complex 5 (COG5) 1 Homo sapiens phospholipid scramblase 1 (PLSCR1) 1 Homo sapiens caveolin 2 1 Homo sapiens vacuolar protein sorting 52 homolog (VPS52) 1 Homo sapiens ASAP1 intronic transcript (non-protein coding) (ASAP1IT), 1 non-coding RNA Homo sapiens chimerin (chimaerin) 2 (CHN2) 1 Homo sapiens sortilin 1 1 Homo sapiens family with sequence similarity 134, member B (FAM134B) 1 Homo sapiens myosin VI (MYO6) 1 Homo sapiens component of oligomeric golgi complex 1 (COG1) 1 Homo sapiens RAB2B, member RAS oncogene family (RAB2B) 1 Homo sapiens Wilms tumor 1 associated protein (WTAP) 2 Homo sapiens nephronophthisis 1 (juvenile) (NPHP1) 1 Homo sapiens Der1-like domain family, member 2 (DERL2) 1

3.3.2 Confirmation of yeast three hybrid assay interactions by co- immunoprecipitation

From the interactions identified by the Y3H screening, a few were selected for confirmation by co-immunoprecipitation experiments. The selection was based on the number of hits from the screening and also their corresponding protein functions. Genes selected were VPS52, ATP6AP2, and COG5. The genes were inserted into HA-tagged vectors by PCR cloning. The HA-tagged fusion proteins were then transiently expressed in VPS33B-FLAG and VPS16B- FLAG stable HEK293 cell lines by transfections. The cell lysates were collected 24 hrs after transfection and used in an anti-FLAG immunoprecipitation, where bound proteins were analyzed by SDS-PAGE.

Figure 16. VPS52 as a potential binding partner of VPS33B or VPS16B. VPS52 interacts with VPS16B strongly; and weakly with VPS33B when it is over expressed in stable HEK293 cells expressing VPS33B-FLAG or VPS16B-FLAG (A). VPS52 also appears to interact with VPS16B in the 720 kDa complex (B). Endogenous VPS52 does not interact with VPS16B-FLAG in HEK293 stable cells using anti-VPS52 antibody as a probe (C).

Based on the immunoprecipitation experiments, HA-tagged COG5, ATP6AP2 and VPS52 all appear to interact with VPS33B and VPS16B when they are transiently expressed in HEK293 cells (Figure 16A and Figure 17). VPS52 appears to interact more strongly with VPS16B compared to VPS33B, and this interaction appears to be specific since nothing is detected in HEK293 cell control. To assess whether VPS52 could be part of the larger molecular weight complexes, I transiently expressed VPS52 in the VPS16B-FLAG stable cells, performed an anti-FLAG IP, and resolved the elution on a BN-PAGE. The immunoblot with an anti-HA antibody shows that HA-VPS52 is found in a complex of 720 kDa, but this band was also present in the HEK293 cell controls (Figure 16B). To further test this interaction, we obtained an antibody against endogenous VPS52. When tested on a blot with elution from an IP, a very faint band was detected when over-exposed; suggesting that the interaction between VPS52 and VPS16B is very weak, if there is any. More studies are needed to confirm the interactions of VPS33B-VPS16B with VPS52, COG5 and ATP6AP2 as well as other potential hits from the Y3H screen.

IP: Anti‐FLAG IB: Anti‐HA Figure 17. COG5 and ATP6AP2 as potential binding partners of VPS33B or VPS16B. HA-COG5 and HA-ATP6AP2 fusion proteins were transiently expressed in stable HEK293 cells expressing VPS33B-FLAG or VPS16B-FLAG. Anti-FLAG immunoprecipitation experiments were performed and elutions were probed with an anti-HA antibody. COG5 and ATP6AP2 were found to co-immunoprecipitate with both VPS33B and VPS16B.

3.4 Interaction with Rab proteins

Rab proteins are GTPases that function in regulating all steps of vesicle trafficking. HOPS/CORVET complexes interact with Rab5 and Rab7 orthologues in yeast, but the mammalian Rab interaction for VPS33B-VPS16B has yet to be identified. I obtained various GFP-Rab constructs (Rab5A, Rab5B, Rab5C, Rab6A, Rab7, Rab8A, Rab11A, Rab11B, and Rab27A) from Dr. John Brumell and over expressed them by co-transfection with VPS16B- FLAG constructs. From the initial screening assays, everything appeared to bind with VPS16B, and this could be due to over-expression of both proteins promoting non-specific interactions (Results not shown). I chose to focus on Rab5, Rab7 and Rab11 as they are markers for early endosome, late endosome and recycling endosome trafficking respectively, which are where VPS33B-VPS16B are hypothesized to function at. I obtained the GST-Rab fusion constructs from Dr. Brumell and purified GST-Rab5, -Rab7 and -Rab11 and performed GST-pulldown assays using the HEK293 stable cell lines expressing VPS33B- FLAG and VPS16B-FLAG.

3.4.1 GST-fusion protein pulldown assay

GST pulldown experiment results using both VPS33B-FLAG stable cell lysates and VPS16B stable cell lysates show that there is significant binding between Rab5 with VPS33B/

VPS16B, and weaker binding between Rab7 and VPS16B. The interaction with Rab11A was insignificant (Figure 18).

Figure 18. GST-pulldown assay using Rab5, 7 and 11 with stable cell line lysates expressing VPS33B-FLAG or VPS16B-FLAG. Lyastes from HEK293 cells stably expressing VPS33B-FLAG (A) or VPS16B-FLAG (B) were incubated with either GST- beads as negative control or with GST-Rab5, -Rab7, -Rab11 beads to test for binding. Elutions were analyzed by SDS-PAGE and Western blotting with anti-FLAG antibody to probe for pulldown of VPS33B or VPS16B. VPS33B and VPS16B interact with Rab5 and/or Rab7, but not with Rab11.

3.4.2 Rab Co-immunoprecipitation experiments in mammalian cells

To confirm Rab5, Rab7 interactions, I transfected GFP-Rab constructs into the stable cell lines expressing VPS33B-FLAG or VPS16B-FLAG, performed anti-FLAG IP’s, and probed for the GFP-Rabs. The interactions of Rab5 or Rab7 with VPS16B or VPS33B are very weak and inconsistent. However, there is no interaction detected between Rab11 and VPS33B or VPS16B (Figure 19).

Figure 19. Co-immunoprecipitations of GFP-Rab5A, - Rab7, and - Rab11A with VPS33B and VPS16B. GFP-Rab fusion proteins were transiently expressed in HEK293 cells stably expressing VPS33B and VPS16 FLAG fusion proteins. Immunoprecipitations were performed using an anti-FLAG antibody and elutions were probed with an anti-GFP antibody. Rab5 and Rab7 interacts with VPS33B and VPS16B. Rab11 does not bind to either VPS33B or VPS16B.

3.5 Hetero-oligomer interaction analysis

Although I have identified potential interactions of VPS52, COG5, ATP6AP2, Rab5, and Rab7 with VPS33B/VPS16B using the Y3H screen, GST-pulldown and co-IP experiments, preliminary data in our lab suggested that there could be more than one copy of VPS33B- VPS16B in the 480 kDa and 720 kDa complexes. Furthermore, the most frequent peptides detected from the BN-PAGE complexes using mass spectrometry were VPS33B and VPS16B (Figure 15).

3.5.1 Verifying multiple copies of VPS33B/16B by co-immunoprecipitation experiments

Previous studies done in our lab have shown that VPS16B can co-immunoprecipitate with VPS16B and VPS33B can co-immunoprecipitate with VPS33B. Based on my mass- spectrometry results (Figure 15), I wanted to test whether this interaction is present in the 480 kDa and the 720 kDa complexes. To do this, I used HA-VPS16B and HA-VPS33B constructs to transfect the VPS16B-FLAG stable and VPS33B-FLAG HEK293 stable cell lines respectively. Transfected cell lysates were used for affinity purification using anti- FLAG beads, and elutions were analyzed by running a BN-PAGE.

From Figure 20, one can see that HA-VPS16B was co-immunoprecipitated by VPS16B- FLAG in the 480 kDa complex in HEK293 cells stably expressing VPS16B-FLAG, but not in the HEK293 control cells. No complex was detected at the 720 kDa size marker. In co- immunoprecipitation performed using VPS33B-FLAG stable HEK293 cell lysates, HA- VPS33B was detected in 240 kDa and 146 kDa complexes that were not present in the HEK293 control cells. This confirms that the 146 kDa complex contains 2 copies of VPS33B (72 kDa) and the 240 kDa complex also contains >2 copies of VPS33B. The absence of VPS16B in the 240 kDa complex using mass spectrometry (Figure 15) suggests that there are 3 or 4 copies of VPS33B in this complex. Loss of high molecular weight complex (720 kDa for VPS16B-FLAG cell line, and 480/720 kDa for VPS33B-FLAG cell line) could be due to over-expression of exogenous fusion proteins (Figure 15), or interference of the tag on VPS33B in the assembly of the complex. Nevertheless, these results suggest that there are at least two copies of VPS33B and at least two copies VPS16B in one large molecular weight complex.

Figure 20. VPS16B interacts with VPS16B and VPS33B interacts with VPS33B. Stable VPS33B-FLAG HEK293 cells and stable VPS16B-FLAG HEK293 cells are transfected with HA-VPS33B and HA-VPS16B respectively. Anti-FLAG immunoprecipitation was performed and elutions were run on SDS-PAGE (A) and BN-PAGE (B). HA-VPS16B interacts with VPS16B-FLAG and is found to interact in the 480 kDa complex. HA-VPS33B also interacts with VPS33B-FLAG but only in the 242 kDa and 146 kDa complex.

3.5.2 VPS16B oligomerization is not phosphorylation dependent

To test whether phosphorylation or point mutations can affect VPS16B oligomerization, I utilized the Y11F mutant and C341R mutant forms of VPS16B. The Y11F mutant is a VPS16B mutant that has lost the ability to be phosphorylated at Tyrosine 11, and the C341R mutation was identified in a patient with ARC syndrome (Smith, Galmes et al., 2012). I obtained GFP-VPS16B fusion constructs for both of these mutants and transfected them into the FLAG-VPS16B stable cells. The elutions from anti-FLAG immunoprecipitation experiments were subjected to both SDS-PAGE and BN-PAGE analysis.

Figure 21. Phosphorylation mutation Y11F and ARC mutation C341R in VPS16B does not affect its interaction with itself or the assembly of the 480 kDa complex. HEK293 cells stably expressing VPS16B-FLAG were transfected with GFP-VPS16B wild type (WT), Y11F mutant or C341R mutant constructs. Anti-FLAG IP’s were performed and elutions were resolved on either SDS-PAGE (A) or BN-PAGE (B). The blots were probed with anti-

GFP antibody to test for GFP-VPS16B.VPS16B is co-immunoprecipitated in the 480 kDa complex with wild type as well as VPS16B (Y11F) and VPS16B (C341R).

It was found that GFP-VPS16B (Y11F) and GFP-VPS16B (C341R) can be co- immunoprecipitated with VPS16-FLAG, and that the mutations do not disrupt the formation of the 480 kDa complex (Figure 21). Furthermore, no 720 kDa complexes were detected.

3.5.3 VPS33B-VPS16B co-purification from SF21 insect cells

Purified proteins are required to confirm whether there are more than two copies of VPS16B and VPS33B in the 480 kDa and 720 kDa complexes. Previous attempts to express VPS16B and VPS33B in E. coli BL21 cells always led to aggregation in inclusion bodies, and solubilization usually required high concentrations of urea (8M) that resulted in denaturation of proteins. Since complex analysis using proteins purified from bacteria cells was not a viable option, I used the baculovirus expression system using SF21 insect cells. I generated baculovirus encoding VPS16B-FLAG and used this with previously generated His-VPS33B viruses to co-infect SF21 cells and performed anti-FLAG co-immunoprecipitations.

Purified proteins were analyzed using SDS-PAGE. His-VPS33B purified from infected insect cells alone produced large amounts of protein, but the expression was significantly reduced when it was co-expressed with VPS16B-FLAG. His-pulldown using co-infected cells usually generated high level of non-specific binding. As a result, anti-FLAG IP’s were performed to co-purify the VPS16B and VPS33B complex with low levels of background at the cost of reduced binding efficiency, and therefore decreased yield.

From the Coomassie blue stained gel (Figure 22A), it can be seen that His-VPS33B was purified in much larger amounts compared to anti-FLAG immunoprecipitation of His- VPS33B and VPS16B-FLAG complexes. This gel also shows that His-VPS33B and VPS16B-FLAG expressed in insect cells are capable of interacting with each other as His- VPS33B was co-immunoprecipitated by VPS16B-FLAG. The Coomassie stain also reveals that the co-immunoprecipitation is clean with very low levels of non-specific binding, and the sample could be pure enough for Blue native analysis to test whether these two proteins could form complexes by themselves.

Figure 22. VPS16B-FLAG co-purifies with His-VPS33B in SF21 insect cells. SF21 cells doubly infected with His-VPS33B viruses and VPS16B-FLAG viruses were subjected to anti-FLAG IP. Three elution fractions were run on SDS-PAGE and stained with Coomassie blue, showing that VPS33B co-purifies with VPS16B-FLAG (A). VPS16B co-purification with His-Septin 12 as a control was done in parallel, where VPS16B was detected in the elution of the His-VPS33B pull down but not in the His-Septin12 pulldown (B).

First, to test whether the interaction between VPS33B and VPS16B in SF21 insect cells is specific, I used another virus encoding genes for His-Septin 12 as a control. I infected SF21 cells with VPS16B-FLAG, and co-infected with either His-VPS33B or His-Septin 12. Infected cells were used in His-pulldown assays using Ni-NTA beads and bound proteins were analyzed using SDS-PAGE followed by Western blot analysis using an anti-FLAG antibody. Results are shown in Figure 22B, VPS16B-FLAG was pulled down by His- VPS33B but not His-Septin 12, confirming that the interaction is specific.

For examining the types of complexes formed by co-purified VPS33B and VPS16B, I ran the anti-FLAG IP elution on a BN-PAGE and stained the gel with Coomassie blue. Two bands were detected on the gel at the sizes that correspond to 480 kDa and 720 kDa (Figure 23A), suggesting that the purified protein can form complexes by themselves, and that they are the same complexes as the ones found in mammalian cells. This was verified by running the

45 purified proteins from SF21 cells in parallel with an anti-FLAG IP from HEK293 VPS16B- FLAG stable cells. The samples were run on BN-PAGE and probed with anti-FLAG antibody, and the results show two bands in both lanes at the size of 480 kDa and 720 kDa (Figure 23B), suggesting that the complex formed in insect cells is the same as the one formed in mammalian cells.

Figure 23. VPS16B-FLAG co-purifies with His-VPS33B in SF21 insect cells in a similar 480 kDa and the 720 kDa complex as in mammalian cells. SF21 cells doubly infected with His-VPS33B virus and VPS16B-FLAG virus were subjected to anti-FLAG IP. Elutions were resolved on Blue native-PAGE and either stained with Coomassie blue (A) or probed for FLAG on a Western blot (B). Complexes of 720 kDa and 480 kDa were observed on the Coomassie stained gel and Western blotting show a similar pattern in both the immunoprecipitation from the HEK293 stable cell line and the pulldown from SF21 cells.

To verify that the co-purified proteins contained only VPS16B and VPS33B, a mass spectrometry analysis was performed on the 480 kDa and 720 kDa band excised from the BN-PAGE gel. By performing analysis against both human library and SF21 cell library, I was able to examine whether there were human proteins or insect cell protein contaminants in the complexes. Results from the mass-spectrometry are shown in Figure 24, confirming that the predominant peptides were VPS33B and VPS16B. These results suggest that the 480 kDa complex and the 720 kDa complex consisted of mainly VPS33B and VPS16B, and that the proteins with low peptide counts, detected from HEK293 VPS16B-FLAG stable cell IPs (Figure 15) are likely non-specific interactions.

Figure 24. VPS33B-VPS16B complexes purified from SF21 insect cells contained only VPS33B and VPS16B. Purified proteins were sent for mass spectrometry and BLASTed against both a human (A) and a SF21 (B) library. The predominant peptides were VPS33B and VPS16B with few peptides from human or SF21 sources. Note: The small numbers of peptides detected are not significant.

3.5.4 Ultrastructure analysis using electron microscopy

The results using co-purified proteins from SF21 insect cells revealed that VPS33B and VPS16B are the only constituents of the 480 kDa and 720 kDa complexes. I wanted to further examine these complexes by means of electron microscopy to obtain some structural information. In collaboration with Dr. John Rubinstein, I looked at the complex structures using negative staining with uranyl acetate, followed by electron microscopy.

Figure 25. Electron micrographs of purified VPS33B-VPS16B complexes from SF21 cells. When examined under EM, VPS33B-VPS16B complexes appeared to have two populations of structures: dumbbells (A) and rings (B), highlighted in white boxes.

After analyzing the data collected (Figure 25), there were two conformations that stood out, one was in the shape of a ring, and the other in the shape of a dumbbell. These structures

48 could correspond to the 480 kDa and 720 kDa complexes. I then attempted to separate the complexes by purifying them on a gel filtration column (Appendix Figure A 2), but the complexes eluted in the low molecular weight fractions, where the size corresponded to single VPS33B-VPS16B complexes, and monomers.

Chapter 4 Discussion

4.1 The VPS33B-VPS16B complex does not include other HOPS components

VPS33B, a member of the Sec1/Munc18 protein family, is important in α-granule biogenesis in megakaryocytes. When VPS33B is deleted or contains specific missense mutations, it can cause ARC syndrome, an autosomal recessive disorder that impairs multiple organ systems and causes a mild to moderate bleeding phenotype. VPS33B interacts with VPS16B to form a 480 kDa and a 720 kDa complex based on Blue native PAGE analysis; and mutations in VPS16B also causes ARC syndrome. Little is known about other components of the VPS33B-VPS16B complex.

The Y2H screen did not yield any useful information (Table 1). Even with VPS33B as an established interacting partner of VPS16B, only 2 of the 23 colonies identified were VPS33B. This is very different from the Y2H assay performed using VPS33B as bait, where over 50% of the hits were VPS16B (Urban, Li et al., 2012). This could be due to the use of a library with reduced efficiency, as the total number of colonies identified was very low in comparison to other Y2H screens. Other hits from the screen did not seem to relate to the function of VPS33B-VPS16B, based on their localization: ATP synthase F1 subunit alpha is found on mitochondria (Vinogradov, 1999; Nath, 2002); and Thyroid receptor interacting protein 6 localizes to focal adhesions (Lin and Lin, 2011). Taken together, the Y2H screens using VPS16B and VPS33B as bait did not yield any interesting hits other than each other.

Even though VPS11 and VPS18 are known to be part of the core VPS-C complex in both the yeast HOPS/CORVET complex and the mammalian HOPS complex, they are not associated with VPS33B-VPS16B complexes as they were not detected in the mass-spectrometry and yeast hybrid assays. This is in contrast to the observation made by another group where they co-immunoprecipitated VPS18 with VPS33B in THP1 cells (Wong, Bach et al., 2011). This experiment did not work when we tried to repeat the same immunoprecipitation experiment in our lab with the same antibody used in their experiment using HEK293 cells (data not

shown). This raises the possibility that VPS33B has the capacity to form different complexes in different cell types. I have not yet tested VPS11’s interaction with VPS33B or VPS16B by co-IP experiments. Overall, my results using Dami cells and HEK293 cells suggest that VPS33B and VPS16B form a distinct complex that is different in composition compared to the VPS33A/VPS16A HOPS complex.

4.2 VPS52 as a potential interacting partner of VPS16B and VPS33B

Based on the Y3H screen and the co-immunoprecipitation experiments, VPS52 appears to be a binding partner of VPS16B and VPS33B. VPS52 is a member of the highly conserved multisubunit tethering complex called Golgi-associated retrograde protein (GARP) complex. Along with VPS51, VPS53 and VPS54, VPS52 regulates the retrograde transport from endosomes to the trans-Golgi network (Liewen, Meinhold-Heerlein et al., 2005; Bonifacino and Hierro, 2011) and also transport of proteins to lysosomes (Perez-Victoria, Mardones et al., 2008). Although GARP is mainly associated with the trans-Golgi network, there also exists an endosomal pool (Perez-Victoria, Abascal-Palacios et al., 2010). Since VPS33B- VPS16B partially co-localizes with markers of the trans-Golgi network and the late endosome (Lo, Li et al., 2005; Urban, Li et al., 2012), VPS52 presents itself as a potential interacting partner. It was co-immunoprecipitated with VPS33B and VPS16B when transiently expressed as a HA-VPS52 fusion protein in the stable HEK293 cell lines (Figure 16A). However, this interaction was not observed in HEK293 cells using an antibody against endogenous VPS52 (Figure 16C). Although VPS52 was found to be present in the 720 kDa complex in HEK293 cells stably expressing VPS16B, it was also detected in HEK293 control cells. Even though there is only trace levels of VPS52 in HEK293 control cells, they are at the exact same size as those found in the IP (Figure 16B). In addition, it was established with both mass spectrometry and insect cell purified protein studies, that the 720 kDa and the 480 kDa complex contained only VPS33B and VPS16B. Nevertheless, VPS52 and the GARP complex components could be transient interacting partners in controlling traffic between the trans-Golgi network and the endosomes.

4.3 COG1/COG5 as potential interacting partners of VPS16B and VPS33B

Both COG1 and COG5 were identified in the Y3H screen (Table 2), and COG5 was found to co-immunoprecipitate with both VPS16B and VPS33B when transiently expressed (Figure 17A). COG1 and COG5 are both subunits of a multisubunit tethering complex called Conserved Oligomeric Golgi (COG) complex. There are eight subunits (COG1-8) in the complex arranged into two lobes, lobe A (COG5-8) and lobe B (COG1-4) (Ungar, Oka et al., 2002; Ungar, Oka et al., 2005). The COG complex plays three distinct roles in the secretory pathway: 1) protein sorting on exit from the ER, 2) targeting ER derived vesicle to Golgi and 3) retention and retrieval of Golgi derived proteins (Oka and Krieger, 2005). Retrieval of Golgi derived proteins indicate that the COG complex also regulates trafficking from endosome to the trans-Golgi network. In addition, it was found that Golgin-84, a protein containing the conserved Golgin A5 domain found in VPS16B, also interacts with the COG complex through COG7 subunit (Sohda, Misumi et al., 2010). There is an interaction between COG5 and VPS33B and VPS16B based on the co-immunoprecipitation experiments, however, they could be caused by transient over-expression of the fusion protein. Further testing with an antibody against endogenous protein or a HEK293 cell line stably expressing HA-COG5 at near endogenous level should be used to confirm the interaction.

The identification of interactions between both components of the GARP complex and the COG complex with VPS33B-VPS16B suggests that there could be cross-talk between the multisubunit tethering complexes. This is not uncommon as there have been data showing interactions between different multisubunit tethering complexes (Friedmann, Salzberg et al., 2002; Oka and Krieger, 2005; Sohda, Misumi et al., 2007).

4.4 ATP6AP2 as a potential interacting partner of VPS16B and VPS33B

ATP6AP2 is a protein that was identified three times in the Y3H screen (Table 2), and it co- immunoprecipitated with VPS16B and VPS33B when transiently expressed (Figure 17B). ATP6AP2 (V-ATPase lysosomal accessory protein 2) was first identified as a receptor for

renin and pro-renin in activation of the Renin-Angiotensin Signaling (RAS) pathway (Nguyen, Delarue et al., 2002). It was later found that a pro-renin receptor fragment associates with the mammalian vacuolar H+-ATPase (V-ATPase) (Figure 26), and is essential in controlling the vesicular acid compartments and cell survival (Ludwig, Kerscher et al., 1998; Nishi and Forgac, 2002). In addition, RNA interference of ATP6AP2 in Xenopus causes defects in melanocytes and eye pigmentation in addition to other impairments (Ichihara and Kinouchi, 2011). ATP6AP2 is important for V-ATPase function in that it

stabilizes the Vo subunit of the V-ATPase. When expression of ATP6AP2 is abolished, there

is also a suppression of Vo subunit expression, resulting in de-acidification of intracellular vesicles (Kinouchi, Ichihara et al., 2010).

It has been previously shown that VPS33B is important in Mycobacterium Tuberculosis (Mtb) invasion (Bach, Papavinasasundaram et al., 2008; Wong, Bach et al., 2011). Infection with Mtb results in decreased acidification of phagosomes due to the absence of V-ATPase on phagosomal membranes. It was found that Mtb secretes phosphatase PtpA, which dephosphorylates and inactivates VPS33B, leading to inhibition of phagosome-lysosome fusion. The same group also showed that V-ATPase transiently interacts with VPS33B to regulate normal endosome-lysosome fusion, where they propose that VPS33B directly associates with the H, B and E subunit of the V-ATPase as part of the HOPS complex with VPS18 (Wong, Bach et al., 2011). Our VPS33B and VPS18 interaction experiments by co- immunoprecipitation were inconclusive. However, we have not looked at the interaction with the V-ATPase. It could be possible that VPS33B or VPS16B interacts with this accessory protein in addition to other subunits of the V-ATPases. Although the co-immunoprecipitation suggests a positive interaction between VPS33B-VPS16B and ATP6AP2, the binding between ATP6AP2 and VPS33B-VPS16B could be due to over-expression of ATP6AP2, similar to the COG5 subunit and VPS33B-VPS16B interactions. Co-immunoprecipitation experiments with an antibody against endogenous ATP6AP2 or with endogenous expression of tagged proteins need to be done to confirm the finding.

Figure 26. ATP6AP2 is part of the V-ATPase complex. ATP6AP2 is a transmembrane accessory protein to V-ATPase complex. Figure is adapted from Kinouchi, Ichihara et al. (2010).

4.5 Rab5 and Rab7 as potential binding partners of VPS33B and VPS16B, but not Rab11A.

Despite not having Rab proteins as a hit in any of the mass spectrometry experiments or the yeast hybrid screens, both the GST pulldown experiment and the co-immunoprecipitation assay suggest that Rab5 and/or 7 likely interact with VPS33B and VPS16B, whereas Rab11A does not. From the GST pulldown assays (Figure 18), both VPS33B and VPS16B interacted strongly with Rab5, and weakly with Rab7. There is no interaction with Rab11A with either VPS33B or VPS16B. In transient expression experiments using GFP- Rab5, - Rab7 and - Rab11A in HEK293 cells stably expressing VPS16B-FLAG or VPS33B-FLAG, co- immunoprecipitation of Rab5 and Rab7 with VPS33B or VPS16B was detected, but Rab11A failed to co-immunoprecipitate when over-expressed (Figure 19).

Rab proteins are a family of GTPases that are essential for vesicle budding, trafficking and fusion processes. For their function in fusion, Rabs in their GTP-bound form activate the tethering proteins. Tethering complexes are multi-subunit complexes such as the HOPS and the CORVET complex that act throughout the endomembrane system to aid in the recognition of membrane via Rab-GTPases that coat vesicles. The cooperation between Rab

proteins and the tethering complexes is required for vesicle capture and fusion to their target membrane.

In yeast, the HOPS complex and the CORVET complex interact with the Rab7 orthologue Ypt7 and the Rab5 orthologue Vps21 respectively, but the Rab interactions with VPS33B have yet to be confirmed. VPS16B contains a Golgin A5 domain, which is found in Golgi localized proteins that contain a coiled-coil motif. Many members of the Golgin family are found to interact with small GTPases in tethering membranes in intracellular trafficking steps, linking vesicles to cytoskeletons (Short, Haas et al., 2005). Based on immunofluorescence studies done in megakaryocytic Dami cell lines, VPS33B and VPS16B was found to not co-localize with markers of dense granules, but partially co-localizes with markers of trans-Golgi network, late endosome/lysosome, and α-granules (Lo, Li et al., 2005; Urban, Li et al., 2012). In studies done using other cell lines, hSPE-39 (VPS16B) was found to co-localize with Rab5, Rab7 and Rab11 compartments in HEK293 cells (Zhu, Salazar et al., 2009); VIPAR (VPS16B) was also shown to co-localize and co- immunoprecipitate with Rab11A in mIMCD-3 and HEK293 cells (Cullinane, Straatman- Iwanowska et al., 2010).

Although my results contradict the published co-immunoprecipitation result for VPS16B and Rab11A (Cullinane, Straatman-Iwanowska et al., 2010), it is in agreement with the localization studies, where VPS33B and VPS16B localize to Rab7-rich late endosome compartments but not the Rab11A enriched recycling endosome (Urban, Li et al., 2012). The inability to detect these Rabs by mass-spectrometry could be due to masking by abundant peptides (from both VPS33B-VPS16B and other non-specific interactions such as ribosomal proteins, transcription factors and nuclear proteins). It could also be that there is a loss of the weak or transient interaction between VPS33B-VPS16B and Rab proteins through the washing steps prior to mass-spectrometry.

4.6 VPS33B and VPS16B can associate to form multimeric complexes

Mass spectrometry experiments performed on isolated complexes from immunoprecipitation experiments showed that the 480 kDa complex and the 720 kDa complex contained mainly VPS33B and VPS16B (Figure 15). The majority of the peptides detected were only from these two proteins. Other proteins were also present but at much lower levels in comparison to these two (based on the total number of peptides detected), making them likely contaminants. Both Y2H results and mass spectrometry data suggest that VPS33B and VPS16B predominantly interact with each other, and this was confirmed by co- immunoprecipitation and SF21 expression experiments. Our lab has previously found that VPS33B can co-immunoprecipitate itself, and VPS16B can also co-immunoprecipitate itself (unpublished data). This has not yet been observed for VPS33A and VPS16A interactions. I have shown that not only do these protein co-immunoprecipitate with each other (Figure 20A), they are found in the same large molecular weight complex (Figure 20B), confirming that there can be more than one copy of VPS33B and VPS16B in the large complexes.

To test the possibility that there are more than two copies of VPS33B and VPS16B in the complexes was challenging as these proteins do not express well in bacteria due to the formation of inclusion bodies. Addition of 8M urea was required in order to solubilize the proteins, but this destroyed any possibility of complex formation due to complete denaturation of the proteins. Luckily, using insect cells, I was able to express these two proteins in reasonable quantities and in soluble form. When co-expressed in insect cells, the two proteins seemed to co-purify in an equal amount (Figure 22A and Appendix Figure A3). When resolved on Blue native gels, they formed the same 480 kDa and 720 kDa complexes as those found in mammalian cells (Figure 23B), further supporting the idea that VPS33B and VPS16B form a distinct complex compared to the VPS33A-VPS16A HOPS complex. Mass spectrometry results also revealed that there are no other proteins from either human sources or insect cells in the purified complexes.

Interestingly, this oligomerization was not disrupted with either ARC mutation or tyrosine- phosphorylation mutation in VPS16B. The phosphorylation site was chosen to be mutated based on both PhosphoSite predictions (www.phosphosite.org) and mass spectrometry data.

This suggests that the formation of the large complex is not tyrosine-phosphorylation dependent, however there are other phosphorylatable residues in the protein, such as Serine 121, that may influence the interaction. Since the C341R ARC mutation in VPS16B does not affect its interaction with VPS33B (unpublished data) or with itself, it likely affects VPS16B’s interaction with other proteins to cause the observed phenotype. This does not rule out the importance of the forming complexes or interactions during megakaryocyte α-granule biogenesis, it may be that mutations that affect the interactions that lead to ARC syndrome (https://grenada.lumc.nl/LOVD2/ARC) have not yet been identified.

The EM structural analysis performed on the purified proteins showed two distinct structural populations, a more common dumbbell form and a ring form. These two forms could correspond to the 480 kDa complex and the 720 kDa complex respectively, as the 720 kDa is found at lower concentration compared to the 480 kDa complex. These two structures could alternate by addition of a pair of VPS33B-VPS16B dimers (128 kDa x2) to serve distinct functions, as the 720 kDa complex was only found in the membrane fraction and not the cytosolic fraction. The separation of the two complexes by gel filtration led to smaller molecular weight complexes, suggesting that the complexes fell apart during the filtration process. This could be due to dilution effects as they move through the column. Further optimization is required to isolate these complexes.

Chapter 5 Conclusion and Future Directions

Yeast-hybrid assays and mass spectrometry experiments revealed that VPS33B and VPS16B do not interact with other components of the HOPS complex. Instead, VPS33B and VPS16B form large complexes (480 kDa and 720 kDa) by oligomerization. This conclusion was supported by the mass-spectrometry data from mammalian immunoprecipitation experiments, as well as protein analysis performed from SF21 insect cell purified VPS33B- VPS16B complexes. It was also found that tyrosine phosphorylation does not affect the formation of this complex. One of the mutations found in ARC patients (C341R) also had no effect on the ability of VPS16B to form large complexes. However, phosphorylation could be important in regulating the formation of complex, as there are other phosphorylated residues on VPS16B and VPS33B. Preliminary examination of the ultrastructures of the VPS33B- VPS16B complexes revealed two types of structures – dumbbells and rings.

In identifying interactions with the Y3H assay, I unveiled some potential interactions that were confirmed by co-immunoprecipitation experiments under transient expression. Many of the proteins identified are part of large multisubunit tethering complexes, such as COG1/5 and VPS52. The accessory protein of the V-ATPase, ATP6AP2, was also found to interact with both VPS33B and VPS16B. All these interactions were tested using transient transfection of exogenous tagged protein. Although no Rab proteins were detected in either the yeast hybrid screens or the mass-spectrometry analysis, both GST pulldown assays and co-immunoprecipitation experiments showed that VPS33B and VPS16B may interact with Rab5 and/or Rab7, but not Rab11A. Although the Rab5 and Rab7 interactions are weak, Rab11A consistently did not co-immunoprecipitate with either VPS33B or VPS16B.

A number of candidate proteins were identified to be interacting partners of VPS33B and VPS16B. Even though VPS52, COG5 and ATP6AP2 all interact with VPS33B and VPS16B, the experiments were performed by transient over-expression of tagged proteins in co- immunoprecipitation experiments. In the case of VPS52, when an antibody against endogenous protein was used, no significant binding was detected. Non-specific interactions

often occur when proteins are expressed at much higher levels, and further confirmation using antibodies against endogenous proteins, or stable cell lines should be used. Co- immunoprecipitation experiments may also be sensitive to the buffers used and the washing conditions, and some interactions may be missed based on non-binding in an immunoprecipitation. To test whether these proteins could interact with VPS33B-VPS16B, knock down each of the protein using RNA interference could be done to observe potential defects in biogenesis of α-granules. Ones exhibiting similar phenotypes as VPS33B/VPS16B knockdown cells would suggest a potential role in α-granule biogenesis and a meaningful interaction.

In addition to VPS52, COG1/COG5 and ATP6AP2, there are other hits that look promising as they are also involved in vesicular trafficking pathways. First, Sortilin1 (SORT1) is a member of the VPS10 family of receptors that is responsible for sorting lysosomal proteins and also exocytic trafficking (Zeng, Racicott et al., 2009; Herda, Raczkowski et al., 2012; Lane, St George-Hyslop et al., 2012). It is possible for SORT1 to interact with the VPS33B- VPS16B complex given its role in vesicular trafficking. There are also two motor proteins that were identified in the Y3H screen, Kinesin 5B (KIF5B) and Myosin VI (MYO6). KIF5B is one of the most abundant kinesins bound to microtubules in macrophages (Patel, Fisher et al., 2009), and it has been found that KIF5B is required for delivery of key membranes and receptors required for FcγR-mediated phagocytosis (Silver and Harrison, 2011). In addition, KIF5B is required for migration of lysosomes along microtubules (Cardoso, Groth-Pedersen et al., 2009). MYO6 on the other hand is an actin based motor, it is different from most of the Myosin family members in that it travels towards the minus end rather than plus end (Wells, Lin et al., 1999). MYO6 has been found to stabilize protein complexes in Drosophila (Geisbrecht and Montell, 2002) and to maintain organelle distribution in C. elegans (Kelleher, Mandell et al., 2000). In mammalian cells, it is localized to endocytic vesicles, cytosol and the Golgi complex (Nishikawa, Homma et al., 2002; Schott, Collins et al., 2002). Both of these motor proteins could potentially interact with VPS33B-VPS16B complex in regulating organelle movement during vesicular trafficking. To validate these interactions, co-immunoprecipitation experiments would be done by exogenously expressing tagged fusion proteins in the established VPS33B-FLAG and VPS16B-FLAG HEK293 stable cell lines. Further confirmation with antibodies against endogenous protein would be done if they

59 are available. Another candidate protein Chorein is also known as VPS13A, and it is mutated in patients with chorea-acanthocytosis (Rampoldi, Dobson-Stone et al., 2001; Ueno, Maruki et al., 2001). It is the orthologue of Vps13p in yeast that is involved in regulation of vesicular trafficking between the trans-Golgi network and the pre-vacuolar compartment (corresponding to multivesicular bodies and late endosomes in mammalian cells) (Redding, Brickner et al., 1996; Brickner and Fuller, 1997). One problem with studying this protein is that VPS13A is 360 kDa in size, and cloning may be difficult with such a large gene. However, one can use antibodies (generated against VPS13A peptides) for co- immunoprecipitation and co-localization experiments.

Since it is still unclear which Rab proteins interact with VPS33B and VPS16B, further experiments are required. Sucrose gradient experiments using HEK293 cell lysates, and Western blot analysis on the fractions using various Rab antibodies could be done to see which Rab proteins co-fractionate with VPS33B and VPS16B. In addition, pulldown assays using purified His-VPS33B-VPS16B complexes from SF21 insect cells could be performed with lysates from HEK293 cells transfected with different GFP-Rab constructs. To test the biological significance of the Rab interaction, knock down using RNA interference could be done to observe potential defects in biogenesis of α-granules.

Any identified interactions with VPS33B-VPS16B could be significant for the formation of α-granules in megakaryocytes and platelets, and determining the significance of these interaction will be important in further understanding megakaryocyte platelet development.

There are still many unanswered questions regarding VPS33B and VPS16B. First, the VPS33B-VPS16B complexes are unique, and distinct from the traditional HOPS or CORVET complexes. Further structural studies should be done to characterize the complexes. The EM ultrastructure analyses were performed on a heterogeneous mixture of purified proteins. To characterize the two complexes, I would need to separate the mixture of the 480 kDa and the 720 kDa complexes before performing EM analysis. This could be done by performing either sucrose gradients or size exclusion chromatography with the purified proteins from SF21 insect cells (Appendix Figure A 3). To prevent dissociation of complex due to dilution, cross-linking of the complexes could be done prior to size exclusion

60 chromatography. There are also different potential ways for the VPS33B and VPS16B proteins to arrange into larger molecular structures. Since VPS33B and VPS16B could both self-associate, it is possible for them to bind as either homodimers or heterodimers. In order to distinguish between the different possibilities, immuno-gold labeling of the EM structures could provide information on the spatial arrangement of individual proteins. The method that could provide the most structural information would be X-ray crystallography, and this could be done by utilizing the large quantities of purified VPS33B and VPS16B from SF21 insect cells. In addition, Blue native PAGE only presents an estimate of the size of the complexes; one could obtain more accurate molecular weights by performing analytical centrifugation experiments,

In addition to the structural analysis by EM and X-ray crystallography, biochemical assays could also be used to characterize the complexes. Mutagenesis experiments could be done to generate different truncation mutants of each VPS33B and VPS16B to delineate the binding site between VPS33B and itself, and between VPS16B and itself. Although the binding site between VPS33B and VPS16B has been identified (unpublished data), it is unclear whether the same binding site could support binding of more than one copy of VPS16B to VPS33B. Identifying the site of interaction could also shed light on the spatial arrangement of these molecules in the large complex.

In addition, the functional importance of forming such a large complex is still not characterized. The complex could act as a scaffold for recruitment of other proteins involved in vesicular trafficking. Even though one of the ARC mutations (C341R) on VPS16B does not affect the formation of the 480 kDa complex, there are others on both VPS16B and VPS33B that haven’t been tested (Gissen, Johnson et al., 2004; Gissen, Tee et al., 2006; Cullinane, Straatman-Iwanowska et al., 2010). Some of these mutations could disrupt the interaction between VPS33B and VPS16B, or amongst themselves, and in turn disrupt the formation of the large molecular weight complexes. If true, that would implicate the requirement for complex formation in the biogenesis of α-granules. To study this, one would first generate all the VPS33B and VPS16B mutants by mutagenesis PCR. Co- immunoprecipitation experiments using mutant VPS16B or mutant VPS33B with wild-type VPS33B or wild type VPS16B can be done to test for loss of interactions. Even if these

61 mutations do not disrupt the complex, they could still affect transient interactions with other proteins.

Any important residues identified in the binding assay screen listed above could be assessed for megakaryocyte and platelet α-granule production. Knockdown of endogenous VPS33B or VPS16B via siRNA in Dami cells would be rescued with the mutant versions of the protein by lentiviral transduction. A comparison between wild type and the mutant expression could provide insights on whether the formation of the complex is crucial for biogenesis of α- granules in megakaryocytes.

References

Akbar, M. A., S. Ray and H. Kramer 2009. The SM protein Car/Vps33A regulates SNARE- mediated trafficking to lysosomes and lysosome-related organelles. Mol Biol Cell 20(6): 1705-1714.

Akbar, M. A., C. Tracy, W. H. Kahr, et al. 2011. The full-of-bacteria gene is required for phagosome maturation during immune defense in Drosophila. J Cell Biol 192(3): 383-390.

Bach, H., K. G. Papavinasasundaram, D. Wong, et al. 2008. Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B. Cell Host Microbe 3(5): 316-322.

Behnke, O. 1992. Degrading and non-degrading pathways in fluid-phase (non-adsorptive) endocytosis in human blood platelets. J Submicrosc Cytol Pathol 24(2): 169-178.

Blair, P. and R. Flaumenhaft 2009. Platelet alpha-granules: basic biology and clinical correlates. Blood Rev 23(4): 177-189.

Bonifacino, J. S. and A. Hierro 2011. Transport according to GARP: receiving retrograde cargo at the trans-Golgi network. Trends Cell Biol 21(3): 159-167.

Brett, C. L., R. L. Plemel, B. T. Lobingier, et al. 2008. Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase. J Cell Biol 182(6): 1141-1151.

Brickner, J. H. and R. S. Fuller 1997. SOI1 encodes a novel, conserved protein that promotes TGN-endosomal cycling of Kex2p and other membrane proteins by modulating the function of two TGN localization signals. J Cell Biol 139(1): 23-36.

Brocker, C., S. Engelbrecht-Vandre and C. Ungermann 2010. Multisubunit tethering complexes and their role in membrane fusion. Curr Biol 20(21): R943-952.

Cardoso, C. M., L. Groth-Pedersen, M. Hoyer-Hansen, et al. 2009. Depletion of kinesin 5B affects lysosomal distribution and stability and induces peri-nuclear accumulation of autophagosomes in cancer cells. PLoS One 4(2): e4424.

Collins, K. M., N. L. Thorngren, R. A. Fratti, et al. 2005. Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion. EMBO J 24(10): 1775-1786.

Coppinger, J. A., G. Cagney, S. Toomey, et al. 2004. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood 103(6): 2096-2104.

Cox, K., V. Price and W. H. Kahr 2011. Inherited platelet disorders: a clinical approach to diagnosis and management. Expert Rev Hematol 4(4): 455-472.

Cramer, E. M., P. Harrison, G. F. Savidge, et al. 1990. Uncoordinated expression of alpha- granule proteins in human megakaryocytes. Prog Clin Biol Res 356: 131-142.

Cullinane, A. R., A. Straatman-Iwanowska, A. Zaucker, et al. 2010. Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization. Nat Genet 42(4): 303-312.

Dell'Angelica, E. C., V. Shotelersuk, R. C. Aguilar, et al. 1999. Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor. Mol Cell 3(1): 11-21.

Di Rocco, M., F. Callea, B. Pollice, et al. 1995. Arthrogryposis, renal dysfunction and cholestasis syndrome: report of five patients from three Italian families. Eur J Pediatr 154(10): 835-839.

Ducharme, N. A., J. A. Williams, A. Oztan, et al. 2007. Rab11-FIP2 regulates differentiable steps in transcytosis. Am J Physiol Cell Physiol 293(3): C1059-1072.

Flaumenhaft, R. 2012. alpha-granules: a story in the making. Blood 120(25): 4908-4909.

Friedmann, E., Y. Salzberg, A. Weinberger, et al. 2002. YOS9, the putative yeast homolog of a gene amplified in osteosarcomas, is involved in the endoplasmic reticulum (ER)- Golgi transport of GPI-anchored proteins. J Biol Chem 277(38): 35274-35281.

Geddis, A. E. and K. Kaushansky 2007. Immunology. The root of platelet production. Science 317(5845): 1689-1691.

Geisbrecht, E. R. and D. J. Montell 2002. Myosin VI is required for E-cadherin-mediated border cell migration. Nat Cell Biol 4(8): 616-620.

George, J. N. and S. Saucerman 1988. Platelet IgG, IgA, IgM, and albumin: correlation of platelet and plasma concentrations in normal subjects and in patients with ITP or dysproteinemia. Blood 72(1): 362-365.

Gissen, P., C. A. Johnson, D. Gentle, et al. 2005. Comparative evolutionary analysis of VPS33 homologues: genetic and functional insights. Hum Mol Genet 14(10): 1261- 1270.

Gissen, P., C. A. Johnson, N. V. Morgan, et al. 2004. Mutations in VPS33B, encoding a regulator of SNARE-dependent membrane fusion, cause arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome. Nat Genet 36(4): 400-404.

Gissen, P., L. Tee, C. A. Johnson, et al. 2006. Clinical and molecular genetic features of ARC syndrome. Hum Genet 120(3): 396-409.

Gunay-Aygun, M., M. Huizing and W. A. Gahl 2004. Molecular defects that affect platelet dense granules. Semin Thromb Hemost 30(5): 537-547.

Handagama, P. J., J. N. George, M. A. Shuman, et al. 1987. Incorporation of a circulating protein into megakaryocyte and platelet granules. Proc Natl Acad Sci U S A 84(3): 861-865.

Harrison, P. and E. M. Cramer 1993. Platelet alpha-granules. Blood Rev 7(1): 52-62.

Hegyi, E., L. K. Heilbrun and A. Nakeff 1990. Immunogold probing of platelet factor 4 in different ploidy classes of rat megakaryocytes sorted by flow cytometry. Exp Hematol 18(7): 789-793.

Heijnen, H. F., N. Debili, W. Vainchencker, et al. 1998. Multivesicular bodies are an intermediate stage in the formation of platelet alpha-granules. Blood 91(7): 2313- 2325.

Herda, S., F. Raczkowski, H. W. Mittrucker, et al. 2012. The sorting receptor Sortilin exhibits a dual function in exocytic trafficking of interferon-gamma and granzyme A in T cells. Immunity 37(5): 854-866.

Huizing, M., J. M. Parkes, A. Helip-Wooley, et al. 2007. Platelet alpha granules in BLOC-2 and BLOC-3 subtypes of Hermansky-Pudlak syndrome. Platelets 18(2): 150-157.

Ichihara, A. and K. Kinouchi 2011. Current knowledge of (pro)renin receptor as an accessory protein of vacuolar H+-ATPase. J Renin Angiotensin Aldosterone Syst 12(4): 638- 640.

Italiano, J. E., Jr., S. Patel-Hett and J. H. Hartwig 2007. Mechanics of proplatelet elaboration. J Thromb Haemost 5 Suppl 1: 18-23.

Italiano, J. E., Jr., J. L. Richardson, S. Patel-Hett, et al. 2008. Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released. Blood 111(3): 1227-1233.

Jonnalagadda, D., L. T. Izu and S. W. Whiteheart 2012. Platelet secretion is kinetically heterogeneous in an agonist-responsive manner. Blood 120(26): 5209-5216.

Junt, T., H. Schulze, Z. Chen, et al. 2007. Dynamic visualization of thrombopoiesis within bone marrow. Science 317(5845): 1767-1770.

Kaushansky, K. and J. G. Drachman 2002. The molecular and cellular biology of thrombopoietin: the primary regulator of platelet production. Oncogene 21(21): 3359- 3367.

Kelleher, J. F., M. A. Mandell, G. Moulder, et al. 2000. Myosin VI is required for asymmetric segregation of cellular components during C. elegans spermatogenesis. Curr Biol 10(23): 1489-1496.

Kim, B. Y., H. Kramer, A. Yamamoto, et al. 2001. Molecular characterization of mammalian homologues of class C Vps proteins that interact with syntaxin-7. J Biol Chem 276(31): 29393-29402.

Kim, S. M., H. K. Chang, J. W. Song, et al. 2010. Agranular platelets as a cardinal feature of ARC syndrome. J Pediatr Hematol Oncol 32(4): 253-258.

Kinouchi, K., A. Ichihara, M. Sano, et al. 2010. The (pro)renin receptor/ATP6AP2 is essential for vacuolar H+-ATPase assembly in murine cardiomyocytes. Circ Res 107(1): 30-34.

Kitano, M., M. Nakaya, T. Nakamura, et al. 2008. Imaging of Rab5 activity identifies essential regulators for phagosome maturation. Nature 453(7192): 241-245.

Lane, R. F., P. St George-Hyslop, B. L. Hempstead, et al. 2012. Vps10 family proteins and the retromer complex in aging-related neurodegeneration and diabetes. J Neurosci 32(41): 14080-14086.

Li, W., M. E. Rusiniak, S. Chintala, et al. 2004. Murine Hermansky-Pudlak syndrome genes: regulators of lysosome-related organelles. Bioessays 26(6): 616-628.

Liewen, H., I. Meinhold-Heerlein, V. Oliveira, et al. 2005. Characterization of the human GARP (Golgi associated retrograde protein) complex. Exp Cell Res 306(1): 24-34.

Lin, V. T. and F. T. Lin 2011. TRIP6: an adaptor protein that regulates cell motility, antiapoptotic signaling and transcriptional activity. Cell Signal 23(11): 1691-1697.

Lo, B., L. Li, P. Gissen, et al. 2005. Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet alpha-granule biogenesis. Blood 106(13): 4159-4166.

Ludwig, J., S. Kerscher, U. Brandt, et al. 1998. Identification and characterization of a novel 9.2-kDa membrane sector-associated protein of vacuolar proton-ATPase from chromaffin granules. J Biol Chem 273(18): 10939-10947.

Markgraf, D. F., K. Peplowska and C. Ungermann 2007. Rab cascades and tethering factors in the endomembrane system. FEBS Lett 581(11): 2125-2130.

Nath, S. 2002. The molecular mechanism of ATP synthesis by F1F0-ATP synthase: a scrutiny of the major possibilities. Adv Biochem Eng Biotechnol 74: 65-98.

Nguyen, G., F. Delarue, C. Burckle, et al. 2002. Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J Clin Invest 109(11): 1417- 1427.

Nickerson, D. P., C. L. Brett and A. J. Merz 2009. Vps-C complexes: gatekeepers of endolysosomal traffic. Curr Opin Cell Biol 21(4): 543-551.

Nishi, T. and M. Forgac 2002. The vacuolar (H+)-ATPases--nature's most versatile proton pumps. Nat Rev Mol Cell Biol 3(2): 94-103.

Nishikawa, S., K. Homma, Y. Komori, et al. 2002. Class VI myosin moves processively along actin filaments backward with large steps. Biochem Biophys Res Commun 290(1): 311-317.

Nurden, A. T. 2011. Platelets, inflammation and tissue regeneration. Thromb Haemost 105 Suppl 1: S13-33.

Nurden, A. T., P. Nurden, M. Sanchez, et al. 2008. Platelets and wound healing. Front Biosci 13: 3532-3548.

Oka, T. and M. Krieger 2005. Multi-component protein complexes and Golgi membrane trafficking. J Biochem 137(2): 109-114.

Ostrowicz, C. W., C. Brocker, F. Ahnert, et al. 2010. Defined subunit arrangement and rab interactions are required for functionality of the HOPS tethering complex. Traffic 11(10): 1334-1346.

Patel, P. C., K. H. Fisher, E. C. Yang, et al. 2009. Proteomic analysis of microtubule- associated proteins during macrophage activation. Mol Cell Proteomics 8(11): 2500- 2514.

Patel, S. R., J. H. Hartwig and J. E. Italiano, Jr. 2005. The biogenesis of platelets from megakaryocyte proplatelets. J Clin Invest 115(12): 3348-3354.

Peplowska, K., D. F. Markgraf, C. W. Ostrowicz, et al. 2007. The CORVET tethering complex interacts with the yeast Rab5 homolog Vps21 and is involved in endolysosomal biogenesis. Dev Cell 12(5): 739-750.

Perez-Victoria, F. J., G. Abascal-Palacios, I. Tascon, et al. 2010. Structural basis for the wobbler mouse neurodegenerative disorder caused by mutation in the Vps54 subunit of the GARP complex. Proc Natl Acad Sci U S A 107(29): 12860-12865.

Perez-Victoria, F. J., G. A. Mardones and J. S. Bonifacino 2008. Requirement of the human GARP complex for mannose 6-phosphate-receptor-dependent sorting of cathepsin D to lysosomes. Mol Biol Cell 19(6): 2350-2362.

Peterson, M. R. and S. D. Emr 2001. The class C Vps complex functions at multiple stages of the vacuolar transport pathway. Traffic 2(7): 476-486.

Pulipparacharuvil, S., M. A. Akbar, S. Ray, et al. 2005. Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules. J Cell Sci 118(Pt 16): 3663-3673.

Rampoldi, L., C. Dobson-Stone, J. P. Rubio, et al. 2001. A conserved sorting-associated protein is mutant in chorea-acanthocytosis. Nat Genet 28(2): 119-120.

Raymond, C. K., I. Howald-Stevenson, C. A. Vater, et al. 1992. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell 3(12): 1389-1402.

Redding, K., J. H. Brickner, L. G. Marschall, et al. 1996. Allele-specific suppression of a defective trans-Golgi network (TGN) localization signal in Kex2p identifies three genes involved in localization of TGN transmembrane proteins. Mol Cell Biol 16(11): 6208-6217.

Reed, G. L. 2004. Platelet secretory mechanisms. Semin Thromb Hemost 30(4): 441-450.

Richardson, J. L., R. A. Shivdasani, C. Boers, et al. 2005. Mechanisms of organelle transport and capture along proplatelets during platelet production. Blood 106(13): 4066-4075.

Richardson, S. C., S. C. Winistorfer, V. Poupon, et al. 2004. Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton. Mol Biol Cell 15(3): 1197-1210.

Rojas, R., T. van Vlijmen, G. A. Mardones, et al. 2008. Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7. J Cell Biol 183(3): 513-526.

Rondina, M. T., A. S. Weyrich and G. A. Zimmerman 2013. Platelets as cellular effectors of inflammation in vascular diseases. Circ Res 112(11): 1506-1519.

Sato, T. K., P. Rehling, M. R. Peterson, et al. 2000. Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion. Mol Cell 6(3): 661-671.

Savina, A., C. M. Fader, M. T. Damiani, et al. 2005. Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner. Traffic 6(2): 131-143.

Schott, D. H., R. N. Collins and A. Bretscher 2002. Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length. J Cell Biol 156(1): 35-39.

Seals, D. F., G. Eitzen, N. Margolis, et al. 2000. A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion. Proc Natl Acad Sci U S A 97(17): 9402-9407.

Sehgal, S. and B. Storrie 2007. Evidence that differential packaging of the major platelet granule proteins von Willebrand factor and fibrinogen can support their differential release. J Thromb Haemost 5(10): 2009-2016.

Semple, J. W., J. E. Italiano, Jr. and J. Freedman 2011. Platelets and the immune continuum. Nat Rev Immunol 11(4): 264-274.

Sevrioukov, E. A., J. P. He, N. Moghrabi, et al. 1999. A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila. Mol Cell 4(4): 479- 486.

Short, B., A. Haas and F. A. Barr 2005. Golgins and GTPases, giving identity and structure to the Golgi apparatus. Biochim Biophys Acta 1744(3): 383-395.

Silver, K. E. and R. E. Harrison 2011. Kinesin 5B is necessary for delivery of membrane and receptors during FcgammaR-mediated phagocytosis. J Immunol 186(2): 816-825.

Simonsen, A., R. C. Cumming, K. Lindmo, et al. 2007. Genetic modifiers of the Drosophila blue cheese gene link defects in lysosomal transport with decreased life span and altered ubiquitinated-protein profiles. Genetics 176(2): 1283-1297.

Smith, H., R. Galmes, E. Gogolina, et al. 2012. Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome. Hum Mutat 33(12): 1656-1664.

Sohda, M., Y. Misumi, A. Yamamoto, et al. 2010. Interaction of Golgin-84 with the COG complex mediates the intra-Golgi retrograde transport. Traffic 11(12): 1552-1566.

Sohda, M., Y. Misumi, S. Yoshimura, et al. 2007. The interaction of two tethering factors, p115 and COG complex, is required for Golgi integrity. Traffic 8(3): 270-284.

Sriram, V., K. S. Krishnan and S. Mayor 2003. deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster. J Cell Biol 161(3): 593-607.

Stenmark, H. 2009. Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10(8): 513-525.

Stenmark, H. and V. M. Olkkonen 2001. The Rab GTPase family. Genome Biol 2(5): REVIEWS3007.

Subramanian, S., C. A. Woolford and E. W. Jones 2004. The Sec1/Munc18 protein, Vps33p, functions at the endosome and the vacuole of Saccharomyces cerevisiae. Mol Biol Cell 15(6): 2593-2605.

Suzuki, T., N. Oiso, R. Gautam, et al. 2003. The mouse organellar biogenesis mutant buff results from a mutation in Vps33a, a homologue of yeast vps33 and Drosophila carnation. Proc Natl Acad Sci U S A 100(3): 1146-1150.

Thon, J. N. and J. E. Italiano 2012. Platelets: production, morphology and ultrastructure. Handb Exp Pharmacol(210): 3-22.

Toonen, R. F. and M. Verhage 2003. Vesicle trafficking: pleasure and pain from SM genes. Trends Cell Biol 13(4): 177-186.

Ueno, S., Y. Maruki, M. Nakamura, et al. 2001. The gene encoding a newly discovered protein, chorein, is mutated in chorea-acanthocytosis. Nat Genet 28(2): 121-122.

Ungar, D., T. Oka, E. E. Brittle, et al. 2002. Characterization of a mammalian Golgi- localized protein complex, COG, that is required for normal Golgi morphology and function. J Cell Biol 157(3): 405-415.

Ungar, D., T. Oka, E. Vasile, et al. 2005. Subunit architecture of the conserved oligomeric Golgi complex. J Biol Chem 280(38): 32729-32735.

Urban, D., L. Li, H. Christensen, et al. 2012. The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet alpha-granule biogenesis. Blood 120(25): 5032-5040.

van Nispen tot Pannerden, H., F. de Haas, W. Geerts, et al. 2010. The platelet interior revisited: electron tomography reveals tubular alpha-granule subtypes. Blood 116(7): 1147-1156.

Vinogradov, A. D. 1999. Mitochondrial ATP synthase: fifteen years later. Biochemistry (Mosc) 64(11): 1219-1229.

Wells, A. L., A. W. Lin, L. Q. Chen, et al. 1999. Myosin VI is an actin-based motor that moves backwards. Nature 401(6752): 505-508.

White, J. G. and C. C. Clawson 1980. The surface-connected canalicular system of blood platelets--a fenestrated membrane system. Am J Pathol 101(2): 353-364.

Wilcke, M., L. Johannes, T. Galli, et al. 2000. Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans- golgi network. J Cell Biol 151(6): 1207-1220.

Wittig, I., H. P. Braun and H. Schagger 2006. Blue native PAGE. Nat Protoc 1(1): 418-428.

Wong, D., H. Bach, J. Sun, et al. 2011. Mycobacterium tuberculosis protein tyrosine phosphatase (PtpA) excludes host vacuolar-H+-ATPase to inhibit phagosome acidification. Proc Natl Acad Sci U S A 108(48): 19371-19376.

Woodman, P. G. and C. E. Futter 2008. Multivesicular bodies: co-ordinated progression to maturity. Curr Opin Cell Biol 20(4): 408-414.

Wurmser, A. E., T. K. Sato and S. D. Emr 2000. New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion. J Cell Biol 151(3): 551-562.

Youssefian, T. and E. M. Cramer 2000. Megakaryocyte dense granule components are sorted in multivesicular bodies. Blood 95(12): 4004-4007.

Zeng, J., J. Racicott and C. R. Morales 2009. The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes. Exp Cell Res 315(18): 3112-3124.

Zhu, G. D., G. Salazar, S. A. Zlatic, et al. 2009. SPE-39 family proteins interact with the HOPS complex and function in lysosomal delivery. Mol Biol Cell 20(4): 1223-1240.

Zucker, M. B. and V. T. Nachmias 1985. Platelet activation. Arteriosclerosis 5(1): 2-18.

Appendices A.1 Complete mass-spectrometry results from individual complexes

Figure A 1. Complete mass spectrometry results using the isolated complex from BN- PAGE.

A.2 Gel Filtration Profile of SF21 purified VPS33B-VPS16B complex

Figure A 2. Gel filtration profile of VPS33B-VPS16B complex from SF21 cells. VPS33B- VPS16B elutes in low molecular weight complexes (~140 kDa and ~70kDa).

A.3 His-VPS16B and VPS33B purification from SF21 insect cells

Figure A 3. His-VPS16B and VPS33B co-purifies from infected SF21 insect cells. VPS33B co-purified by His-pulldown of His-VPS16B in approximately equal molar amounts.