THE ROLE OF THE N-TERMINAL EXTENSION DOMAIN OF VAMP4 IN THE REGULATION OF ITS RECYCLING TO THE TRANS-GOLGI NETWORK TRAN THI TON HOAI (B.SC.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 THE ROLE OF THE N-TERMINAL EXTENSION DOMAIN OF VAMP4 IN THE REGULATION OF ITS RECYCLING TO THE TRANS-GOLGI NETWORK TRAN THI TON HOAI INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgements I would like to express my heartfelt gratitude to my supervisor, Hong Wanjin, for his supervision, guidance and constant support through out my research project; and to the members of my supervisory committee: Cai Mingjie and Walter Hunziker, for their encouragement and invaluable discussions and advices on my work. I would also like to thank my past and present colleagues of the Membrane Biology Laboratory (Institute of Molecular and Cell Biology, Singapore) for making it a great environment for work. I am most grateful to Jill Tham, Tang Bor Luen, and Paramjeet Singh for their critical and careful reading of this thesis, as well as all the helpful comments; and to Jill Tham, Ong Yan Shan, and Eva Loh, for their constant encouragement throughout the writing process. I would also like to express my sincere gratitude to Tang Bor Luen, Wong Siew Heng, and Bui Dinh Thuan, for teaching me basic molecular, cell biological and biochemical techniques without reservations; to Zhou Zhi Hong for her help with the flow cytometry analysis in Figure 30; to Jill Tham, Lu Lei, Tang Bor Luen, Tai Guihua, Wang Tuanlao, Ong Yan Shan, Seet Lifong, Lim Koh Pang, Chan Siew Wee, and Li Hongyu for sharing critical reagents for this study; and to Jill Tham, Lu Lei, Tang Bor Luen, Paramjeet Singh and all other lab members for the advices, discussions and support given. My special thanks also go to: my collaborator, Zeng Qi (IMCB), for providing the expression constructs of VAMP4-EGFP, VAMP5-EGFP and V4nV5-EGFP and the stable NRK cells expressing these EGFP-fusion proteins; to Alexandre Benmerah (Inserm, Paris, France) for supplying me the expression constructs of EGFP-Eps15, i EGFP-EH29, EGFP-DIII and EGFP-D3Δ2; and to Frederick Maxfield (Cornell University Medical College, New York, USA) for providing the stable cell line CHOTacTGN38 and mouse-anti-Tac antibody. My appreciation also goes to the DNA sequencing unit of IMCB and the Flow Cytometry Facility (Biopolis Scientific and Facility Services) for their excellent services. And to all people in IMCB, who have contributed their support, either directly or indirectly, to my research life in IMCB, please accept my most sincere thanks. Special thanks also to my teachers in Hanoi National University (Vietnam), who had given me the chance to come to IMCB. Last but not least, I would like to express my deepest gratitude to my family, living far away in Vietnam, for their encouragement, patience, understanding and most important, their love. Tran Thi Ton Hoai 2009 ii Table of contents Thesis title Acknowledgement i Table of contents iii Summary viii List of Tables x List of Figures xi Abreviations xiii Chapter 1.1 1.2 1.3 1.4 Introduction Intracellular vesicular transport pathways 1.1.1 The endocytic pathway 1.1.2 The biosynthetic/secretory pathway 1.1.3 Retrograde transport to the TGN Vesicular transport 1.2.1 Vesicle formation 11 1.2.2 Vesicle movement 15 1.2.3 Vesicle tethering 17 1.2.4 Vesicle docking-fusion 22 SNAREs in vesicular transport 23 1.3.1 General structure of SNAREs 25 1.3.2 General mode of action of SNAREs 37 1.3.3 SNARE localization and the specificity of transport 41 44 VAMPs and the focus of this study iii 1.4.1 The mammalian R-SNARE subfamily 44 1.4.2 Previous studies on VAMP4 48 1.4.3 Rationale of the study 52 Materials and Methods 55 Chapter 2.1 2.2 Materials 55 2.1.1 Antibodies 55 2.1.2 Cell lines 56 Plasmid constructs 56 2.2.1 Vectors 57 2.2.2 Expression constructs 57 2.3 Expression of constructs in mammalian cells 63 2.4 Flow cytometry and cell sorting 64 2.5 Immunofluorescence (IF) microscopy 64 2.6 Immunoprecipitation (IP) 65 2.7 SDS-PAGE 66 2.8 Western Blot 66 2.9 Selective surface biotinylation and analysis 67 2.10 Internalization assay 68 2.10.1 Continuous internalization of antibodies and/or ligands 68 2.10.2 Discontinuous internalization of antibodies 68 Inhibition of endocytosis 68 2.11.1 Potassium depletion 68 2.11.2 Hypertonic treatment 69 2.11.3 Inhibition of endocytosis using Eps15 mutants 69 2.11.4 Clathrin knock-down 70 2.11 iv Recycling perturbation 70 2.12.1 Thermal perturbation 70 2.12.2 Pharmacological perturbation 71 2.12.3 Nocodazole treatment 71 2.13 Acidic stripping 71 2.14 siRNA 72 2.14.1 Sequences for siRNAs 72 2.14.2 siRNA transfection 72 Brefeldin A (BFA) treatment 73 2.12 2.15 Chapter 74 Role(s) of the N-terminal extension of VAMP4 in its targeting 3.1 Dissection of targeting signals at the N-terminal extension of VAMP4 74 3.2 Discussion 79 Chapter 84 VAMP4-EGFP recycles from the plasma membrane to the TGN 4.1 VAMP4-EGFP is faithfully targeted to the TGN 84 4.2 VAMP4-EGFP is incorporated into an authentic SNARE complex 86 4.3 The N-terminal region of VAMP4 participates in regulating its recycling 88 from the plasma membrane to the TGN 4.4 93 Low but detectable amounts of VAMP4-EGFP and V4nV5-EGFP are present on the cell surface 4.5 97 VAMP4-EGFP and V4nV5-EGFP are transported to the TGN via vesicular intermediates 4.6 Discussion Chapter 101 VAMP4-EGFP recycles from the plasma membrane to the 104 TGN primarily through clathrin-dependent endocytosis and via the SE/RE v compartments 5.1 VAMP4-EGFP is transported primarily via clathrin-dependent 104 endocytosis 5.1.1 The endocytosis of VAMP4-EGFP in cells under either 104 potassium depletion or hypertonic treatment 5.1.2 The endocytosis of VAMP4-EGFP in cells overexpressing 108 dominant negative mutant of Eps15, a regulator of clathrin-mediated endocytosis 5.1.3 5.2 113 The endocytosis of VAMP4-EGFP in cells depleted of clathrin VAMP4-EGFP is recycled to the TGN through the SE/RE 116 compartments 5.2.1 The colocalization of internalized anti-GFP antibody with SE 116 and RE markers 5.2.2 Accumulation of anti-GFP antibody at SE/RE compartments 124 when endosome-TGN recycling pathway is blocked 5.2.2.1 Anti-GFP antibody is blocked at the RE at 18oC 124 5.2.2.2 Disruption of microtubular network by nocodazole does 127 not affect the traffic of VAMP4-EGFP 5.2.2.3 BFLA1 and conA inhibit the recycling of VAMP4-EGFP 129 at the level of peri-Golgi RE 5.3 Discussion Chapter 132 Role(s) of the N-terminal extension of VAMP4 in the 137 recycling of the protein 6.1 The recycling of V4nV5-EGFP mutants 137 6.2 The recycling of VAMP4-EGFP mutants 140 vi 6.2.1 The Double-Leu motif and the second acidic cluster take part in 140 mediating the transport of VAMP4-EGFP from the PM back to the TGN 6.2.2 The Ser-30 takes part in mediating the transport of VAMP4- 144 EGFP from the TGN back to the PM 6.3 Discussion Chapter 148 153 General discussion and future perspective 167 References vii Summary SNAREs (soluble N-ethylamaleimide sensitive factor attachment protein receptor) are central players in the last stage of vesicle docking and subsequent fusion in diverse intracellular membrane transport events. 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Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes with multiple topologies. EMBO J. 26, 9–18. 209 THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 278, No. 25, Issue of June 20, pp. 23046 –23054, 2003 Printed in U.S.A. The Cytoplasmic Domain of Vamp4 and Vamp5 Is Responsible for Their Correct Subcellular Targeting THE N-TERMINAL EXTENSION OF VAMP4 CONTAINS A DOMINANT AUTONOMOUS TARGETING SIGNAL FOR THE TRANS-GOLGI NETWORK* Received for publication, March 28, 2003 Published, JBC Papers in Press, April 6, 2003, DOI 10.1074/jbc.M303214200 Qi Zeng, Thi Ton Hoai Tran, Hui-Xian Tan, and Wanjin Hong‡ From the Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore SNAREs represent a superfamily of proteins responsible for the last stage of docking and subsequent fusion in diverse intracellular membrane transport events. The Vamp subfamily of SNAREs contains members (Vamp1, Vamp2, Vamp3/cellubrevin, Vamp4, Vamp5, Vamp7/Ti-Vamp, and Vamp8/endobrevin) that are distributed in various post-Golgi structures. Vamp4 and Vamp5 are distributed predominantly in the trans-Golgi network (TGN) and the plasma membrane, respectively. When C-terminally tagged with enhanced green fluorescent protein, the majority of Vamp4 and Vamp5 is correctly targeted to the TGN and plasma membrane, respectively. Swapping the N-terminal cytoplasmic region and the C-terminal membrane anchor domain between Vamp4 and Vamp5 demonstrates that the N-terminal cytoplasmic region of these two SNAREs contains the correct subcellular targeting information. As compared with Vamp5, Vamp4 contains an N-terminal extension of 51 residues. Appending this 51-residue N-terminal extension onto the N terminus of Vamp5 results in targeting of the chimeric protein to the TGN, suggesting that this N-terminal extension of Vamp4 contains a dominant and autonomous targeting signal for the TGN. Analysis of deletion mutants of this N-terminal region suggests that this TGN-targeting signal is encompassed within a smaller region consisting of a di-Leu motif followed by two acidic clusters. The essential role of the di-Leu motif and the second acidic cluster was then established by site-directed mutagenesis. Intracellular traffic between different membrane compartments involves diverse membrane-enclosed intermediates in the form of small transport vesicles and/or larger containers/ carriers (1–5). These transport intermediates are generated at a given donor compartment by the concerted action of membrane proteins as well as cytosolic coat proteins and then delivered to the vicinity of a specific acceptor compartment. After faithful tethering of the intermediates with the target compartment, a process catalyzed by the action of Rab small GTPases and their effectors (6 –7), the final short range docking of the intermediates with the acceptor membrane is mediated by the * This work was supported by the Agency for Science, Technology, and Research (A*Star), Singapore. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore. Tel.: 65-6778-6827; Fax: 65-6779-1117; E-mail: mcbhwj@imcb.astar.edu.sg. interaction of vesicle-associated SNAREs1 with target SNAREs to form trans-SNARE complexes that may mediate the subsequent physical fusion between the intermediates and the target compartment (8 –10). Combined efforts using approaches of genetics, biochemistry, molecular biology, and bioinformatics have led to the identification of about 35 distinct members of the SNARE superfamily characterized by the presence of a common structural domain referred to as the SNARE motif (11). Based on whether the residue in the center of the SNARE motif is an Arg or a Gln and the relatedness of SNARE sequences, SNAREs have been roughly classified into four families: the R-SNAREs, QaSNAREs, Qb-SNAREs, and Q-syntaxins (10, 12). For SNAP-23, SNAP-25, and SNAP29/GS32, both Qa-SNARE and QbSNARE motifs coexist in the same protein (10). For a given transport event, it is generally believed that each family contributes one SNARE motif so that the four SNARE motifs will form a parallel, highly twisted, four helices bundle that catalyzes the fusion (8). The emerging theme is that the combinatorial use of these SNAREs will give rise to a wide spectrum of trans-SNARE complexes that mediate various transport events (13–14). A given SNARE may participate in several different transport processes by incorporating into different SNARE complexes. The availability of a specific set of SNAREs is thus important in membrane traffic. The function of SNAREs is regulated primarily through targeting/sorting of specific SNAREs to defined subcellular compartments and/or transport intermediates (8 –10). Additional regulations may be mediated by interacting with sequestering/inhibitory factors (15) and/or phosphorylation (16). The physiological relevance of SNARE targeting/trafficking is reflected by several studies (17–20) showing the importance of SNARE targeting motifs in various cellular functions of SNARE. The demonstration that a spectrum of transport machinery proteins participates in SNARE trafficking further sustains the importance of SNARE targeting/trafficking in cellular physiology (21–23). For example, the trafficking signals of Vamp2/synaptobrevin2 are well defined (24 –25), and only wild-type Vamp2, but not mutants defective in proper trafficking, could support regulated secretion when the endogenous protein was inactivated (17–18). Similarly, only wild-type but not endocytosis-defective Vamp2 could support axonal polarization of developing neurons (19). Snc1p is a yeast Vamp involved in exocytic transport from the late Golgi to the plasma membrane as well as in endocytosis (26 –27). The trafficking of Snc1p is crucial for its function and involves The abbreviations used are: SNAREs, soluble NSF attachment protein receptors; NSF, N-ethylmaleimide-sensitive factor; BFA, brefeldin A; TGN, trans-Golgi network; EGFP, enhanced green fluorescent protein; NRK, normal rat kidney; MTOC, microtubular organizing center. 23046 This paper is available on line at http://www.jbc.org 1028 Research Article VAMP4 cycles from the cell surface to the trans-Golgi network via sorting and recycling endosomes Ton Hoai Thi Tran, Qi Zeng and Wanjin Hong* Institute of Molecular and Cell Biology, Proteos Building, 61 Biopolis Drive, 138673 Singapore *Author for correspondence (e-mail: mcbhwj@imcb.a-star.edu.sg) Accepted January 2007 Journal of Cell Science 120, 1028-1041 Published by The Company of Biologists 2007 doi:10.1242/jcs.03387 Journal of Cell Science Summary VAMP4 is enriched in the trans-Golgi network (TGN) and functions in traffic from the early and recycling endosomes to the TGN, but its trafficking itinerary is unknown. Cells stably expressing TGN-enriched VAMP4 C-terminallytagged with EGFP (VAMP4-EGFP) are able to internalize and transport EGFP antibody efficiently to the TGN, suggesting that VAMP4-EGFP cycles between the cell surface and the TGN. The N-terminal extension of VAMP4 endows a chimeric VAMP5 with the ability to cycle from the surface to the TGN. Detailed time-course analysis of EGFP antibody transport to the TGN as well as pharmacological and thermal perturbation experiments suggest that VAMP4-EGFP is endocytosed by clathrindependent pathways and is delivered to the sorting and then recycling endosomes. This is followed by a direct transport to the TGN, without going through the late endosome. The di-Leu motif of the TGN-targeting signal is important for internalization, whereas the acidic cluster is crucial for efficient delivery of internalized antibody from the endosome to the TGN. These results suggest that the TGN-targeting signal of VAMP4 mediates the efficient recycling of VAMP4 from the cell surface to the TGN via the sorting and recycling endosomes, thus conferring steady-state enrichment of VAMP4 at the TGN. Introduction Protein transport in the secretory and endocytic pathways is mediated by membrane-enclosed intermediates in the form of small vesicles or larger containers. These shuttling vehicles are generated from a donor compartment and delivered to the target compartment. After a process referred to as tethering, these intermediates are docked and then fused with the target compartment in a process catalyzed by SNARE (soluble Nethyl maleimide sensitive factor adaptor protein receptor) (Söllner et al., 1993; McNew et al., 2000; Bonifacino and Glick, 2004; Hong, 2005). SNARE-mediated fusion of vesicles/containers with a target compartment is evolutionally conserved from yeast to human and is used in diverse transports in the secretory and endocytic pathways (Weimbs et al., 1997; Fasshauer et al., 1998; Hong, 2005). For a given transport, v-SNARE associated with the vesicle interacts with t-SNARE on the target compartment to form a trans-SNARE complex between two apposing membranes (Sutton et al., 1998; Weber et al., 1998; Antonin et al., 2002). t-SNARE generally consists of a heavy chain and two light chains, thus providing three SNARE motifs (Fukuda et al., 2000; Hong, 2005). During the formation of a typical trans-SNARE complex, the four SNARE motifs (one from v-SNARE and three from t-SNARE) assemble into a four-helical bundle to catalyze the fusion event (Söllner et al., 1993; Weber et al., 1998; Bock et al., 2001; Chen and Scheller, 2001; Antonin et al., 2002; Jahn et al., 2003). The function of SNAREs is best defined for exocytosis of synaptic vesicles. Fusion of synaptic vesicles with the presynaptic membrane of neurons is mediated by interaction of VAMP2/synaptobrevin as a v-SNARE with a t-SNARE assembled from syntaxin1 and SNAP-25 (Söllner et al., 1993; Sutton et al., 1998; Jahn et al., 2003; Sudhof, 2004). Syntaxin1 and VAMP2 have one SNARE motif each, whereas SNAP-25 has two tandem SNARE motifs. The structure resolved by xray crystallography revealed that the four-helical bundle assembled from the four SNARE motifs is characterized by 16 layers of mostly hydrophobic interactions between amino acid side chains (Sutton et al., 1998; Antonin et al., 2002). The central zero layer is defined by a cluster of hydrophilic interactions between three Gln (Q) residues (derived from tSNARE) and one Arg (R) residue (derived from v-SNARE). Based on the residue predicted to reside at the zero layer, SNAREs can be classified into either Q-SNAREs or RSNAREs (Fasshauer et al., 1998). After membrane fusion, the SNARE complex becomes a cis complex in the target compartment and is disassembled into free v-SNARE and tSNARE subunits via the action of NSF (N-ethylmaleimidesensitive factor) and ␣-SNAP (soluble N-ethylmaleimidesensitive factor attachment protein). The v-SNARE is generally recycled back to the donor compartment, whereas the tSNARE subunits are reused for the next rounds of fusion (Hohl et al., 1998; Wimmer et al., 2001; Marz et al., 2003). Most of the nine known R-SNAREs (Sec22b, Ykt6, VAMP1, VAMP2, VAMP3/cellubrevin, VAMP4, VAMP5, VAMP7/Ti-VAMP, VAMP8/endobrevin) function as v- Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/6/929/DC1 Key words: VAMP4, SNARE, Endosome, Golgi complex, TGN, Membrane targeting [...]... is transported (internalized) into cells In this process, small portions of the PM surround the material to be internalized, invaginate and then pinch off to form endocytic vesicles containing the ingested material There are two main types of endocytosis categorized based on the size of the endocytic vesicles: pinocytosis (‘cellular drinking’), in which fluid and soluble matter are transported into 3... the pathway at the 5 ER The ER has many ribosomes bound to its cytosolic side, which are involved in producing all the transmembrane proteins and soluble proteins destined for the biosynthetic/secretory pathway All of these proteins contain specific signal sequences that allow them to be translocated into the ER co-translationally by transmembrane transport Subsequent transport from the ER to the Golgi. .. specifying the traffic route of that protein at certain stages of transport The combined effects of these signals would eventually determine the localization of that protein (Rothman and Wieland, 1996) A sorting signal that restricts the entry of a protein into a vesicle is termed a ‘retention signal’ These signals are compartment specific and they have been found in the transmembrane domains of a number... abrin) These toxins are usually composed of two subunits: a subunit that binds to the cell surface, and an enzymatic subunit that causes the actual toxic effects by inhibiting essential cytosolic reactions The toxins bind to the cell surface and are internalized into the cells They then undergo retrograde transport to the TGN, where some may be activated by furin (Molloy et al., 1999) These toxins can be... responsible for the incorporation of a protein into a budding vesicle is termed a ‘transport signal’ (Rothman and Wieland, 1996) For membrane proteins, transport signals that are located in the cytoplasmic domain of cargo proteins bind directly to coat proteins (Pearse, 1988) For example, the tyrosine-based signal present in the cytoplasmic tail of the low-density lipoprotein (LDL) receptor interacts... trans- Golgi network (TGN), proteins face several possible destinations: the extracellular space, different domains of the PM, secretory vesicles and the endosomal-lysosomal system According to the conventional, TGN-based model for Golgi sorting, proteins destined for different destinations are sorted in the TGN into specific sets of membrane-enclosed carriers that ferry them to their specific destination... 2008) The synthesis of most proteins occurs in the cytosol Their subsequent fate depends on whether their amino acid sequence contains specific signals (sorting signals) that direct their delivery to certain intracellular locations There are three types of transport by which proteins move from one compartment to another Proteins move between the cytosol and the nucleus through nuclear pore complexes in. .. Syntaxin Sulfo-NHS-biotin: Sulfo -N- hydroxysuccinimidobiotin t-SNARE: target compartment-associated SNARE Tac : T cell antigen Tf-AF555: AlexaFluor 555-conjugated transferring Tf-AF647: AlexaFluor 647-conjugated transferrin Tf-FITC: FITC-conjugated transferrin TfR : Transferrin receptor TGN : trans- Golgi network TGN38/46: rat TGN protein of 38 kD or its human orthologue of 46 kD xviii Thr : Threonine... of a number of integral membrane proteins (Tang et al., 1992; Machamer, 1993; Nilsson et al., 1991; Nilsson and Warren, 1994) It has been proposed that these signals may either cause protein aggregation or participate in the interaction between the transmembrane domains and the lipid bilayers; thus leading to the retention of proteins in certain compartments (Pfeffer and Rothman, 1987; Baranski et al.,... efficient TGN targeting of V4nV5-EGFP 78 Figure 8 : VAMP4- EGFP is faithfully targeted to the TGN 85 Figure 9 : VAMP4- EGFP is incorporated into an authentic SNARE complex 87 Figure 10 : The N- terminal extension of VAMP4 mediates its recycling from the PM to the TGN 89 Figure 11 : VAMP4- EGFP recycles between the PM and the TGN in MDCK cells 92 Figure 12 : Study the transport of proteins that recycle between . THE ROLE OF THE N-TERMINAL EXTENSION DOMAIN OF VAMP4 IN THE REGULATION OF ITS RECYCLING TO THE TRANS-GOLGI NETWORK TRAN THI TON HOAI (B.SC.) A THESIS SUBMITTED FOR THE DEGREE OF. conA inhibit the recycling of VAMP4- EGFP at the level of peri-Golgi RE 5.3 Discussion Chapter 6 Role( s) of the N-terminal extension of VAMP4 in the recycling of the protein 6.1 The recycling. transfection 2.15 Brefeldin A (BFA) treatment Chapter 3 Role( s) of the N-terminal extension of VAMP4 in its targeting 3.1 Dissection of targeting signals at the N-terminal extension of VAMP4 3.2 Discussion