The role of the n terminal extension domain of vamp4 in the regulation of its recycling to the TRANS GOLGI network

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. The pairing between the vesicle-associated SNARE (v-SNARE) and its cognate target membrane-associated SNARE (t-SNARE) facilitates the fusion of these opposing membranes and confers specificity to vesicular transport. Since the function of SNAREs is regulated primarily by their localization, it is important to understand their targeting mechanisms. The mammalian VAMP subfamily (vesicle-associated membrane protein) contains nine SNAREs (VAMP1, 2, 3, 4, 5, and 8, Ykt6 and Sec22b), most of which function as v-SNAREs. VAMP4 is the only VAMP that is located mainly in the trans-Golgi network (TGN). It functions in membrane traffic from the sorting and recycling endosomes to the TGN, but its trafficking itinerary is unknown. The N-terminal domain preceding the SNARE motif of VAMP4 contains an autonomous targeting signal for the TGN, which resides in a region consisting of a double-Leu motif followed by two acidic clusters. To find which residue(s) within this region participates in regulating the targeting of VAMP4, Ala-mutagenesis screening was performed. Immunofluorescence study shows that the double-Leu motif and an acidic cluster play essential roles in mediating efficient TGN targeting. An antibody internalization assay using C-terminally EGFP-tagged VAMP4 also shows that VAMP4-EGFP cycles between the PM and the TGN and that its Nterminal domain participate in regulating this recycling. Results from detailed timecourse analysis of anti-GFP antibody transport to the TGN as well as pharmacological and thermal perturbation experiments indicate that VAMP4-EGFP is endocytosed by clathrin-dependent pathways. The protein is then transported to the TGN via the viii Pfeffer, S.R., and Rothman, J.E. (1987). Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu. Rev. Biochem. 56, 829-852. Pfeffer, S.R. (1999). Transport-vesicle targeting: tethers before SNAREs. Nat. Cell Biol. 1, E17-E22. Pfeffer, S.R, and Aivazian, D. (2004). Targeting Rab GTPases to distinct membrane compartments. Nature Rev. Mol. Cell Biol. 5, 886–896. Piguet, V., Wan, L., Borel, C., Mangasarian, A., Demaurex, N., Thomas, G., and Trono, D. (2000). HIV-1 Nef protein binds to the cellular protein PACS-1 to downregulate class I major histocompatibility complexes. Nat. Cell. Biol. 2, 163–167. Poussu, A., Lohi, O., and Lehto, V.P. (2000). Vear, a novel Golgi-associated protein with VHS and γ-adaptin “ear” domains. J. Biol. Chem. 275, 7176-7183. Powers, J., and Barlowe, C. (2002). Erv14p directs a transmembrane secretory protein into COPII-coated transport vesicles. Mol. Biol. Cell 13, 880-891. Prekeris, R., Klumperman, J., and Scheller, R.H. (2000). Syntaxin 11 is an atypical SNARE abundant in the immune system. Eur. J. Cell Biol. 79, 771–780. Pryer, N.K., Wuestehube, L.J., and Schekman, R. (1992). Vesicle-mediated protein sorting. Annu. Rev. Biochem. 61, 471-516. Pryor, P.R., Mullock, B.M., Bright, N.A., Lindsay, M.R., Gray, S.R., Richardson, S.C., Stewart, A., James, D.E., Piper, R.C., and Luzio, J.P. (2004). Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events. EMBO Rep. 5, 590–595 Puthenveedu, M.A., and Linstedt, A.D. (2001). Evidence that Golgi structure depends on a p115 activity that is independent of the vesicle tether components giantin and GM130. J. Cell Biol. 155, 227– 238. Puthenveedu, M.A., and Linstedt, A.D. (2004). Gene replacement reveals that p115/SNARE interactions are essential for Golgi biogenesis. Proc. Natl. Acad. Sci. USA 101, 1253–1256. 196 Rao, S.K., Huynh, C., Proux-Gillardeaux, V., Galli, T., and Andrews, N.W. (2004). Identification of SNAREs involved in STXaptotagmin VII-regulated lysosomal exocytosis. J. Biol. Chem. 279, 20471–20479. Reaves, B., and Banting, G. (1992). Perturbation of the morphology of the transGolgi network following brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38. J. Cell Biol. 116, 85–94. Reaves, B., and Banting, G. (1994). Vacuolar ATPase inactivation blocks recycling to the trans-Golgi network from the plasma membrane. FEBS Lett. 345, 61-66. Reddy, A., Caler, E.V., and Andrews, N.W. (2001). Plasma membrane repair is mediated by Ca2+- regulated exocytosis of lysosomes. Cell 106, 157–169 Rein, U., Andag, U., Duden, R., Schmitt, H.D., and Spang, A. (2002). ARF-GAPmediated interaction between the ER-Golgi v-SNAREs and the COPI coat. J. Cell Biol. 157, 395–404. Remacle, A., Murphy, G., and Roghi, C. (2003). Membrane type I-matrix metalloproteinase (MT1-MMP) is internalised by two different pathways and is recycled to the cell surface. J. Cell Sci. 116, 3905-3916. Riederer, M.A., Soldati, T., Shapiro, A.D., Lin, J., and Pfeffer, S.R. (1994). Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network. J. Cell Biol. 125, 573-582. Rodriguez-Boulan, E., and Musch, A. (2005). Protein sorting in the Golgi complex: shifting paradigms. Biochim. Biophys. Acta 1744, 455–464. Rohde, J., Dietrich, L., Langosch, D. and Ungermann, C. (2003). The transmembrane domain of Vam3 affects the composition of cis- and trans- SNARE complexes to promote homotypic vacuole fusion. J. Biol. Chem. 278, 1656-1662. Rohn, W.M., Rouille, Y., Waguri, S., and Hoflack, B. (2000). Bi-directional trafficking between the trans-Golgi network and the endosomal/lysosomal system. J. Cell Sci. 113, 2093–2101. Rossi, V., Banfield, D.K., Vacca, M., Dietrich, L.E., Ungermann, C., D’Esposito, M., Galli, T., and Filippini, F. (2004). Longins and their longin domains: regulated SNAREs and multifunctional SNARE regulators. Trends Biochem. Sci. 29, 682– 688. 197 Rothman, J.E., and Schmid, S.L. (1986). Enzymatic recycling of clathrin from coated vesicles. Cell 46, 5-9. Rothman, J.E. (1994). Intracellular membrane fusion. Adv. Second Messenger Phosphoprotein Res. 29, 81-96. Rothman, J.E., and Wieland, F.T. (1996). Protein sorting by transport vesicles. Science 272, 227-234. Rothman, J.E. (2002). Lasker basic medical research award. The machinery and principles of vesicle transport in the cell. Nat. Med. 8, 1059–1062. Sacher, M., Stone, S., and Ferro-Novick, S. (1997). The synaptobrevin-related domains of Bos1p and Sec22p bind to the syntaxin-like region of Sed5p. J. Biol. Chem. 272, 17134–17138 Sacher, M., Jiang, Y., Barrowman, J., Scarpa, A., Burston, J., Zhang, L., Schieltz, D., Yates, J.R., 3rd, Abeliovich, H., and Ferro-Novick, S. (1998). TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion. EMBO J. 17, 2494–2503. Sacher, M., Barrowman, J., Wang, W., Horecka, J., Zhang, Y., Pypaert, M., and Ferro-Novick, S. (2001). TRAPP I implicated in the specificity of tethering in ER-toGolgi transport. Mol. Cell 7, 433–442. Saint-Pol, A., Yelamos, B., Amessou, M., Mills, I.G., Dugast, M., Tenza, D., Schu, P., Antony, C., McMahon, H.T., Lamaze, C., and Johannes, L. (2004). Clathrin adaptor epsinR is required for retrograde sorting on early endosomal membranes. Dev. Cell 6, 525-538. Sakai, T., Yamashina, S., and Ohnishi, S. (1991). Microtubule-disrupting drugs blocked delivery of endocytosed transferrin to the cytocenter, but did not affect return of transferrin to plasma membrane. J. Biochem. 109, 528-533. Sandvig, K., and van Deurs, B. (2000). Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. EMBO J. 19, 5943-5950. Sandvig, K., and van Deurs, B. (2002). Transport of protein toxins into cells: pathways used by ricin, cholera toxin and Shiga toxin. FEBS Lett. 529, 49-53. 198 Sandvig, K., and van Deurs, B. (2005). Delivery into cells: lessons learned from plant and bacterial toxins. Gene Ther.12, 865–872. Sapperstein, S.K., Walter, D.M., Grosvenor, A.R., Heuser, J.E., and Waters, M.G. (1995). p115 is a general vesicular transport factor related to the yeast endoplasmic reticulum to Golgi transport factor Uso1p. Proc. Natl. Acad. Sci. USA 92, 522–526. Sapperstein, S.K., Lupashin, V.V., Schmitt, H.D., and Waters, M.G. (1996). Assembly of the ER to Golgi SNARE complex requires Uso1p. J. Cell Biol. 132, 755767. Saraste, J., and Kuismanen, E. (1992). Pathways of protein sorting and membrane traffic between the rough endoplasmic reticulum and the Golgi complex. Semin. Cell Biol. 3, 343-355. Scales, S.J., Chen, Y.A., Yoo, B.Y., Patel, S.M., Doung, Y.C., and Scheller, R.H. (2000). SNAREs contribute to the specificity of membrane fusion. Neuron 26, 457464. Schafer, W., Stroh, A., Berghofer, S., Seiler, J., Vey, M., Kruse, M.L., Kern, H.F., Klenk, H.D., and Garten, W. (1995). Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin. EMBO J. 14, 2424–2435. Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B.R. and Montecucco, C. (1992) Tetanus and botulinum-B neurotoxins blockneurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359, 832-835. Schiavo, G., Rossetto, O., Catsicas, S., Polverino de Laureto, P., DasGupta, B.R., Benfenati, F., and Montecucco, C. (1993). Identification of the nerve terminal targets of botulinum neurotoxin serotypes A, D, and E. J. Biol. Chem. 268, 23784– 23787. Seabra, M.C., and Coudrier, E. (2004). Rab GTPases and myosin motors in organelle motility. Traffic 5, 393-399. Seals, D.F., Eitzen, G., Margolis, N., Wickner, W.T., and Price, A. (2000). A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion. Proc. Natl. Acad. Sci. USA 97, 9402–9407. 199 Seaman, M.N. (2004). Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer. J. Cell Biol. 165, 111-122. Seaman, M.N. (2005). Recycle your receptors with retromer. Trends Cell Biol. 15, 68-75. Serafini, T., Orci, L., Amherdt, M., Brunner, M., Kahn, R.A., and Rothman, J.E. (1991). ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a novel role for a GTP-binding protein. Cell 67, 239-253. Shewan, A.M., van Dam, E.M., Martin, S., Tang, B.L., Hong, W., Bryant, N.J., and James, D.E. (2003). GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in Syntaxins and 16 but not TGN38: involvement of an acidic targeting motif. Mol. Biol. Cell 14, 973-986. Short, B., Preisinger, C., Schaletzky, J., Kopajtich, R., and Barr, F.A. (2002). The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes. Curr. Biol. 12, 1792-1795. Shorter, J., and Warren, G. (1999). A role for the vesicle tethering protein, p115, in the postmitotic stacking of reassembling Golgi cisternae in a cell‐free system. J. Cell Biol. 146, 57–70. Shorter, J., Beard, M. B., Seemann, J., Dirac‐Svejstrup, A. B., and Warren, G. (2002). Sequential tethering of Golgins and catalysis of SNAREpin assembly by the vesicle-tethering protein p115. J. Cell Biol. 157, 45–62. Silver, P.A. (1991). How proteins enter the nucleus. Cell 64, 489-497. Simon, S.M. (1993). Translocation of proteins across the endoplasmic reticulum. Curr. Opin. Cell Biol. 5, 581-588. Simonsen, A., Lippe, R., Christoforidis, S., Gaullier, J. M., Brech, A., Callaghan, J., Toh, B. H., Murphy, M., Zerial, M., and Stenmark, H. (1998). EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature 394, 494–498. Sögaard, M., Tani, K., Ye, R.B., Geromanos, S., Tempst, P., Kirchhausen, T., Rothman, J.E., and Söllner, T. (1994). A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles. Cell 78, 937-948. 200 Sohda, M., Misumi, Y., Yoshimura, S.I., Nakamura, N., Fusano, T., Sakisaka, S., Ogata, S., Jujimoto, N., Kiyokawa, N., and Ikehara, Y. (2005). Depletion of vesicle‐tethering factor p115 causes mini‐stacked Golgi fragments with delayed protein transport. Biochem. Biophys. Res. Commun. 338, 1268–1274. Söllner, T., Bennett, M.K., Whiteheart, S.W., Scheller, R.H. and Rothman, J.E. (1993a). A protein assembly–disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75, 409–418. Söllner, T., Whiteheart, S.W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J.E. (1993b). SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324. Sonnichsen, B., Lowe, M., Levine, T., Jamsa, E., Dirac-Svejstrup, B., and Warren, G. (1998). A role for giantin in docking COPI vesicles to Golgi membranes. J. Cell Biol. 140, 1013– 1021. Stamnes, M.A., and Rothman, J.E. (1993). The binding of AP-1 clathrin adaptor particles to Golgi membranes requires ADP-ribosylation factor, a small GTP-binding protein. Cell 73, 999-1005. Stanley, K.K., and Howell, K.E. (1993). TGN38/41: a molecule on the move. Trends Cell Biol. 3, 252–255. Steegmaier, M., Klumperman, J., Foletti, D.L., Yoo, J.S., and Scheller, R.H. (1999). Vesicle-associated membrane protein is implicated in trans-Golgi network vesicle trafficking. Mol. Biol. Cell 10, 1957–1972. Steegmaier, M., Lee, K.C., Prekeris, R., and Scheller, R.H. (2000). SNARE protein trafficking in polarized MDCK cells. Traffic 1, 553–560. Storey, H., Stewart, A., Vandenabeele, P., and Luzio, J.P. (2002). The p55 tumour necrosis factor receptor TNFR1 contains a trans-Golgi network localization signal in the C-terminal region of its cytoplasmic tail. Biochem. J. 366, 15–22. Stow, J.L., Manderson, A.P., and Murray, R.Z. (2006). SNAREing immunity: the role of SNAREs in the immune system. Nat. Rev. Immunol. 6, 919-929. Straley, K.S., and Green, S.A. (2000). Rapid transport of internalized P-selectin to late endosomes and the TGN: roles in regulating cell surface expression and recycling to secretory granules. J. Cell Biol. 151, 107-116. 201 Stroupe, C., Collins, K.M., Fratti, R.A., and Wickner, W. (2006). Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p. EMBO J. 25, 1579–1589. Subramaniam, V.N., Loh, E., Horstmann, H., Habermann, A., Xu, Y., Coe, J., Griffiths, G., and Hong, W. (2000). Preferential association of syntaxin with the early endosome. J. Cell Sci. 113, 997–1008. Sudhof, T.C., Baumert, M., Perin, M.S. and Jahn, R. (1989) A synaptic vesicle membrane protein is conserved from mammals to Drosophila. Neuron 2, 1475–1481. Sudhof, T.C. (2004). The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509-547. Sun, W., Yan, Q., Vida, T.A., and Bean, A.J. (2003). Hrs regulates early endosome fusion by inhibiting formation of an endosomal SNARE complex. J. Cell Biol. 162, 125– 137. Suvorova, E.S., Duden, R., and Lupashin, V.V. (2002). The Sec34/ Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins. J. Cell Biol. 157, 631–643. Sutton, R.B., Fasshauer, D., Jahn, R., and Brunger, A.T. (1998). Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, 347-353. Sztul, E., and Lupashin, V. (2006). Role of tethering factors in secretory membrane traffic. Am. J. Physiol. Cell Physiol. 290, C11–C26. Tai, G., Lu, L., Wang, T.L., Tang, B.L., Goud, B., Johannes, L. and Hong, W. (2004). Participation of the syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the TGN. Mol. Biol. Cell 15, 40114022. Takahashi, S., Nakagawa, T., Banno, T., Watanabe, T., Murakami, K., and Nakayama, K. (1995). Localization of furin to the trans-Golgi network and recycling from the cell surface involves Ser and Tyr residues within the cytoplasmic domain. J. Biol. Chem. 270, 28397–28401 202 Takatsu, H., Yoshino, K., and Nakayama, K. (2000). Adaptor γ ear homology domain conserved in γ-adaptin and GGA proteins that interact with γ-synergin. Biochem. Biophys. Res. Commun. 271, 719-725. Takeuchi, Y., Yanagishita, M., Hascall, V.C. (1992). Recycling of transferrin receptors and heparan sulfate proteoglycans in a rat parathyroid cell line. J. Biol. Chem. 267, 14685-14690. Tang, B.L., Wong, S.H., Low, S.H., and Hong, W. (1992). The transmembrane domain of N-glucosaminyltransferase I contains a Golgi retention signal. J. Biol. Chem. 267, 10122-10126. Tang, B.L., Low, S.H., Wong, S.H., and Hong, W. (1995). Cell type differences in Golgi retention signals for transmembrane proteins. Eur. J. Cell Biol. 66, 365-374. Tanigawa, G., Orci, L., Amherdt, M., Ravazzola, M., Helms, J.B., and Rothman, J.E. (1993). Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgiderived COP-coated vesicles. J. Cell Biol. 123, 1365-1371. Tawfeek, H.A., and Abou-Samra, A.B. (2004). Important role for the V-type H(+)ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor. Am. J. Physiol. Endocrinol. Metab. 286, 704-710. Tebar, F., Sorkina, T., Sorkin, A., and Kirchhausen, T. (1996). Eps15 Is a component of clathrin-coated pits and vesicles and is located at the rim of coated pits. J. Biol. Chem. 271, 28727-28730. TerBush, D.R., Maurice, T., Roth, D., and Novick, P. (1996). The Exocyst is amultiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15, 6483–6494. Tochio, H., Tsui, M.M., Banfield, D.K., and Zhang, M. (2001). An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p. Science 293, 698– 702. Toonen, R.F., and Verhage, M. (2003). Vesicle trafficking: pleasure and pain from SM genes. Trends Cell Biol. 13, 177–186. Tran, T. H., Zeng, Q., and Hong, W. (2007). VAMP4 cycles from the cell surface to the trans-Golgi network via sorting and recycling endosomes. J. Cell Sci. 120, 1028-1041. 203 Traub, L.M., Ostrom J.A., and Kornfeld, S. (1993). Biochemical dissection of AP1 recruitment onto Golgi membranes. J. Cell Biol. 123, 561-573. Traub, L.M., and Kornfeld, S. (1997). The trans-Golgi network: a late secretory sorting station. Curr. Opin. Cell Biol. 9, 527-533. Tscherne, D.M., Jones, C.T., Evans, M.J., Lindenbach, B.D., McKeating, J.A., and Rice, C.M. (2006). Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J. Virol. 80, 1734-1741. Tsui, M.M., and Banfield, D.K. (2000). Yeast Golgi SNARE interactions are promiscuous. J. Cell Sci. 113, 145-152. Ungar, D., Oka, T., Brittle, E.E., Vasile, E., Lupashin, V.V., Chatterton, J.E., Heuser, J.E., Krieger, M., and Waters, M.G. (2002). Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function. J. Cell Biol. 157, 405–415. Ungermann, C., and Langosch, D. (2005). Functions of SNAREs in intracellular membrane fusion and lipid bilayer mixing. J. Cell Sci. 118, 3819-3828. Ungewickell, E., Ungewickell, H., Holstein, S.E., Lindner, R., Prasad, K., Barouch, W., Martin, B., Greene, L.E., and Eisenberg, E. (1995). Role of auxilin in uncoating clathrin-coated vesicles. Nature 378, 632-635. Vale, R.D. (2003). The molecular motor toolbox for intracellular transport. Cell 112, 467-480. Van Komen, J.S., Bai, X., Scott, B.L. and McNew, J.A. (2006). An intramolecular t-SNARE complex functions in vivo without the syntaxin NH2-terminal regulatory domain. J. Cell Biol. 172, 295–307. van Weert, A.W., Dunn, K.W., Gueze, H.J., Maxfield, F.R., and Stoorvogel, W. (1995). Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump. J. Cell Biol. 130, 821-834. Varlamov, O., and Fricker, L.D. (1998). Intracellular trafficking of metallocarboxypeptidase D in AtT-20 cells: localization to the trans-Golgi network and recycling from the cell surface. J. Cell Sci. 111, 877–885. 204 Veit, M., Sollner, T.H., and Rothman, J.E. (1996). Multiple palmitoylation of synaptotagmin and the t-SNARE SNAP-25. FEBS Lett. 385, 119– 123. Veit, M., Laage, R., Dietrich, L., Wang, L., and Ungermann, C. (2001). Vac8p release from the SNARE complex and its palmitoylation are coupled and essential for vacuole fusion. EMBO J. 20, 3145–3155 Veit, M. (2004). The human SNARE protein Ykt6 mediates its own palmitoylation at C-terminal cysteine residues. Biochem. J. 384, 233–237. Vogel, K. and Roche, P.A. (1999). SNAP-23 and SNAP-25 are palmitoylated in vivo. Biochem. Biophys. Res. Commun. 258, 407-410. Vogel, K., Cabaniols, J.P., and Roche, P.A. (2000). Targeting of SNAP-25 to membranes is mediated by its association with the target SNARE syntaxin. J. Biol. Chem. 275, 2959–2965. Voorhees, P., Deignan, E., Vandonselaar, E., Humphrey, J., Marks, M.S., Peters, P.J., and Bonifacino, J.S. (1995). An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface. EMBO J. 14, 4961–4975. Walker, J.K., Premont, R.T., Barak, L.S., Caron, M.G., and Shetzline, M.A. (1999). Properties of secretin receptor internalization differ from those of the beta(2)adrenergic receptor. J. Biol. Chem. 274, 31515-31523. Walther, K., Krauss, M., Diril, M.K., Lemke, S., Ricotta, D., Honing, S., and Haucke, V. (2001). Human Stoned B interacts with AP-2 and synaptotagmin and facilitates clathrin-coated vesicle uncoating. EMBO Rep. 2, 634-640. Wan, L., Molloy, S.S., Thomas, L., Liu, G., Xiang, Y., Rybak, S.L, and Thomas, G. (1998). PACS-1 defines a novel gene family of cytosolic sorting proteins required for trans-Golgi network localization. Cell 94, 205–216. Wang, C.C., Ng, C.P., Lu, L., Atlashkin, V., Zang, W., Seet, L.F., and Hong, W. (2004). A role of VAMP8/endobrevin in regulated exocytosis of pancreatic acinar cells. Dev. Cell 7, 359-371. Wang, X., Ma, D., Keski-Oja, J., and Pei, D. (2004). Co-recycling of MT1-MMP and MT3-MMP through the tran-Golgi network. Identification of DKV582 as a recycling signal. J. Biol. Chem 279, 9331-9336. 205 Wang, Y., Dulubova, I., Rizo, J. and Südhof, T.C. (2001). Functional analysis of conserved structural elements in yeast syntaxin Vam3p. J. Biol. Chem. 276, 28598– 28605. Wang, Y.J., Wang, J., Sun, H.Q., Martinez, M., Sun, Y.X., Macia, E., Kirchhausen, T., Albanesi, J.P., Roth, M.G., and Yin, H.L. (2003). Phosphatidylinositol phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi. Cell 114, 299-310. Ward, D.M., Pevsner, J., Scullion, M.A., Vaughn, M., and Kaplan, J. (2000). Syntaxin and VAMP-7 are soluble N-ethylmaleimide-sensitive factor attachment protein receptors required for late endosome–lysosome and homotypic lysosome fusion in alveolar macrophages. Mol. Biol. Cell 11, 2327–2333. Warren, G. (1990). Intracellular transport: vesicular consumption. Nature 345, 382383. Waters, M.G., Serafini, T., and Rothman, J.E. (1991). ‘Coatomer’: a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature 349, 248-251. Waters, M.G., Clary, D.O., and Rothman, J.E. (1992). A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack. J. Cell Biol. 118, 1015– 1026. Waters, M.G., and Pfeffer, S.R. (1999). Membrane tethering in intracellular transport. Curr. Opin. Cell Biol. 11, 453–459. Watson, R.T., Kanzaki, M., and Pessin, J.E. (2004). Regulated membrane trafficking of the insulin-responsive glucose transporter in adipocytes. Endocr. Rev. 25, 177– 204. Watts, C., and Marsh, M. (1992). Endocytosis: what goes in and how? J. Cell Sci. 103, 1-8. Weber, T., Zemelman, B.V., McNew, J.A., Westermann, B., Gmachl, M., Parlati, F., Söllner, T.H., and Rothman, J.E. (1998). SNAREpins: minimal machinery for membrane fusion. Cell 92, 759-772. 206 Weidman, P.J., Melancon, P., Block, M.R., and Rothman, J.E. (1989). Binding of an N-ethylmaleimide-sensitive fusion protein to Golgi membranes requires both a soluble protein(s) and an integral membrane receptor. J. Cell Biol. 708, 1589-1596. Weimbs, T., Low, S.H., Chapin, S.J., Mostov, K.E., Bucher, P., and Hoffmann, K. (1997). A conserved domain is present in different families of vesicular fusion proteins: a new superfamily. Proc. Natl. Acad. Sci. USA. 94, 3046-3051. Weninger, K., Bowen, M.E., Chu, S. and Brunger, A.T. (2003). Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations. Proc. Natl. Acad. Sci. USA 100, 14800–14805. Werbonat, Y., Kleutges, N., Jakobs, K.H, and van Koppen, C.J. (2000). Essential role of dynamin in internalization of M2 muscarinic acetylcholine and angiotensin AT1A receptors. J. Biol. Chem. 275, 21969-21974. Whiteheart S.W., Brunner, M., Wilson, D.W., Wiedmann, M. and Rothman, J.E. (1992) Soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAPs) bind to a multi-SNAP receptor complex in Golgi membranes. J. Biol. Chem. 267, 12239– 12243. Whyte, J.R., and Munro, S. (2001). The Sec34/35 Golgi transport complex is related to the exocyst, defining a family of complexes involved in multiple steps of membrane traffic. Dev. Cell 1, 527–537. Whyte, J.R. and Munro, S. (2002). Vesicle tethering complexes in membrane traffic, J. Cell Sci. 115, 2627-2637. Wilson, D.W., Wilcox, C.A., Flynn, G.C., Chen, E., Kuang, W.J., Henzel, W.J., Block, M.R., Ullrich, A., and Rothman, J.E. (1989). A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast. Nature 339, 355-359. Wilson, D.W., Whiteheart, S.W., Wiedmann, M., Brunner, M., and Rothman, J.E. (1992). A multisubunit particle implicated in membrane fusion. J. Cell Biol. 117, 531-538. Wimmer, C., Hohl, T.M., Hughes, C.A, Muller, S.A., Söllner, T.H., Engel, A., and Rothman, J.E. (2001). Molecular mass, stoichimestry, and assembly of 20S particles. J. Biol. Chem. 276, 29091-29097. 207 Wong, S.H., Zhang, T., Xu, Y., Subramaniam, V.N., Griffiths, G., and Hong, W. (1998). Endobrevin, a novel synaptobrevin/VAMP-like protein preferentially associated with the early endosome. Mol. Biol. Cell 9, 1549-1563. Wong, W.T., Kraus, M.H., Carlomagno, F., Zelano, A., Druck, T., Croce, C.M., Huebner, K., and Di Fiore, P.P. (1994). The human eps15 gene, encoding a tyrosine kinase substrate, is conserved in evolution and maps to 1p31-p32. Oncogene 9, 15911597. Wong, W.T., Schumacher, C., Salcini, A.E., Romano, A., Castagnino, P., Pelicci, P.G., and Di Fiore, P.P. (1995). A protein-binding domain, EH, Identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution. Proc. Nat. Acad. Sci. USA 92, 9530-9534. Woodward, M.P., and Roth, T.F. (1978). Coated vesicles: characterization, selective dissociation, and reassembly. Proc. Natl. Acad. Sci. USA. 75, 4394-4398. Wu, M., Lu, L., Hong, W., and Song, H. (2004). Structural recruitment of GRIP domain golgin‐245 by small GTPase Arl1. Nat. Struct. Biol. 11, 86–94. Xiang, Y., Molloy, S.S., Thomas, L., and Thomas, G. (2000) The PC6B cytoplasmic domain contains two acidic clusters that direct sorting to distinct transGolgi network/endosomal compartments. Mol. Biol. Cell 11, 1257–1273. Xu, Y., Wong, S. H., Zhang, T., Subramaniam, V. N., and Hong, W. (1997). GS15, a 15-kilodalton Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) homologous to rbet1. J. Biol. Chem. 272, 20162–20166. Xu, Y., Martin, S., James, D.E., and Hong, W. (2002). GS15 forms a SNARE complex with syntaxin 5, GS28, and Ykt6 and is implicated in traffic in the early cisternae of the Golgi apparatus. Mol. Biol. Cell 13, 3493–3507. Yamaguchi, T., Dulubova, I., Min, S.W., Chen, X., Rizo, J., and Sudhof, T.C. (2002). Sly1 binds to Golgi and ER syntaxins via a conserved N-terminal peptide motif, Dev. Cell 2, 295– 305. Yang, B., Gonzalez, L. Jr., Prekeris, R., Steegmaier, M., Advani, and R.J., Scheller, R.H. (1999). SNARE interactions are not selective. Implications for membrane fusion specificity. J. Biol. Chem. 274, 5649–5653. 208 Yang, C., Mora, S., Ryder, J.W., Coker, K.J., Hansen, P., Allen, L.A., and Pessin, J.E. (2001). VAMP3 null mice display normal constitutive, insulin and exerciseregulated vesicle trafficking. Mol. Cell. Biol. 21, 1573– 1580. Zeng, Q., Subramaniam, V.N., Wong, S.H., Tang, B.L., Parton, R.G., Rea, S., James, D.E., and Hong, W. (1998). A novel synaptobrevin/VAMP homologous protein (VAMP5) is increased during in vitro myogenesis and present in the plasma membrane. Mol. Biol. Cell 9, 2423–2437. Zeng, Q., Tran, T.T., Tan, H.X., and Hong, W. (2003). The cytoplasmic domain of Vamp4 and Vamp5 is responsible for their correct subcellular targeting: the Nterminal extension of VAMP4 contains a dominant autonomous targeting signal for the trans-Golgi network. J. Biol. Chem. 278, 23046-23054. Zerial, M., and McBride, H. (2001). Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2, 107-117. Zhang, T., Wong, S.H., Tang, B.L., Xu, Y., Peter, F., Subramaniam, V.N., and Hong, W. (1997). The mammalian protein (rbet1) homologous to yeast Bet1p is primarily associated with the pre-Golgi intermediate compartment and is involved in vesicular transport from the endoplasmic reticulum to the Golgi apparatus, J. Cell Biol. 139, 1157–1168. Zhang, T., Wong, S. H., Tang, B. L., Xu, Y., and Hong, W. (1999). Morphological and functional association of Sec22b/ERS-24 with the pre-Golgi intermediate compartment. Mol. Biol. Cell 10, 435-453. Zhang, T., and Hong, W. (2001). Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport. J. Biol. Chem. 276, 27480-27487. Zolov, S.N., and Lupashin, V.V. (2005). Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells. J. Cell Biol. 168, 747–759. Zwilling, D., Cypionka, A., Pohl, W.H., Fasshauer, D., Walla, P.J., Wahl, M.C., and Jahn, R. (2007). 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