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Regulation of caveolar endocytosis by syntaxin 6-dependent delivery of membrane components to the cell surface

Nature Cell Biology volume 8, pages 317–328 (2006)Cite this article

A Corrigendum to this article was published on 01 August 2006

Abstract

Caveolar endocytosis has an important function in the cellular uptake of some bacterial toxins, viruses and circulating proteins. However, the molecular machinery involved in regulating caveolar uptake is poorly defined. Here, we demonstrate that caveolar endocytosis is regulated by syntaxin 6, a target membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) involved in membrane fusion events along the secretory pathway. When syntaxin 6 function was inhibited, internalization through caveolae was dramatically reduced, whereas other endocytic mechanisms were unaffected. Syntaxin 6 inhibition also reduced the presence of caveolin-1 and caveolae at the plasma membrane. In addition, syntaxin 6 inhibition decreased the delivery of GM1 ganglioside (GM1) and glycosylphosphatidylinositol (GPI)–GFP (but not vesicular stomatitis virus-glycoprotein G; VSV-G) protein from the Golgi complex to the plasma membrane. Addition of GM1 to syntaxin 6-inhibited cells resulted in the reappearance of caveolin-1 and caveolae at the plasma membrane, and restored caveolar uptake. These results suggest that syntaxin 6 regulates the delivery of microdomain-associated lipids and proteins to the cell surface, which are required for caveolar endocytosis.

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Figure 1: Adenoviral delivery of syntaxin cytosolic domains and morpholino oligonucleotide mediated endogenous-syntaxin knockdown.
Figure 2: Inhibition of syntaxin 6 function blocks caveolar endocytosis without affecting other internalization mechanisms.
Figure 3: Effect of syntaxin 6-cyto and syntaxin 6 MO on Cav1, caveolae and pCav1.
Figure 4: Inhibition of syntaxin 6 function and its effect on GM1 and cholesterol.
Figure 5: Syntaxin 6-cyto blocks the transport of GPI–GFP to the plasma membrane.
Figure 6: Syntaxin 6-cyto does not block the transport of VSV-G to the plasma membrane.
Figure 7: Cav1 and syntaxin 6 colocalize at the Golgi apparatus and coimmunoprecipitate.
Figure 8: Effects of exogenous GM1 ganglioside on syntaxin 6-cyto infected cells.

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References

  1. Cohen, A. W., Hnasko, R., Schubert, W. & Lisanti, M. P. Role of caveolae and caveolins in health and disease. Physiol. Rev. 84, 1341–1379 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Mineo, C. & Anderson, G. W. Potocytosis. Histochem. Cell Biol. 116, 109–118 (2001).

    CAS  PubMed  Google Scholar 

  3. Parton, R. G. & Richards, A. A. Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms. Traffic 4, 724–738 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Duncan, M. J., Shin, J. S. & Abraham, S. N. Microbial entry through caveolae: variations on a theme. Cell Microbiol. 4, 783–791 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Lencer, W. I., Hirst, T. R. & Holmes, R. K. Membrane traffic and the cellular uptake of cholera toxin. Biochim. Biophys. Acta. 1450, 177–190 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Marjomäki, V. S., Huovila, A. -P. J., Surkka, M. A., Jokinen, I. & Salminen, A. Lysosomal trafficking in rat cardiac myocytes. J. Histochem. Cytochem. 38, 1155–1164 (1990).

    Article  PubMed  Google Scholar 

  7. Norkin, L. C. Caveolae in the uptake and targeting of infectious agents and secreted toxins. Adv. Drug Deliv. Rev. 49, 301–315 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Pelkmans, L., Kartenbeck, J. & Helenius, A. Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nature Cell Biol. 3, 473–483 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Richterova, Z. et al. Caveolae are involved in the trafficking of mouse polyomavirus virions and artificial VP1 pseudocapsids toward cell nuclei. J. Virol. 75, 10880–10891 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Shin, J. -S., Gao, Z. & Abraham, N. Involvement of cellular caveolae in bacterial entry into mast cells. Science 289, 785–788 (2000).

    Article  CAS  PubMed  Google Scholar 

  11. Kim, Y. N. & Bertics, P. J. The endocytosis-linked protein dynamin associates with caveolin-1 and is tyrosine phosphorylated in response to the activation of a noninternalizing epidermal growth factor receptor mutant. Endocrinology 143, 1726–1731 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. Mineo, C., Gill, G. N. & Anderson, R. G. Regulated migration of epidermal growth factor receptor from caveolae. J. Biol. Chem. 274, 30636–30643 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Shajahan, A. N. et al. Role of Src-induced dynamin-2 phosphorylation in caveolae-mediated endocytosis in endothelial cells. J. Biol. Chem. 279, 20392–20400 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Sharma, D. K. et al. Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol. Mol. Biol. Cell 15, 3114–3122 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Volonte, D. et al. Flotillins–cavatellins are differentially expressed in cells and tissues and form a hetero-oligomeric complex with caveolins in vivo. Characterization and epitope-mapping of a novel flotillin-1 monoclonal antibody probe. J. Biol. Chem. 274, 12702–12709 (1999).

    Article  CAS  PubMed  Google Scholar 

  16. Chen, Y. A. & Scheller, R. H. SNARE-mediated membrane fusion. Nature Rev. Mol. Cell Biol. 2, 98–106 (2001).

    Article  CAS  Google Scholar 

  17. McNew, J. A. et al. Compartmental specificity of cellular membrane fusion encoded in SNARE proteins. Nature 407, 153–159 (2000).

    Article  CAS  PubMed  Google Scholar 

  18. Ungar, D. & Hughson, F. M. SNARE protein structure and function. Annu. Rev. Cell Dev. Biol. 19, 493–517 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. McIntosh, D. P. & Schnitzer, J. E. Caveolae require intact VAMP for targeted transport in vascular endothelium. Am. J. Physiol. 277, H2222–H2232 (1999).

    CAS  PubMed  Google Scholar 

  20. Predescu, D., Horvat, R., Predescu, S. & Palade, G. E. Transcytosis in the continuous endothelium of the myocardial microvasculature is inhibited by N-ethylmaleimide. Proc. Natl Acad. Sci. USA 91, 3014–3018 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Schnitzer, J. E., Liu, J. & Oh, P. Endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexins, and GTPases. J. Biol. Chem. 270, 14399–14404 (1995).

    Article  CAS  PubMed  Google Scholar 

  22. Kuliawat, R. et al. Syntaxin-6 SNARE involvement in secretory and endocytic pathways of cultured pancreatic β-cells. Mol. Biol. Cell 15, 1690–1701 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Perera, H. K. I. et al. Syntaxin 6 regulates Glut4 trafficking in 3T3-L1 Adipocytes. Mol. Biol Cell 14, 2946–2958 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wendler, F., Page, L., Urbe, S. & Tooze, S. A. Homotypic fusion of immature secretory granules during maturation requires syntaxin 6. Mol. Biol. Cell 12, 1699–1709 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ros-Baro, A. et al. Lipid rafts are required for GLUT4 internalization in adipose cells. Proc. Natl Acad. Sci. USA 98, 12050–12055 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Shigematsu, S., Watson, R. T., Khan, A. H. & Pessin, J. E. The adipocyte plasma membrane caveolin functional–structural organization is necessary for the efficient endocytosis of GLUT4. J. Biol. Chem. 278, 10683–10690 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Shewan, A. M. et al. GLUT4 recycles via a trans-Golgi network (TGN) subdomain enriched in Syntaxins 6 and 16 but not TGN38: involvement of an acidic targeting motif. Mol. Biol. Cell 14, 973–986 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Puri, V. et al. Clathrin-dependent and independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways. J. Cell Biol. 154, 535–547 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sharma, D. K. et al. Glycosphingolipids internalized via caveolar-related endocytosis rapidly merge with the clathrin pathway in early endosomes and form microdomains for recycling. J. Biol. Chem. 278, 7564–7572 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Singh, R. D. et al. Selective caveolin-1-dependent endocytosis of glycosphingolipids. Mol. Biol. Cell 14, 3254–3265 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Olson, A. L., Knight, J. B. & Pessin, J. E. Syntaxin 4, VAMP2, and/or VAMP3–cellubrevin are functional target membrane and vesicle SNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes. Mol. Cell Biol. 17, 2425–2435 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tellam, J. T. et al. Characterization of Munc-18c and syntaxin-4 in 3T3-L1 adipocytes. Putative role in insulin-dependent movement of GLUT-4. J. Biol. Chem. 272, 6179–6186 (1997).

    Article  CAS  PubMed  Google Scholar 

  33. Shubert, W. et al. Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. J. Biol. Chem. 276, 48619–48622 (2001).

    Article  Google Scholar 

  34. Prekeris, R., Klumperman, J., Chen, Y. A. & Scheller, R. H. Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes. J. Cell Biol. 143, 957–971 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. del Pozo, M. A. et al. Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization. Nature Cell Biol. 7, 901–908 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Orlichenko, L., Huang, B., Krueger, E. & McNiven, M. A. EGF-induced phosphorylation of caveolin 1 at tyrosine 14 stimulates caveolae formation in epithelial cells. J. Biol. Chem. 281, 4570–4579 (2006).

    Article  CAS  PubMed  Google Scholar 

  37. Chamberlain, L. H., Burgoyne, R. D. & Gould, G. W. SNARE proteins are highly enriched in lipid rafts in PC12 cells: implications for the spatial control of exocytosis. Proc. Natl Acad. Sci. USA 98, 5619–5624 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Puri, V. et al. Sphingolipid storage induces accumulation of intracellular cholesterol by stimulating SREBP-1 cleavage. J. Biol. Chem. 278, 20961–20970 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Martin, L. B., Shewan, A., Millar, C. A., Gould, G. W. & James, D. E. Vesicle-associated membrane protein 2 plays a specific role in the insulin-dependent trafficking of the facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes. J. Biol. Chem. 273, 1444–1452 (1998).

    Article  CAS  PubMed  Google Scholar 

  40. Volchuk, A. et al. Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport. Mol. Biol. Cell 7, 1075–1082 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Morcos, P. A. Achieving efficient delivery of morpholino oligos in cultured cells. Genesis 30, 94–102 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Wendler, F. & Tooze, S. Syntaxin 6: The promiscuous behaviour of a SNARE protein. Traffic 2, 606–611 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Martin-Martin, B., Nabokina, S. M., Blasi, J., Lazo, P. A. & Mollinedo, F. Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis. Blood 96, 2574–2583 (2000).

    CAS  PubMed  Google Scholar 

  44. Sharma, D. K. et al. The glycosphingolipid, lactosylceramide, regulates β1-integrin clustering and endocytosis. Cancer Res. 65, 8233–8241 (2005).

    Article  CAS  PubMed  Google Scholar 

  45. Choudhury, A. et al. Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells. J. Clin. Invest. 109, 1541–1550 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Martin, O. C. & Pagano, R. E. Internalization and sorting of a fluorescent analog of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms. J. Cell Biol. 125, 769–781 (1994).

    Article  CAS  PubMed  Google Scholar 

  47. Sabharanjak, S., Sharma, P., Parton, R. G. & Mayor, S. GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Develop. Cell 2, 411–423 (2002).

    Article  CAS  Google Scholar 

  48. Henley, J. R., Krueger, E. W., Oswald, B. J. & McNiven, M. A. Dynamin-mediated internalization of caveolae. J. Cell Biol. 141, 85–99 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Supported by U.S. Public Health Service grant (GM-22942) to R.E.P. and by The Wellcome Trust (studentship to K.P. and Research Leave Award to G.W.G.). A.C. was supported by a fellowship from the American Heart Association. We thank L.Chamberlain (University of Glasgow, Glasgow, UK) and E. Krueger (Mayo Foundation, Rochester, MN) for helpful advice.

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  1. Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street, S.W., Rochester, 55905, MN, USA

    Amit Choudhury, David L. Marks & Richard E. Pagano

  2. Division of Biochemistry and Molecular Biology, Henry Wellcome Laboratory of Cell Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, Scotland

    Kirsty M. Proctor & Gwyn W. Gould

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Choudhury, A., Marks, D., Proctor, K. et al. Regulation of caveolar endocytosis by syntaxin 6-dependent delivery of membrane components to the cell surface. Nat Cell Biol 8, 317–328 (2006). https://doi.org/10.1038/ncb1380

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