Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion

Abstract

Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1–SNX2 and SNX5–SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: SNX3 is required for EGL-20 (Wnt) signalling and MIG-14 (Wls) recycling in C. elegans.
Figure 2: DSnx3 is required for Wg secretion and Wls recycling in the Drosophila wing imaginal disc.
Figure 3: Co-localization and physical interaction of SNX3 with the cargo-selective subcomplex of the retromer.
Figure 4: SNX3 co-localizes with Wls and facilitates membrane association of the cargo-selective subcomplex of the retromer.
Figure 5: Wls is contained within SNX3-positive vesicular carriers but is absent from SNX1 retromer-decorated tubular carriers.

References

  1. Carlton, J. et al. Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of high- curvature membranes and 3-phosphoinositides. Curr. Biol. 14, 1791–1800 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Carlton, J. G. et al. Sorting nexin-2 is associated with tubular elements of the early endosome, but is not essential for retromer-mediated endosome-to-TGN transport. J. Cell Sci. 118, 4527–4539 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Wassmer, T. et al. A loss-of-function screen reveals SNX5 and SNX6 as potential components of the mammalian retromer. J. Cell Sci. 120, 45–54 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Wassmer, T. et al. The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network. Dev. Cell 17, 110–122 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Seaman, M. N. Recycle your receptors with retromer. Trends Cell Biol. 15, 68–75 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Attar, N. & Cullen, P. J. The retromer complex. Adv. Enzyme Regul. 50, 216–236 (2009).

    Article  PubMed  Google Scholar 

  7. Seaman, M. N. Identification of a novel conserved sorting motif required for retromer-mediated endosome-to-TGN retrieval. J. Cell Sci. 120, 2378–2389 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Cullen, P. J. Endosomal sorting and signalling: an emerging role for sorting nexins. Nat. Rev. Mol. Cell Biol. 9, 574–582 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Seaman, M. N., McCaffery, J. M. & Emr, S. D. A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast. J. Cell Biol. 142, 665–681 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Seaman, M. N. Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer. J. Cell Biol. 165, 111–122 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Arighi, C. N., Hartnell, L. M., Aguilar, R. C., Haft, C. R. & Bonifacino, J. S. Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor. J. Cell Biol. 165, 123–133 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Banziger, C. et al. Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125, 509–522 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Bartscherer, K., Pelte, N., Ingelfinger, D. & Boutros, M. Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell 125, 523–533 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Goodman, R. M. et al. Sprinter: a novel transmembrane protein required for Wg secretion and signaling. Development 133, 4901–4911 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Belenkaya, T. Y. et al. The retromer complex influences Wnt secretion by recycling Wntless from endosomes to the trans-Golgi network. Dev. Cell 14, 120–131 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. Franch-Marro, X. et al. Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex. Nat. Cell Biol. 10, 170–177 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Pan, C. L. et al. C. elegans AP-2 and retromer control Wnt signaling by regulating mig-14/Wntless. Dev. Cell 14, 132–139 (2008).

    Article  CAS  PubMed  Google Scholar 

  18. Port, F. et al. Wingless secretion promotes and requires retromer-dependent cycling of Wntless. Nat. Cell Biol. 10, 178–185 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Yang, P. T. et al. Wnt signaling requires retromer-dependent recycling of MIG-14/Wntless in Wnt-producing cells. Dev. Cell 14, 140–147 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Nothwehr, S. F. & Hindes, A. E. The yeast VPS5/GRD2 gene encodes a sorting nexin-1-like protein required for localizing membrane proteins to the late Golgi. J. Cell Sci. 110 (Pt 9), 1063–1072 (1997).

    Google Scholar 

  21. Hettema, E. H., Lewis, M. J., Black, M. W. & Pelham, H. R. Retromer and the sorting nexins Snx4/41/42 mediate distinct retrieval pathways from yeast endosomes. EMBO J. 22, 548–557 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Mari, M. et al. SNX1 defines an early endosomal recycling exit for sortilin and mannose 6-phosphate receptors. Traffic 9, 380–393 (2008).

    Article  CAS  PubMed  Google Scholar 

  23. Coudreuse, D. Y., Roel, G., Betist, M. C., Destree, O. & Korswagen, H. C. Wnt gradient formation requires retromer function in Wnt-producing cells. Science 312, 921–924 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Prasad, B. C. & Clark, S. G. Wnt signaling establishes anteroposterior neuronal polarity and requires retromer in C. elegans . Development 133, 1757–1766 (2006).

    Article  CAS  Google Scholar 

  25. Chen, D. et al. Retromer is required for apoptotic cell clearance by phagocytic receptor recycling. Science 327, 1261–1264 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Zecca, M., Basler, K. & Struhl, G. Direct and long-range action of a Wingless morphogen gradient. Cell 87, 833–844 (1996).

    Article  CAS  PubMed  Google Scholar 

  27. Harris, J., Honigberg, L., Robinson, N. & Kenyon, C. Neuronal cell migration in C. elegans: regulation of Hox gene expression and cell position. Development 122, 3117–3131 (1996).

    CAS  PubMed  Google Scholar 

  28. Salser, S. J. & Kenyon, C. Activation of a C. elegans Antennapedia homologue in migrating cells controls their direction of migration. Nature 355, 255–258 (1992).

    Article  CAS  PubMed  Google Scholar 

  29. Korswagen, H. C. et al. The Axin-like protein PRY-1 is a negative regulator of a canonical Wnt pathway in C. elegans . Genes Dev. 16, 1291–1302 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Maloof, J. N., Whangbo, J., Harris, J. M., Jongeward, G. D. & Kenyon, C. A Wnt signaling pathway controls Hox gene expression and neuroblast migration in C. elegans . Development 126, 37–49 (1999).

    CAS  PubMed  Google Scholar 

  31. Hidalgo, A. & Ingham, P. Cell patterning in the Drosophila segment: spatial regulation of the segment polarity gene patched. Development 110, 291–301 (1990).

    CAS  PubMed  Google Scholar 

  32. Entchev, E. V., Schwabedissen, A. & Gonzalez-Gaitan, M. Gradient formation of the TGFβ homolog Dpp. Cell 103, 981–991 (2000).

    Article  CAS  PubMed  Google Scholar 

  33. Teleman, A. A. & Cohen, S. M. Dpp gradient formation in the Drosophila wing imaginal disc. Cell 103, 971–980 (2000).

    Article  CAS  PubMed  Google Scholar 

  34. Seaman, M. N., Harbour, M. E., Tattersall, D., Read, E. & Bright, N. Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-GAP TBC1D5. J. Cell Sci. 122, 2371–2382 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Rojas, R. et al. Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7. J. Cell Biol. 183, 513–526 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Popoff, V. et al. Analysis of articulation between clathrin and retromer in retrograde sorting on early endosomes. Traffic 10, 1868–1880 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. Xu, Y., Hortsman, H., Seet, L., Wong, S. H. & Hong, W. SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P. Nat. Cell Biol. 3, 658–666 (2001).

    Article  CAS  PubMed  Google Scholar 

  38. Skanland, S. S., Walchli, S., Brech, A. & Sandvig, K. SNX4 in complex with clathrin and dynein: implications for endosome movement. PLoS ONE 4, e5935 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Lorenowicz, M. J. & Korswagen, H. C. Sailing with the Wnt: charting the Wnt processing and secretion route. Exp. Cell Res. 315, 2683–2689 (2009).

    Article  CAS  PubMed  Google Scholar 

  40. Port, F. & Basler, K. Wnt trafficking: new insights into Wnt maturation, secretion and spreading. Traffic 11, 1265–1271 (2010).

    Article  CAS  PubMed  Google Scholar 

  41. Voos, W. & Stevens, T. H. Retrieval of resident late-Golgi membrane proteins from the prevacuolar compartment of Saccharomyces cerevisiae is dependent on the function of Grd19p. J. Cell Biol. 140, 577–590 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Nothwehr, S. F., Ha, S. A. & Bruinsma, P. Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains with Vps35p. J. Cell Biol. 151, 297–310 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Strochlic, T. I., Setty, T. G., Sitaram, A. & Burd, C. G. Grd19/Snx3p functions as a cargo-specific adapter for retromer-dependent endocytic recycling. J. Cell Biol. 177, 115–125 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Yu, J. W. & Lemmon, M. A. All phox homology (PX) domains from Saccharomyces cerevisiae specifically recognize phosphatidylinositol 3-phosphate. J. Biol. Chem. 276, 44179–44184 (2001).

    Article  CAS  PubMed  Google Scholar 

  45. Duncan, J. R. & Kornfeld, S. Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus. J. Cell Biol. 106, 617–628 (1988).

    Article  CAS  PubMed  Google Scholar 

  46. Jin, M., Sahagian, G. G. Jr & Snider, M. D. Transport of surface mannose 6-phosphate receptor to the Golgi complex in cultured human cells. J. Biol. Chem. 264, 7675–7680 (1989).

    CAS  PubMed  Google Scholar 

  47. Lin, S. X., Mallet, W. G., Huang, A. Y. & Maxfield, F. R. Endocytosed cation-independent mannose 6-phosphate receptor traffics via the endocytic recycling compartment en route to the trans-Golgi network and a subpopulation of late endosomes. Mol. Biol. Cell 15, 721–733 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Lewis, J. A. & Fleming, J. T. Basic culture methods. Methods Cell Biol. 48, 3–29 (1995).

    Article  CAS  PubMed  Google Scholar 

  49. Ch’ng, Q. et al. Identification of genes that regulate a left-right asymmetric neuronal migration in Caenorhabditis elegans . Genetics 164, 1355–1367 (2003).

    PubMed  PubMed Central  Google Scholar 

  50. Whangbo, J., Harris, J. & Kenyon, C. Multiple levels of regulation specify the polarity of an asymmetric cell division in C. elegans . Development 127, 4587–4598 (2000).

    CAS  PubMed  Google Scholar 

  51. Herman, M. A. & Horvitz, H. R. The Caenorhabditis elegans gene lin-44 controls the polarity of asymmetric cell divisions. Development 120, 1035–1047 (1994).

    CAS  PubMed  Google Scholar 

  52. Mello, C. C. & Fire, A. in Caenorhabditis elegans: Modern biological analysis of an organism, Vol. 48 (eds Epstein, H. F. & Shakes, D. C.) 451–482 (Academic, 1995).

    Book  Google Scholar 

  53. Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003).

    Article  CAS  PubMed  Google Scholar 

  54. Baeg, G. H., Lin, X., Khare, N., Baumgartner, S. & Perrimon, N. Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development 128, 87–94 (2001).

    CAS  PubMed  Google Scholar 

  55. Kawakami, A. et al. Rab7 regulates maturation of melanosomal matrix protein gp100/Pmel17/Silv. J. Invest. Dermatol. 128, 143–150 (2008).

    Article  CAS  PubMed  Google Scholar 

  56. Bolte, S. & Cordelieres, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank C. Rabouille for critically reading the manuscript, M. Seaman for advice, D. Xue (University of Colorado, Boulder) for smIs34, M. Tabuchi (Kawasaki Medical School, Okayama, Japan) for the bacterial construct expressing 3×Flag-tagged retromer complex, S. Mitani (National Bioresource Project for the Nematode, Tokyo, Japan) for deletion mutants, A. Fire for expression vectors and the Caenorhabditis Genetic Center (University of Minnesota, Minneapolis) for strains. This work was funded by the Dutch Cancer Society (HUBR 2008-4114), the EU FP6 Programme Cells into Organs and NWO VIDI (016.076.317) (H.C.K.), an NWO VENI fellowship (M.J.L.), a Boehringer Ingelheim Foundation fellowship (M.H.), the Swiss National Science Foundation and the Forschungskredit of the University of Zürich (F.P. and K.B.) and the Wellcome Trust (089928/Z/09/Z and 085743) (P.J.C.). I.J.M. is a Wellcome Trust-funded PhD student (086777/Z/08/Z).

Author information

Authors and Affiliations

Authors

Contributions

M.H., M.S., T.C.M., M.C.B., R.G.H.P.H. and H.C.K. designed and carried out the C. elegans experiments, F.P. and K.B. designed and carried out the Drosophila experiments, M.J.L., I.J.M., J.R.T.W., H.C.K. and P.J.C. designed and carried out the cell biological analysis of SNX3 function in tissue culture cells and M.H., K.B., P.J.C. and H.C.K. wrote the paper.

Corresponding authors

Correspondence to Peter J. Cullen or Hendrik C. Korswagen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1846 kb)

Supplementary Table 1

Supplementary Information (XLS 50 kb)

Supplementary Movie 1

Supplementary Information (MOV 148 kb)

Supplementary Movie 2

Supplementary Information (MOV 111 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Harterink, M., Port, F., Lorenowicz, M. et al. A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion. Nat Cell Biol 13, 914–923 (2011). https://doi.org/10.1038/ncb2281

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2281

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing