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A SNARE–adaptor interaction is a new mode of cargo recognition in clathrin-coated vesicles

Nature volume 450, pages 570–574 (2007)Cite this article

Abstract

Soluble NSF attachment protein receptors (SNAREs) are type II transmembrane proteins that have critical roles in providing the specificity and energy for transport-vesicle fusion and must therefore be correctly partitioned between vesicle and organelle membranes1,2,3. Like all other cargo, SNAREs need to be sorted into the forming vesicles by direct interaction with components of the vesicles’ coats. Here we characterize the molecular details governing the sorting of a SNARE into clathrin-coated vesicles, namely the direct recognition of the three-helical bundle Habc domain of the mouse SNARE Vti1b by the human clathrin adaptor epsinR (EPNR, also known as CLINT1). Structures of each domain and of their complex show that this interaction (dissociation constant 22 μM) is mediated by surface patches composed of approximately 15 residues each, the topographies of which are dependent on each domain’s overall fold. Disruption of the interface with point mutations abolishes the interaction in vitro and causes Vti1b to become relocalized to late endosomes and lysosomes. This new class of highly specific, surface–surface interaction between the clathrin coat component and the cargo is distinct from the widely observed binding of short, linear cargo motifs by the assembly polypeptide (AP) complex and GGA adaptors4 and is therefore not vulnerable to competition from standard motif-containing cargoes for incorporation into clathrin-coated vesicles. We propose that conceptually similar but mechanistically different interactions will direct the post-Golgi trafficking of many SNAREs.

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Figure 1: Mapping the binding sites on the Vti1b H abc domain and the EPNR ENTHΔα0 domain on their isolated structures.
Figure 2: The EPNR ENTHΔα0–Vti1b H abc domain complex.
Figure 3: The effect of disrupting the Vti1b–EPNR interaction on the localization of Vti1b in vivo.
Figure 4: Disrupting the EPNR–Vti1b interaction in vivo increases the amount of Vti1b in late endosomes/lysosomes.

References

  1. Hong, W. SNAREs and traffic. Biochim. Biophys. Acta 1744, 493–517 (2005)

    PubMed  Google Scholar 

  2. Fasshauer, D., Sutton, R. B., Brunger, A. T. & Jahn, R. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc. Natl Acad. Sci. USA 95, 15781–15786 (1998)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Bonifacino, J. S. & Traub, L. M. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72, 395–447 (2003)

    Article  CAS  Google Scholar 

  5. Hurley, J. H., Lee, S. & Prag, G. Ubiquitin-binding domains. Biochem. J. 399, 361–372 (2006)

    Article  CAS  Google Scholar 

  6. Peden, A. A., Park, G. Y. & Scheller, R. H. The Di-leucine motif of vesicle-associated membrane protein 4 is required for its localization and AP-1 binding. J. Biol. Chem. 276, 49183–49187 (2001)

    Article  CAS  Google Scholar 

  7. Tran, T. H., Zeng, Q. & Hong, W. VAMP4 cycles from the cell surface to the trans-Golgi network via sorting and recycling endosomes. J. Cell Sci. 120, 1028–1041 (2007)

    Article  CAS  Google Scholar 

  8. Hirst, J., Miller, S. E., Taylor, M. J., von Mollard, G. F. & Robinson, M. S. EpsinR is an adaptor for the SNARE protein Vti1b. Mol. Biol. Cell 15, 5593–5602 (2004)

    Article  CAS  Google Scholar 

  9. Chidambaram, S., Mullers, N., Wiederhold, K., Haucke, V. & von Mollard, G. F. Specific interaction between SNAREs and epsin N-terminal homology (ENTH) domains of epsin-related proteins in trans-Golgi network to endosome transport. J. Biol. Chem. 279, 4175–4179 (2004)

    Article  CAS  Google Scholar 

  10. Nonet, M. L. et al. UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles. Mol. Biol. Cell 10, 2343–2360 (1999)

    Article  CAS  Google Scholar 

  11. Drees, B. L. et al. A protein interaction map for cell polarity development. J. Cell Biol. 154, 549–571 (2001)

    Article  CAS  Google Scholar 

  12. Martinez-Arca, S. et al. A dual mechanism controlling the localization and function of exocytic v-SNAREs. Proc. Natl Acad. Sci. USA 100, 9011–9016 (2003)

    Article  ADS  CAS  Google Scholar 

  13. Pryor, P. R. et al. Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events. EMBO Rep. 5, 590–595 (2004)

    Article  CAS  Google Scholar 

  14. Murray, R. Z., Wylie, F. G., Khromykh, T., Hume, D. A. & Stow, J. L. Syntaxin 6 and Vti1b form a novel SNARE complex, which is up-regulated in activated macrophages to facilitate exocytosis of tumor necrosis factor-α. J. Biol. Chem. 280, 10478–10483 (2005)

    Article  CAS  Google Scholar 

  15. Song, J., Lee, M. H., Lee, G. J., Yoo, C. M. & Hwang, I. Arabidopsis EPSIN1 plays an important role in vacuolar trafficking of soluble cargo proteins in plant cells via interactions with clathrin, AP-1, VTI11, and VSR1. Plant Cell 18, 2258–2274 (2006)

    Article  CAS  Google Scholar 

  16. Mossessova, E., Bickford, L. C. & Goldberg, J. SNARE selectivity of the COPII coat. Cell 114, 483–495 (2003)

    Article  CAS  Google Scholar 

  17. Ford, M. G. et al. Curvature of clathrin-coated pits driven by epsin. Nature 419, 361–366 (2002)

    Article  ADS  CAS  Google Scholar 

  18. Krissinel, E. & Henrick, K. Detection of Protein Assemblies in Crystals (ed. Berthold, M.) (Springer, Berlin/Heidelberg, 2005)

    Book  Google Scholar 

  19. Fridmann-Sirkis, Y., Kent, H. M., Lewis, M. J., Evans, P. R. & Pelham, H. R. Structural analysis of the interaction between the SNARE Tlg1 and Vps51. Traffic 7, 182–190 (2006)

    Article  CAS  Google Scholar 

  20. Munson, M., Chen, X., Cocina, A. E., Schultz, S. M. & Hughson, F. M. Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly. Nature Struct. Biol. 7, 894–902 (2000)

    Article  CAS  Google Scholar 

  21. Lawrence, M. C. & Colman, P. M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, 946–950 (1993)

    Article  CAS  Google Scholar 

  22. Honing, S. et al. Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2. Mol. Cell 18, 519–531 (2005)

    Article  Google Scholar 

  23. Owen, D. J. & Evans, P. R. A structural explanation for the recognition of tyrosine-based endocytotic signals. Science 282, 1327–1332 (1998)

    Article  ADS  CAS  Google Scholar 

  24. Kato, Y., Misra, S., Puertollano, R., Hurley, J. H. & Bonifacino, J. S. Phosphoregulation of sorting signal–VHS domain interactions by a direct electrostatic mechanism. Nature Struct. Biol. 9, 532–536 (2002)

    CAS  PubMed  Google Scholar 

  25. Edeling, M. A., Smith, C. & Owen, D. Life of a clathrin coat: insights from clathrin and AP structures. Nature Rev. Mol. Cell Biol. 7, 32–44 (2006)

    Article  CAS  Google Scholar 

  26. Traub, L. M. Common principles in clathrin-mediated sorting at the Golgi and the plasma membrane. Biochim. Biophys. Acta 1744, 415–437 (2005)

    Article  CAS  Google Scholar 

  27. Di Paolo, G. & De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 443, 651–657 (2006)

    Article  ADS  CAS  Google Scholar 

  28. Mancias, J. D. & Goldberg, J. The transport signal on Sec22 for packaging into COPII-coated vesicles is a conformational epitope. Mol. Cell 26, 403–414 (2007)

    Article  CAS  Google Scholar 

  29. Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L. & Clardy, J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124 (1993)

    Article  CAS  Google Scholar 

  30. Stahelin, R. V. et al. Contrasting membrane interaction mechanisms of AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH) domains. J. Biol. Chem. 278, 28993–28999 (2003)

    Article  CAS  Google Scholar 

  31. Leslie, A. G. W. in Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography No. 26 (SERC, Daresbury Laboratory, Warrington, 1992)

    Google Scholar 

  32. Evans, P. R. Scaling and assessment of data quality. Acta Crystallogr. D 62 72–82. (2006)

    Article  Google Scholar 

  33. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2006)

    Google Scholar 

  34. de la Fortelle, E. & Bricogne, G. in Methods in Enzymology (eds Carter, C. W. Jr & Sweet, R. M.). 472–494 (1997)

    Google Scholar 

  35. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  36. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  37. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C., Adams, P. D. & Read, R. J. PHASER crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  38. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

Download references

Acknowledgements

We thank PX beamline staff at SRS Daresbury and ESRF ID23-1, J. Connell for assistance with microscopy, and P. Luzio and P. Evans for discussions. This work was supported by a Wellcome Trust SRF to D.J.O., a PRF to M.S.R., an MRC studentship to S.E.M. and an Australian NHMRC Career Development Award to B.M.C.

Coordinates have been deposited under PDB codes 2QYW, 2QY7 and 2V8S for Vti1b Habc, epsin ENTH domain and the Vti1b/epsinR complex, respectively.

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Authors and Affiliations

  1. University of Cambridge, CIMR, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK ,

    Sharon E. Miller, Airlie J. McCoy, Margaret S. Robinson & David J. Owen

  2. IMB, University of Queensland, Queensland Bioscience Precinct, Building 80, Services Road, St Lucia, Queensland 4075, Australia ,

    Brett M. Collins

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  1. Sharon E. Miller
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Correspondence to Margaret S. Robinson or David J. Owen.

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The file contains Supplementary Notes, Supplementary Tables S1-S2 and Supplementary Figures S1-S9 with Legends. (PDF 14768 kb)

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Miller, S., Collins, B., McCoy, A. et al. A SNARE–adaptor interaction is a new mode of cargo recognition in clathrin-coated vesicles. Nature 450, 570–574 (2007). https://doi.org/10.1038/nature06353

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