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Phosphoinositide-mediated clathrin adaptor progression at the trans-Golgi network

Abstract

Clathrin-coated vesicles mediate endocytosis and transport between the trans-Golgi network (TGN) and endosomes in eukaryotic cells. Clathrin adaptors play central roles in coat assembly, interacting with clathrin, cargo and membranes. Two main types of clathrin adaptor act in TGN–endosome traffic: GGA proteins and the AP-1 complex. Here we characterize the relationship between GGA proteins, AP-1 and other TGN clathrin adaptors using live-cell and super-resolution microscopy in yeast. We present evidence that GGA proteins and AP-1 are recruited sequentially in two waves of coat assembly at the TGN. Mutations that decrease phosphatidylinositol 4-phosphate (PtdIns(4)P) levels at the TGN slow or uncouple AP-1 coat assembly from GGA coat assembly. Conversely, enhanced PtdIns(4)P synthesis shortens the time between adaptor waves. Gga2p binds directly to the TGN PtdIns(4)-kinase Pik1p and contributes to Pik1p recruitment. These results identify a PtdIns(4)P-based mechanism for regulating progressive assembly of adaptor-specific clathrin coats at the TGN.

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Figure 1: Sequential assembly of clathrin adaptors.
Figure 2: Spatial relationships of clathrin adaptors by SIM.
Figure 3: Adaptor and clathrin dynamics at the TGN.
Figure 4: TGN PtdIns(4)P dynamics depend on Arf1p and Gga proteins.
Figure 5: Depletion of PtdIns(4)P alters localization of AP-1 and Ent5p.
Figure 6: Pik1p/Frq1p overexpression enhances co-localization of sequentially recruited clathrin adaptors with functional consequences.
Figure 7: Gga2p acts in Pik1p recruitment and binds Pik1p.

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References

  1. 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  PubMed  Google Scholar 

  2. Duncan, M. C. & Payne, G. S. ENTH/ANTH domains expand to the Golgi. Trends Cell Biol. 13, 211–215 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Black, M. W. & Pelham, H. R. B. A selective transport route from Golgito late endosomes that requires the yeast GGA proteins. J. Cell Biol. 151, 587–600 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Costaguta, G., Duncan, M. C., Fernandez, G. E., Huang, G. H. & Payne, G. S. Distinct roles for TGN/endosome epsin-like adaptors Ent3p and Ent5p. Mol. Biol. Cell 17, 3907–3920 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Costaguta, G., Stefan, C. J., Bensen, E. S., Emr, S. D. & Payne, G. S. Yeast Gga coat proteins function with clathrin in Golgi to endosome transport. Mol. Biol. Cell 12, 1885–1896 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Duncan, M. C., Costaguta, G. & Payne, G. S. Yeast epsin-related proteins required for Golgi-endosome traffic define a γ-adaptin ear-binding motif. Nat. Cell Biol. 5, 77–81 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Fernandez, G. E. & Payne, G. S. Laa1p, a conserved AP-1 accessory protein important for AP-1 localization in yeast. Mol. Biol. Cell 17, 3304–3317 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ha, S. A. et al. The synaptojanin-like protein Inp53/Sjl3 functions with clathrin in a yeast TGN-to-endosome pathway distinct from the GGA protein-dependent pathway. Mol. Biol. Cell 14, 1319–1333 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Graham, T. R. & Burd, C. G. Coordination of Golgi functions by phosphatidylinositol 4-kinases. Trends Cell Biol. 21, 113–121 (2011).

    Article  CAS  PubMed  Google Scholar 

  10. Boman, A. L. et al. ADP-ribosylation factor (ARF) interaction is not sufficient for yeast GGA protein function or localization. Mol. Biol. Cell 13, 3078–3095 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Newpher, T. M., Smith, R. P., Lemmon, V. & Lemmon, S. K. In vivo dynamics of clathrin and its adaptor-dependent recruitment to the actin-based endocytic machinery in yeast. Dev. Cell 9, 87–98 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Schermelleh, L. et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 1332–1336 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Franzusoff, A., Redding, K., Crosby, J., Fuller, R. S. & Schekman, R. Localization of components involved in protein transport and processing through the yeast Golgi apparatus. J. Cell Biol. 112, 27–37 (1991).

    Article  CAS  PubMed  Google Scholar 

  14. Rossanese, O. W. et al. A role for actin, Cdc1p, and Myo2p in the inheritance of late Golgi elements in Saccharomyces cerevisiae. J. Cell Biol. 153, 47–62 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Losev, E. et al. Golgi maturation visualized in living yeast. Nature 441, 1002–1006 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Stearns, T., Kahn, R. A., Botstein, D. & Hoyt, M. A. ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes. Mol. Cell Biol. 10, 6690–6699 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Gaynor, E. C., Chen, C. Y., Emr, S. D. & Graham, T. R. ARF is required for maintenance of yeast Golgi and endosome structure and function. Mol. Biol. Cell 9, 653–670 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wang, J. et al. PI4P promotes the recruitment of the GGA adaptor proteins to the trans-Golgi network and regulates their recognition of the ubiquitin sorting signal. Mol. Biol. Cell 18, 2646–2655 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wang, Y. J. et al. Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi. Cell 114, 299–310 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Demmel, L. et al. The clathrin adaptor Gga2p is a phosphatidylinositol 4-phosphate effector at the Golgi exit. Mol. Biol. Cell 19, 1991–2002 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Loewen, C. J., Roy, A. & Levine, T. P. A conserved ER targeting motif in three families of lipid binding proteins and in Opi1p binds VAP. EMBO J. 22, 2025–2035 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Audhya, A., Foti, M. & Emr, S. D. Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics. Mol. Biol. Cell 11, 2673–2689 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Walch-Solimena, C. & Novick, P. The yeast phosphatidylinositol-4-OH kinase pik1 regulates secretion at the Golgi. Nat. Cell Biol. 1, 523–525 (1999).

    Article  CAS  PubMed  Google Scholar 

  24. Strahl, T., Hama, H., DeWald, D. B. & Thorner, J. Yeast phosphatidylinositol 4-kinase, Pik1, has essential roles at the Golgi and in the nucleus. J. Cell Biol. 171, 967–979 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Schu, P. V. et al. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science 260, 88–91 (1993).

    Article  CAS  PubMed  Google Scholar 

  26. Hendricks, K. B., Wang, B. Q., Schnieders, E. A. & Thorner, J. Yeast homologue of neuronal frequenin is a regulator of phosphatidylinositol-4-OH kinase. Nat. Cell Biol. 1, 234–241 (1999).

    Article  CAS  PubMed  Google Scholar 

  27. Fuller, R. S., Sterne, R. E. & Thorner, J. Enzymes required for yeast prohormone processing. Annu. Rev. Physiol. 50, 345–362 (1988).

    Article  CAS  PubMed  Google Scholar 

  28. Payne, G. S. & Schekman, R. Clathrin—a role in the intracellular retention of a Golgi membrane protein. Science 245, 1358–1365 (1989).

    Article  CAS  PubMed  Google Scholar 

  29. Phan, H. L. et al. The Saccharomyces cerevisiae APS1 gene encodes a homolog of the small subunit of the mammalian clathrin AP-1 complex: evidence for functional interaction with clathrin at the Golgi complex. EMBO J. 13, 1706–1717 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Dean, N. Asparagine-linked glycosylation in the yeast Golgi. Biochim. Biophys. Acta 1426, 309–322 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Graham, T. R., Seeger, M., Payne, G. S., MacKay, V. L. & Emr, S. D. Clathrin-dependent localization of α1,3 mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae. J. Cell Biol. 127, 667–678 (1994).

    Article  CAS  PubMed  Google Scholar 

  32. Black, M. W. & Pelham, H. R. A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins. J. Cell Biol. 151, 587–600 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chidambaram, S., Zimmermann, J. & von Mollard, G. F. ENTH domain proteins are cargo adaptors for multiple SNARE proteins at the TGN endosome. J. Cell Sci. 121, 329–338 (2008).

    Article  CAS  PubMed  Google Scholar 

  34. Wang, J. et al. Epsin N-terminal homology domains bind on opposite sides of two SNAREs. Proc. Natl Acad. Sci. USA 108, 12277–12282 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Doray, B., Ghosh, P., Griffith, J., Geuze, H. J. & Kornfeld, S. Cooperation of GGAs and AP-1 in packaging MPRs at the trans-Golgi network. Science 297, 1700–1703 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. 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  PubMed  Google Scholar 

  37. Eugster, A. et al. Ent5p is required with Ent3p and Vps27p for ubiquitin-dependent protein sorting into the multivesicular body. Mol. Biol. Cell 15, 3031–3041 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Friant, S. et al. Ent3p is a PtdIns(3,5)P2 effector required for protein sorting to the multivesicular body. Dev. Cell 5, 499–511 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Narayan, K. & Lemmon, M. A. Determining selectivity of phosphoinositide-binding domains. Methods 39, 122–133 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Singer-Kruger, B. et al. Yeast and human Ysl2p/hMon2 interact with Gga adaptors and mediate their subcellular distribution. EMBO J. 27, 1423–1435 (2008).

    PubMed  PubMed Central  Google Scholar 

  41. Hirst, J. et al. A family of proteins with γ-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome. J. Cell Biol. 149, 67–80 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Poussu, A., Lohi, O. & Lehto, V. P. Vear, a novel Golgi-associated protein with VHS and γ-adaptin ‘ear’ domains. J. Biol. Chem. 275, 7176–7183 (2000).

    Article  CAS  PubMed  Google Scholar 

  43. Zoncu, R. et al. A phosphoinositide switch controls the maturation and signaling properties of APPL endosomes. Cell 136, 1110–1121 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Robinson, J. S., Klionsky, D. J., Banta, L. M. & Emr, S. D. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol. Cell Biol. 8, 4936–4948 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Yeung, B. G., Phan, H. L. & Payne, G. S. Adaptor complex-independent clathrin function in yeast. Mol. Biol. Cell 10, 3643–3659 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Janke, C. et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962 (2004).

    Article  CAS  PubMed  Google Scholar 

  47. Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

    Article  CAS  PubMed  Google Scholar 

  48. Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).

    Article  CAS  PubMed  Google Scholar 

  49. Burd, C. G. & Emr, S. D. Phosphatidylinositol(3)-phosphate signaling mediated by specific binding to RING FYVE domains. Mol. Cell 2, 157–162 (1998).

    Article  CAS  PubMed  Google Scholar 

  50. Sikorski, R. S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Vowels, J. J. & Payne, G. S. A role for the lumenal domain in Golgi localization of the Saccharomyces cerevisiae guanosine diphosphatase. Mol. Biol. Cell 9, 1351–1365 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank J.Thorner, S. Emr and T. Levine for plasmids and strains, J. Atkins for help with SIM, and members of the laboratory for helpful discussions. We are especially grateful to K. Kilborn for advice and assistance with confocal microscopy and K. Martin, G. Weinmaster and A. van der Bliek for comments on the manuscript. This work was supported by NIH NRSA T32 GM-007104 and a UCLA Dissertation Year Fellowship to L.D. and NIH GM39040 to G.P.

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L.D. and G.S.P. conceived the experiments. L.D. carried out all microscopy, protein interaction and CPY experiments. G.C. carried out the α-factor experiments, and generated recombinant expression and integration constructs. L.D. and G.S.P. wrote the manuscript.

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Correspondence to Gregory S. Payne.

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Daboussi, L., Costaguta, G. & Payne, G. Phosphoinositide-mediated clathrin adaptor progression at the trans-Golgi network. Nat Cell Biol 14, 239–248 (2012). https://doi.org/10.1038/ncb2427

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