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Protein complexes containing CYFIP/Sra/PIR121 coordinate Arf1 and Rac1 signalling during clathrin–AP-1-coated carrier biogenesis at the TGN

Nature Cell Biology volume 12, pages 330–340 (2010)Cite this article

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A Corrigendum to this article was published on 01 May 2010

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Abstract

Actin dynamics is a tightly regulated process involved in various cellular events including biogenesis of clathrin-coated, AP-1 (adaptor protein 1)-coated transport carriers connecting the trans-Golgi network (TGN) and the endocytic pathway. However, the mechanisms coordinating coat assembly, membrane and actin remodelling during post-TGN transport remain poorly understood. Here we show that the Arf1 (ADP-ribosylation factor 1) GTPase synchronizes the TGN association of clathrin–AP-1 coats and protein complexes comprising CYFIP (cytoplasmic fragile-X mental retardation interacting protein; Sra, PIR121), a clathrin heavy chain binding protein associated with mental retardation. The Rac1 GTPase and its exchange factor β-PIX (PAK-interacting exchange factor) activate these complexes, allowing N-WASP-dependent and Arp2/3-dependent actin polymerization towards membranes, thus promoting tubule formation. These phenomena can be recapitulated with synthetic membranes. This protein-network-based mechanism facilitates the sequential coordination of Arf1-dependent membrane priming, through the recruitment of coats and CYFIP-containing complexes, and of Rac1-dependent actin polymerization, and provides complementary but independent levels of regulation during early stages of clathrin–AP1-coated carrier biogenesis.

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Figure 1: Localization of CYFIP1 and CYFIP2.
Figure 2: CYFIP1 and CYFIP2 interact with CHC, and CYFIP1 recruitment to the TGN is regulated by Arf1.
Figure 3: Rac1 and β-PIX control the recruitment of p21Arc but not CYFIP1 to the TGN.
Figure 4: CYFIP2 depletion disrupts organelle integrity and decreases transport carrier biogenesis.
Figure 5: Reconstitution of clathrin–AP-1-coated carrier biogenesis on model membranes.
Figure 6: Model of clathrin–AP-1-coated carrier biogenesis.

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  • 22 March 2010

    In the version of this article initially published online, 'LAMP-1' was incorrectly written as 'LAMP-2'. The definition of AP-1 has been corrected to 'adaptor protein 1'. An additional sentence has been added to the acknowledgements. These errors have been corrected in both the HTML and PDF versions of the article.

References

  1. Ghosh, P., Dahms, N. M. & Kornfeld, S. Mannose 6-phosphate receptors: new twists in the tale. Nature Rev. Mol. Cell Biol. 4, 202–212 (2003).

    Article  CAS  Google Scholar 

  2. 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 

  3. Bonifacino, J. S. & Glick, B. S. The mechanisms of vesicle budding and fusion. Cell 116, 153–166 (2004).

    Article  CAS  Google Scholar 

  4. Godi, A. et al. ARF mediates recruitment of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex. Nature Cell Biol. 1, 280–287 (1999).

    Article  CAS  Google Scholar 

  5. D'Souza-Schorey, C. & Chavrier, P. ARF proteins: roles in membrane traffic and beyond. Nature Rev. Mol. Cell Biol. 7, 347–358 (2006).

    Article  CAS  Google Scholar 

  6. Lee, I., Doray, B., Govero, J. & Kornfeld, S. Binding of cargo sorting signals to AP-1 enhances its association with ADP ribosylation factor 1-GTP. J. Cell Biol. 180, 467–472 (2008).

    Article  CAS  Google Scholar 

  7. Le Borgne, R. & Hoflack, B. Mannose 6-phosphate receptors regulate the formation of clathrin-coated vesicles in the TGN. J. Cell Biol. 137, 335–345 (1997).

    Article  CAS  Google Scholar 

  8. Baust, T., Czupalla, C., Krause, E., Bourel-Bonnet, L. & Hoflack, B. Proteomic analysis of adaptor protein 1A coats selectively assembled on liposomes. Proc. Natl Acad. Sci. USA 103, 3159–3164 (2006).

    Article  CAS  Google Scholar 

  9. Pelish, H. E. et al. Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro. Nature Chem. Biol. 2, 39–46 (2006).

    CAS  Google Scholar 

  10. Salvarezza, S. B. et al. LIM kinase 1 and cofilin regulate actin filament population required for dynamin-dependent apical carrier fission from the trans-Golgi network. Mol. Biol. Cell 20, 438–451 (2009).

    Article  CAS  Google Scholar 

  11. Egea, G., Lazaro-Dieguez, F. & Vilella, M. Actin dynamics at the Golgi complex in mammalian cells. Curr. Opin. Cell Biol. 18, 168–178 (2006).

    Article  CAS  Google Scholar 

  12. Kondylis, V., van Nispen tot Pannerden, H. E., Herpers, B., Friggi-Grelin, F. & Rabouille, C. The golgi comprises a paired stack that is separated at G2 by modulation of the actin cytoskeleton through Abi and Scar/WAVE. Dev. Cell 12, 901–915 (2007).

    CAS  Google Scholar 

  13. Carreno, S., Engqvist-Goldstein, A. E., Zhang, C. X., McDonald, K. L. & Drubin, D. G. Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network. J. Cell Biol. 165, 781–788 (2004).

    Article  CAS  Google Scholar 

  14. Cao, H. et al. Actin and Arf1-dependent recruitment of a cortactin–dynamin complex to the Golgi regulates post-Golgi transport. Nature Cell Biol. 7, 483–492 (2005).

    Article  CAS  Google Scholar 

  15. Engqvist-Goldstein, A. E. et al. RNAi-mediated Hip1R silencing results in stable association between the endocytic machinery and the actin assembly machinery. Mol. Biol. Cell 15, 1666–1679 (2004).

    Article  CAS  Google Scholar 

  16. Kaksonen, M., Toret, C. P. & Drubin, D. G. Harnessing actin dynamics for clathrin-mediated endocytosis. Nature Rev. Mol. Cell Biol. 7, 404–414 (2006).

    Article  CAS  Google Scholar 

  17. McNiven, M. A. & Thompson, H. M. Vesicle formation at the plasma membrane and trans-Golgi network: the same but different. Science 313, 1591–1594 (2006).

    Article  CAS  Google Scholar 

  18. Welch, M. D., Iwamatsu, A. & Mitchison, T. J. Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes. Nature 385, 265–269 (1997).

    Article  CAS  Google Scholar 

  19. Takenawa, T. & Suetsugu, S. The WASP–WAVE protein network: connecting the membrane to the cytoskeleton. Nature Rev. Mol. Cell Biol. 8, 37–48 (2007).

    Article  CAS  Google Scholar 

  20. Campellone, K. G., Webb, N. J., Znameroski, E. A. & Welch, M. D. WHAMM is an Arp2/3 complex activator that binds microtubules and functions in ER to Golgi transport. Cell 134, 148–161 (2008).

    Article  CAS  Google Scholar 

  21. Schenck, A. et al. CYFIP/Sra-1 controls neuronal connectivity in Drosophila and links the Rac1 GTPase pathway to the fragile X protein. Neuron 38, 887–898 (2003).

    Article  CAS  Google Scholar 

  22. Eden, S., Rohatgi, R., Podtelejnikov, A. V., Mann, M. & Kirschner, M. W. Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 418, 790–793 (2002).

    Article  CAS  Google Scholar 

  23. Kunda, P., Craig, G., Dominguez, V. & Baum, B. Abi, Sra1, and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions. Curr. Biol. 13, 1867–1875 (2003).

    Article  CAS  Google Scholar 

  24. Steffen, A. et al. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J. 23, 749–759 (2004).

    Article  CAS  Google Scholar 

  25. Gautreau, A. et al. Purification and architecture of the ubiquitous Wave complex. Proc. Natl Acad. Sci. USA 101, 4379–4383 (2004).

    Article  CAS  Google Scholar 

  26. Bogdan, S., Stephan, R., Lobke, C., Mertens, A. & Klambt, C. Abi activates WASP to promote sensory organ development. Nature Cell Biol. 7, 977–984 (2005).

    Article  CAS  Google Scholar 

  27. Innocenti, M. et al. Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes. Nature Cell Biol. 7, 969–976 (2005).

    Article  CAS  Google Scholar 

  28. Schenck, A., Bardoni, B., Moro, A., Bagni, C. & Mandel, J. L. A highly conserved protein family interacting with the fragile X mental retardation protein (FMRP) and displaying selective interactions with FMRP-related proteins FXR1P and FXR2P. Proc. Natl Acad. Sci. USA 98, 8844–8849 (2001).

    Article  CAS  Google Scholar 

  29. Waguri, S. et al. Visualization of TGN to endosome trafficking through fluorescently labeled MPR and AP-1 in living cells. Mol. Biol. Cell 14, 142–155 (2003).

    Article  CAS  Google Scholar 

  30. Folsch, H., Pypaert, M., Maday, S., Pelletier, L. & Mellman, I. The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains. J. Cell Biol. 163, 351–362 (2003).

    Article  Google Scholar 

  31. Dubois, T. et al. Golgi-localized GAP for Cdc42 functions downstream of ARF1 to control Arp2/3 complex and F-actin dynamics. Nature Cell Biol. 7, 353–364 (2005).

    Article  CAS  Google Scholar 

  32. Fucini, R. V., Chen, J. L., Sharma, C., Kessels, M. M. & Stamnes, M. Golgi vesicle proteins are linked to the assembly of an actin complex defined by mAbp1. Mol. Biol. Cell 13, 621–631 (2002).

    Article  CAS  Google Scholar 

  33. Kobayashi, K. et al. p140Sra-1 (specifically Rac1-associated protein) is a novel specific target for Rac1 small GTPase. J. Biol. Chem. 273, 291–295 (1998).

    Article  CAS  Google Scholar 

  34. Poupon, V. et al. Clathrin light chains function in mannose phosphate receptor trafficking via regulation of actin assembly. Proc. Natl Acad. Sci. USA 105, 168–173 (2008).

    Article  CAS  Google Scholar 

  35. Kraynov, V. S. et al. Localized Rac activation dynamics visualized in living cells. Science 290, 333–337 (2000).

    Article  CAS  Google Scholar 

  36. Faucherre, A. et al. Lowe syndrome protein OCRL1 interacts with Rac GTPase in the trans-Golgi network. Hum. Mol. Genet. 12, 2449–2456 (2003).

    Article  CAS  Google Scholar 

  37. Jou, T. S. et al. Selective alterations in biosynthetic and endocytic protein traffic in Madin–Darby canine kidney epithelial cells expressing mutants of the small GTPase Rac1. Mol. Biol. Cell 11, 287–304 (2000).

    Article  CAS  Google Scholar 

  38. Premont, R. T. et al. The GIT/PIX complex: an oligomeric assembly of GIT family ARF GTPase-activating proteins and PIX family Rac1/Cdc42 guanine nucleotide exchange factors. Cell Signal. 16, 1001–1011 (2004).

    Article  CAS  Google Scholar 

  39. Kroschewski, R., Hall, A. & Mellman, I. Cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of MDCK cells. Nature Cell Biol. 1, 8–13 (1999).

    Article  CAS  Google Scholar 

  40. Musch, A., Cohen, D., Kreitzer, G. & Rodriguez-Boulan, E. cdc42 regulates the exit of apical and basolateral proteins from the trans-Golgi network. EMBO J. 20, 2171–2179 (2001).

    Article  CAS  Google Scholar 

  41. Egorov, M. V. et al. Faciogenital dysplasia protein (FGD1) regulates export of cargo proteins from the golgi complex via Cdc42 activation. Mol. Biol. Cell 20, 2413–2427 (2009).

    Article  CAS  Google Scholar 

  42. McNiven, M. A. et al. Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape. J. Cell Biol. 151, 187–198 (2000).

    Article  CAS  Google Scholar 

  43. Dawson, J. C., Legg, J. A. & Machesky, L. M. Bar domain proteins: a role in tubulation, scission and actin assembly in clathrin-mediated endocytosis. Trends Cell Biol 16, 493–498 (2006).

    CAS  Google Scholar 

  44. Merrifield, C. J., Feldman, M. E., Wan, L. & Almers, W. Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nature Cell Biol. 4, 691–698 (2002).

    Article  CAS  Google Scholar 

  45. Merrifield, C. J., Qualmann, B., Kessels, M. M. & Almers, W. Neural Wiskott Aldrich syndrome protein (N-WASP) and the Arp2/3 complex are recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts. Eur. J. Cell Biol. 83, 13–18 (2004).

    Article  CAS  Google Scholar 

  46. Merrifield, C. J., Perrais, D. & Zenisek, D. Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 121, 593–606 (2005).

    Article  CAS  Google Scholar 

  47. Schenck, A. et al. WAVE/SCAR, a multifunctional complex coordinating different aspects of neuronal connectivity. Dev. Biol. 274, 260–270 (2004).

    Article  CAS  Google Scholar 

  48. Borck, G. et al. Clinical, cellular, and neuropathological consequences of AP1S2 mutations: further delineation of a recognizable X-linked mental retardation syndrome. Hum. Mutat. 29, 966–974 (2008).

    Article  CAS  Google Scholar 

  49. Tarpey, P. S. et al. Mutations in the gene encoding the Sigma 2 subunit of the adaptor protein 1 complex, AP1S2, cause X-linked mental retardation. Am. J. Hum. Genet. 79, 1119–1124 (2006).

    Article  CAS  Google Scholar 

  50. Allen, K. M. et al. PAK3 mutation in nonsyndromic X-linked mental retardation. Nature Genet. 20, 25–30 (1998).

    Article  CAS  Google Scholar 

  51. Nowicki, S. T. et al. The Prader–Willi phenotype of fragile X syndrome. J. Dev. Behav. Pediatr. 28, 133–138 (2007).

    Google Scholar 

  52. Dwyer, N. D., Adler, C. E., Crump, J. G., L'Etoile, N. D. & Bargmann, C. I. Polarized dendritic transport and the AP-1 mu1 clathrin adaptor UNC-101 localize odorant receptors to olfactory cilia. Neuron 31, 277–287 (2001).

    Article  CAS  Google Scholar 

  53. Sakaguchi-Nakashima, A., Meir, J. Y., Jin, Y., Matsumoto, K. & Hisamoto, N. LRK-1, a C. elegans PARK8-related kinase, regulates axonal-dendritic polarity of SV proteins. Curr. Biol. 17, 592–598 (2007).

    Article  CAS  Google Scholar 

  54. Nakamoto, M. et al. Fragile X mental retardation protein deficiency leads to excessive mGluR5-dependent internalization of AMPA receptors. Proc. Natl Acad. Sci. USA 104, 15537–15542 (2007).

    Article  CAS  Google Scholar 

  55. Wang, H. et al. FMRP acts as a key messenger for dopamine modulation in the forebrain. Neuron 59, 634–647 (2008).

    Article  CAS  Google Scholar 

  56. Park, M., Penick, E. C., Edwards, J. G., Kauer, J. A. & Ehlers, M. D. Recycling endosomes supply AMPA receptors for LTP. Science 305, 1972–1875 (2004).

    Article  CAS  Google Scholar 

  57. Correia, S. S. et al. Motor protein-dependent transport of AMPA receptors into spines during long-term potentiation. Nature Neurosci. 11, 457–466 (2008).

    Article  CAS  Google Scholar 

  58. Margeta, M. A., Wang, G. J. & Shen, K. Clathrin adaptor AP-1 complex excludes multiple postsynaptic receptors from axons in C. elegans. Proc. Natl Acad. Sci. USA 106, 1632–1637 (2009).

    Article  CAS  Google Scholar 

  59. Ehrlich, M. et al. Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell 118, 591–605 (2004).

    Article  CAS  Google Scholar 

  60. Ludwig, T. et al. Differential sorting of lysosomal enzymes in mannose 6-phosphate receptor-deficient fibroblasts. EMBO J. 13, 3430–3437 (1994).

    Article  CAS  Google Scholar 

  61. Baust, T. et al. Protein networks supporting AP-3 function in targeting lysosomal membrane proteins. Mol. Biol. Cell 19, 1942–1951 (2008).

    Article  CAS  Google Scholar 

  62. Bacia, K., Schuette, C. G., Kahya, N., Jahn, R. & Schwille, P. SNAREs prefer liquid-disordered over 'raft' (liquid-ordered) domains when reconstituted into giant unilamellar vesicles. J. Biol. Chem. 279, 37951–37955 (2004).

    Article  CAS  Google Scholar 

  63. Gao, Y., Dickerson, J. B., Guo, F., Zheng, J. & Zheng, Y. Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc. Natl Acad. Sci. USA 101, 7618–7623 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the members of the different laboratories for their helpful discussions and critical comments. D. Thiel provided technical assistance. We thank M. Zerial, T. Wassmer and C. Antos for their critical reading of the manuscript. This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (TRR 13/1-2010, HO 2584/1-1, HO 2584/2-1, HO 2584/6-1, HO 2584/8-1, HO 2584/9-1), the German Ministry of Research (BMBF) (0313815B) and Dresden University of Technology (HWP-1207). T. Kirchhausen's research was supported by the NIH grant GM075252.

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Authors and Affiliations

  1. Biotechnology Center, Dresden University of Technology, Tatzberg 47-51, 01307, Dresden, Germany

    Mihaela Anitei, Christoph Stange, Irina Parshina, Thorsten Baust & Bernard Hoflack

  2. Harvard Medical School/CBRI, W. Alpert Building Room 128, 200 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Thorsten Baust & Tomas Kirchhausen

  3. Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Donders Institute for Brain, Cognition and Behaviour & Radboud University Nijmegen Medical Center, Geert Grooteplein 10, 6525 GA, Nijmegen, The Netherlands

    Annette Schenck

  4. Institut Curie, Centre de Recherche, Paris, F-75248, France

    Graça Raposo

  5. CNRS, UMR144, Paris, F-75248, France

    Graça Raposo

  6. Cell and Tissue Imaging facility (IBiSA), Paris, F-75248, France

    Graça Raposo

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  1. Mihaela Anitei
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Contributions

M.A. designed, performed and analysed the in vivo experiments. C.S. designed, performed and analysed the in vitro recruitment experiments. I.P. performed the quantitative real-time PCR experiments and yeast two-hybrid analyses. G.R. performed the electron microscopy. T.B., A.S. and T.K. provided key reagents. B.H. designed and analysed experiments, and wrote the manuscript with M.A. and C.S.

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Correspondence to Bernard Hoflack.

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Anitei, M., Stange, C., Parshina, I. et al. Protein complexes containing CYFIP/Sra/PIR121 coordinate Arf1 and Rac1 signalling during clathrin–AP-1-coated carrier biogenesis at the TGN. Nat Cell Biol 12, 330–340 (2010). https://doi.org/10.1038/ncb2034

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