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ARF1 is directly involved in dynamin-independent endocytosis

Abstract

Endocytosis of glycosylphosphatidyl inositol (GPI)-anchored proteins (GPI-APs) and the fluid phase takes place primarily through a dynamin- and clathrin-independent, Cdc42-regulated pinocytic mechanism. This mechanism is mediated by primary carriers called clathrin-independent carriers (CLICs), which fuse to form tubular early endocytic compartments called GPI-AP enriched endosomal compartments (GEECs). Here, we show that reduction in activity or levels of ARF1 specifically inhibits GPI-AP and fluid-phase endocytosis without affecting other clathrin-dependent or independent endocytic pathways. ARF1 is activated at distinct sites on the plasma membrane, and by the recruitment of RhoGAP domain-containing protein, ARHGAP10, to the plasma membrane, modulates cell-surface Cdc42 dynamics. This results in the coupling of ARF1 and Cdc42 activity to regulate endocytosis at the plasma membrane. These findings provide a molecular basis for a crosstalk of endocytosis with secretion by the sharing of a key regulator of secretory traffic, ARF1.

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Figure 1: GDP-exchange deficient ARF1 inhibits uptake of GPI-APs and the fluid-phase.
Figure 2: GEEC pathway is inhibited by depletion of ARF1 protein.
Figure 3: RNAi-resistant ARF1 reverts shRNA-mediated inhibition of the GEEC pathway.
Figure 4: Brefeldin A inhibits surface delivery of GPI-APs, but enhances endocytosis via the GEEC pathway.
Figure 5: ARF1 functions via ARHGAP10.
Figure 6: Activated ARF1 is located at the plasma membrane and on fluid-containing nascent endosomes.
Figure 7: ARF1 activation couples to Cdc42 dynamics at plasma membrane.

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References

  1. Conner, S. D. & Schmid, S. L. Regulated portals of entry into the cell. Nature 422, 37–44 (2003).

    Article  CAS  Google Scholar 

  2. Kirkham, M. & Parton, R. G. Clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers. Biochim. Biophys. Acta 1746, 350–363 (2005).

    Article  CAS  Google Scholar 

  3. Mayor, S. & Pagano, R. E. Pathways of clathrin-independent endocytosis. Nature Rev. Mol. Cell Biol. 8, 603–612 (2007).

    Article  CAS  Google Scholar 

  4. 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. Dev. Cell 2, 411–423 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Kirkham, M. et al. Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. J. Cell Biol. 168, 465–476 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kalia, M. et al. Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3′-kinase-dependent machinery. Mol. Biol. Cell 17, 3689–3704 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Guha, A., Sriram, V., Krishnan, K. S. & Mayor, S. Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes. J. Cell Sci. 116, 3373–3386 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Gauthier, N. C. et al. Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes. Mol. Biol. Cell 16, 4852–4866 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chadda, R. et al. Cholesterol-sensitive Cdc42 activation regulates actin polymerization for endocytosis via the GEEC pathway. Traffic 8, 702–717 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Glebov, O. O., Bright, N. A. & Nichols, B. J. Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells. Nature Cell Biol. 8, 46–54 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Bonazzi, M. et al. CtBP3/BARS drives membrane fission in dynamin-independent transport pathways. Nature Cell Biol. 7, 570–580 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Sabharanjak, S. & Mayor, S. Folate receptor endocytosis and trafficking. Adv. Drug Deliv. Rev. 56, 1099–1109 (2004).

    Article  CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

  14. Zhang, C. J. et al. Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo . J. Cell Biol. 124, 289–300 (1994).

    Article  CAS  PubMed  Google Scholar 

  15. Nie, Z., Hirsch, D. S. & Randazzo, P. A. Arf and its many interactors. Curr. Opin. Cell Biol. 15, 396–404 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Naslavsky, N., Weigert, R. & Donaldson, J. G. Convergence of non-clathrin- and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides. Mol. Biol. Cell 14, 417–431 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Tanabe, K. et al. A novel GTPase-activating protein for ARF6 directly interacts with clathrin and regulates clathrin-dependent endocytosis. Mol. Biol. Cell 16, 1617–1628 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Paleotti, O. et al. The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes. J. Biol. Chem. 280, 21661–21666 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Vasudevan, C. et al. The distribution and translocation of the G protein ADP-ribosylation factor 1 in live cells is determined by its GTPase activity. J. Cell Sci. 111, 1277–1285 (1998).

    CAS  PubMed  Google Scholar 

  20. Dascher, C. & Balch, W. E. Dominant inhibitory mutants of ARF1 block endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus. J. Biol. Chem. 269, 1437–1448 (1994).

    CAS  PubMed  Google Scholar 

  21. Lamaze, C. et al. Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway. Mol. Cell 7, 661–671 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Volpicelli-Daley, L. A., Li, Y., Zhang, C. J. & Kahn, R. A. Isoform-selective effects of the depletion of ADP-ribosylation factors 1-5 on membrane traffic. Mol. Biol. Cell 16, 4495–4508 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Macia, E., Chabre, M. & Franco, M. Specificities for the small G proteins ARF1 and ARF6 of the guanine nucleotide exchange factors ARNO and EFA6. J. Biol. Chem. 276, 24925–24930 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Shen, X. et al. Association of brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2) with recycling endosomes during transferrin uptake. Proc. Natl. Acad Sci. USA 103, 2635–2640 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Choudhury, A., Marks, D. L., Proctor, K. M., Gould, G. W. & Pagano, R. E. Regulation of caveolar endocytosis by syntaxin 6-dependent delivery of membrane components to the cell surface. Nature Cell Biol. 8, 317–328 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Ellis, M. A., Miedel, M. T., Guerriero, C. J. & Weisz, O. A. ADP-ribosylation factor 1-independent protein sorting and export from the trans-Golgi network. J. Biol. Chem. 279, 52735–52743 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Yahara, N., Ueda, T., Sato, K. & Nakano, A. Multiple roles of Arf1 GTPase in the yeast exocytic and endocytic pathways. Mol. Biol. Cell 12, 221–238 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Riezman, H. Endocytosis in yeast: several of the yeast secretory mutants are defective in endocytosis. Cell 40, 1001–1009 (1985).

    Article  CAS  PubMed  Google Scholar 

  29. Doms, R. W., Russ, G. & Yewdell, J. W. Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J. Cell Biol. 109, 61–72 (1989).

    Article  CAS  PubMed  Google Scholar 

  30. Donaldson, J. G., Finazzi, D. & Klausner, R. D. Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature 360, 350–352 (1992).

    Article  CAS  PubMed  Google Scholar 

  31. Gu, F. & Gruenberg, J. ARF1 regulates pH-dependent COP functions in the early endocytic pathway. J. Biol. Chem. 275, 8154–8160 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Prydz, K., Hansen, S. H., Sandvig, K. & van Deurs, B. Effects of brefeldin A on endocytosis, transcytosis and transport to the Golgi complex in polarized MDCK cells. J. Cell Biol. 119, 259–272 (1992).

    Article  CAS  PubMed  Google Scholar 

  33. Shin, H. W., Morinaga, N., Noda, M. & Nakayama, K. BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity. Mol. Biol. Cell 15, 5283–5294 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 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  PubMed  Google Scholar 

  35. Sousa, S. et al. ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion. Nature Cell Biol. 7, 954–960 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Hall, A. Rho GTPases and the actin cytoskeleton. Science 279, 509–514 (1998).

    Article  CAS  Google Scholar 

  37. Yang, L., Wang, L. & Zheng, Y. Gene targeting of Cdc42 and Cdc42GAP affirms the critical involvement of Cdc42 in filopodia induction, directed migration, and proliferation in primary mouse embryonic fibroblasts. Mol. Biol. Cell 17, 4675–4685 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Spang, A. ARF1 regulatory factors and COPI vesicle formation. Curr. Opin. Cell Biol. 14, 423–427 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Faundez, V., Horng, J. T. & Kelly, R. B. A function for the AP3 coat complex in synaptic vesicle formation from endosomes. Cell 93, 423–432 (1998).

    Article  CAS  PubMed  Google Scholar 

  40. Beemiller, P., Hoppe, A. D. & Swanson, J. A. A phosphatidylinositol-3-kinase-dependent signal transition regulates ARF1 and ARF6 during Fcγ receptor-mediated phagocytosis. PLoS Biol 4, e162 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Xu, J. & Scheres, B. Dissection of Arabidopsis ADP-RIBOSYLATION FACTOR 1 function in epidermal cell polarity. Plant Cell 17, 525–536 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Teh, O.K. & Moore, I. An Arf–GEF acting at the Golgi and in selective endocytosis in polarized plant cells. Nature 448, 493–496 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Chardin, P. et al. A human exchange factor for Arf contains Sec7- and pleckstrin homology domains. Nature 384, 481–484 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Yang, J. S. et al. Key components of the fission machinery are interchangeable. Nature Cell Biol. 8, 1376–1382 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Erickson, J. W., Zhang, C., Kahn, R. A., Evans, T. & Cerione, R. A. Mammalian Cdc42 is a brefeldin A-sensitive component of the Golgi apparatus. J. Biol. Chem. 271, 26850–26854 (1996).

    Article  CAS  PubMed  Google Scholar 

  46. Kojima, S., Vignjevic, D. & Borisy, G. G. Improved silencing vector co-expressing GFP and small hairpin RNA. Biotechniques 36, 74–79 (2004).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M. Kalia and B. Srinag for help with biochemistry, H. Krishnamurthy and Wellcome Trust-aided imaging and flow-cytometry facility at NCBS for help with confocal imaging and sorting transfected cells. We are indebted to: G. D. Gupta for making ARF6 shRNA; R. Alexander for ARF1 RNAi resistant and mRFP–ABD vectors; N, Sabu for ARHGAP10 shRNA; J. Gruenberg for GFP-tagged wild-type ARF1 and ARF1T31N plasmids; S.G. Ferguson for HA-tagged wild-type ARF1, ARF1T31N and ARF1Q71L constructs; P. Chavrier for GFP–ARHGAP10 domains; R. A. Kahn for ARF1, ARF3, ARF4 and ARF5 shRNA plasmids; R. Vishwakarma for fluorescent folate analogues; S. Bourgoin for ARF6-specific antibodies; and R. E. for C6-LacCer. We thank other members of the Mayor Laboratory and NCBS for generous support and encouragement. S.K. is supported by a pre-doctoral fellowship from Council of Scientific and Industrial Research (Government of India). Work in S.M.'s laboratory is supported by intramural funds from NCBS, and a J. C. Bose fellowship.

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S.K. executed and analysed all experiments. S.K. and S.M. planned all experiments and wrote the manuscript.

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Correspondence to Satyajit Mayor.

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Kumari, S., Mayor, S. ARF1 is directly involved in dynamin-independent endocytosis. Nat Cell Biol 10, 30–41 (2008). https://doi.org/10.1038/ncb1666

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