Skip to main content

Thank you for visiting 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.

  • Letter
  • Published:

The Rab5 effector EEA1 is a core component of endosome docking


Intracellular membrane docking and fusion requires the interplay between soluble factors and SNAREs. The SNARE hypothesis1 postulates that pairing between a vesicular v-SNARE and a target membrane z-SNARE is the primary molecular interaction underlying the specificity of vesicle targeting as well as lipid bilayer fusion. This proposal is supported by recent studies using a minimal artificial system2. However, several observations demonstrate that SNAREs function at multiple transport steps and can pair promiscuously, questioning the role of SNAREs in conveying vesicle targeting3,4,5,6. Moreover, other proteins have been shown to be important in membrane docking or tethering7,8,9. Therefore, if the minimal machinery is defined as the set of proteins sufficient to reproduce in vitro the fidelity of vesicle targeting, docking and fusion as in vivo, then SNAREs are not sufficient to specify vesicle targeting. Endosome fusion also requires cytosolic factors and is regulated by the small GTPase Rab5 (refs 10,11,12,13,14,15,16,17,18,19,20). Here we show that Rab5-interacting soluble proteins can completely substitute for cytosol in an in vivo endosome-fusion assay, and that the Rab5 effector EEA1 is the only factor necessary to confer minimal fusion activity. Rab5 and other associated proteins seem to act upstream of EEA1, implying that Rab5 effectors comprise both regulatory molecules and mechanical components of the membrane transport machinery. We further show that EEA1 mediates endosome docking and, together with SNAREs, leads to membrane fusion.

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

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Twenty-two cytosolic proteins bind to Rab5 and can substitute cytosol in endosome fusion.
Figure 2: Fractionated eluate reveals a core activity of EEA1, regulated by the other effectors.
Figure 3: EEA1-mediated fusion can bypass Rab5 and PtdIns(3)P, but is inhibited by α-SNAP(L294A), ATP-γS and NEM.
Figure 4: Endosome clustering requires EEA1 but not primed SNAREs.

Similar content being viewed by others


  1. Rothman, J. E. Mechanisms of intracellular protein transport. Nature 372, 55–63 (1994).

    Article  ADS  CAS  Google Scholar 

  2. Weber, T. et al. SNAREpins: Minimal machinery for membrane fusion. Cell 92, 759–772 (1998).

    Article  CAS  Google Scholar 

  3. Robinson, L. M. & Martin, T. F. J. Docking and fusion in neurosecretion. Curr. Opin. Cell Biol. 10, 483–492 (1998).

    Article  CAS  Google Scholar 

  4. von Mollard, G. F., Nothwehr, S. F. & Stevens T. H. The yeast v-SNARE Vti1p mediates two vesicle transport pathways through interactions with the t-SNAREs Sed5p and Pep12p. J. Cell Biol. 137, 1511–1524 (1997).

    Article  CAS  Google Scholar 

  5. Holthuis, J. C., Nichols, B. J., Dhruvakumar, S. & Pelham, H. R. Two syntaxin homologues in the TGN/endosomal system of yeast. EMBO J. 17, 113–126 (1998).

    Article  CAS  Google Scholar 

  6. Hay, J. C. et al. Localization, dynamics, and protein interactions reveal distinct roles for ER and Golgi SNAREs. J. Cell Biol. 141, 1489–1502 (1998).

    Article  CAS  Google Scholar 

  7. Sapperstein, S. K., Lupashin, V. V., Schmitt, H. D. & Waters, M. G. Assembly of the ER to Golgi SNARE complex requires Uso1p. J. Cell Biol. 132, 755–767 (1996).

    Article  CAS  Google Scholar 

  8. Cao, X., Ballew, N. & Barlowe, C. Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins. EMBO J. 17, 2156–2165 (1998).

    Article  CAS  Google Scholar 

  9. VanRheenen, S. M., Cao, X., Lupashin, V. V., Barlowe, C. & Waters, G. M. Sec35p, a novel peripheral membrane protein, is required for ER to Golgi vesicle docking. J. Cell Biol. 141, 1107–1119 (1998).

    Article  CAS  Google Scholar 

  10. Novick, P. & Zerial M. The diversity of Rab proteins in vesicle transport. Curr. Opin. Cell Biol. 9, 496–504 (1997).

    Article  CAS  Google Scholar 

  11. Søgaard, M. et al. ARab protein is required for the assembly of SNARE complexes in the docking of transport vesicles. Cell 78, 937–948 (1994).

    Article  Google Scholar 

  12. Lian, J. P., Stone, S., Jiang, Y., Lyons, P. & Ferro-Novick, S. Ypt1p implicated in v-SNARE activation. Nature 372, 698–701 (1994).

    Article  ADS  CAS  Google Scholar 

  13. Mayer, A. & Wickner, W. Docking of yeast vacuoles is catalyzed by the Ras-like GTPase Ypt7p after symmetric priming by Sec18p (NSF). J. Cell Biol. 136, 307–317 (1997).

    Article  CAS  Google Scholar 

  14. Lupashin, V. V. & Waters, M. G. t-SNARE activation through transient interaction with a rab-like guanosine triphosphatase. Science 276, 1255–1258 (1997).

    Article  CAS  Google Scholar 

  15. Gorvel, J. -P., Chavrier, P., Zerial, M. & Gruenberg, J. Rab5 controls early endosome fusion in vitro. Cell 64, 915–925 (1991).

    Article  CAS  Google Scholar 

  16. Horiuchi, H. et al. Anovel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function. Cell 90, 1149–1159 (1997).

    Article  CAS  Google Scholar 

  17. Gournier, H., Stenmark, H., Rybin, V., Lippe, R. & Zerial, M. Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion. EMBO J. 17, 1930–1940 (1998).

    Article  CAS  Google Scholar 

  18. Simonsen, A. et al. EEA1 links phosphatidylinositol 3-kinase function to Rab5 regulation of endosome fusion. Nature 394, 494–498 (1998).

    Article  ADS  CAS  Google Scholar 

  19. Emans, N. et al. Annexin II is a major component of fusogenic endosomal vesicles. J. Cell Biol. 120, 1357–1369 (1993).

    Article  CAS  Google Scholar 

  20. Colombo, M. I., Beron, W. & Stahl, P. D. Calmodulin regulates endosome fusion. J. Biol. Chem. 272, 7707–7712 (1997).

    Article  CAS  Google Scholar 

  21. Rodriguez, L., Stirling, C. J. & Woodman, P. G. Multiple N-ethylmaleimide-sensitive components are required for endosomal vesicle fusion. Mol. Biol. Cell 5, 773–783 (1994).

    Article  CAS  Google Scholar 

  22. Colombo, M. I., Taddese, M., Whiteheart, S. W. & Stahl, P. D. Apossible predocking attachment site for N-ethylmaleimide-sensitive fusion protein. Insights from in vitro endosome fusion. J. Biol. Chem. 271, 18810–18816 (1996).

    Article  CAS  Google Scholar 

  23. Wiedemann, C. & Cockcroft, S. Vesicular transport: sticky fingers grab a lipid. Nature 394, 426–427 (1998).

    Article  ADS  CAS  Google Scholar 

  24. Patki, V. et al. Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase. Proc. Natl Acad. Sci. USA 94, 7326–7330 (1997).

    Article  ADS  CAS  Google Scholar 

  25. Barnard, R. J. O., Morgan, A. & Burgoyne, R. D. Stimulation of NSF ATPase activity by alpha-SNAP is required for SNARE complex disassembly and exocytosis. J. Cell Biol. 139, 875–883 (1997).

    Article  CAS  Google Scholar 

  26. Mayer, A., Wickner, W. & Haas, A. Sec18p (NSF)-driven release of Sec17p (α-SNAP) can precede docking and fusion of yeast vacuoles. Cell 85, 83–94 (1996).

    Article  CAS  Google Scholar 

  27. Sonnichsen, B. et al. Arole for giantin in docking COPI vesicles to Golgi membranes. J. Cell Biol. 140, 1013–1021 (1998).

    Article  CAS  Google Scholar 

  28. Barlowe, C. Coupled ER to golgi transport reconstituted with purified cytosolic proteins. J. Cell Biol. 139, 1097–1108 (1997).

    Article  CAS  Google Scholar 

  29. Mu, F. et al. EEA1, an early endosome-associated protein. J. Biol. Chem. 270, 13503–13511 (1995).

    Article  CAS  Google Scholar 

  30. Stenmark, H., Aasland, R., Toh, B. H. & D'Arringo, A. Endosomal localization of the autoantigen EEA1 is mediated by zinc-binding FYVE finger. J. Biol. Chem. 271, 24048–24054 (1996).

    Article  CAS  Google Scholar 

Download references


We thank R. Lippe for the supply of recombinant Rabaptin-5/Rabex-5 complex; H.Stenmark for the GST–Rab5 cDNA construct; J. Rothman for pQE9 α-SNAP; A. Giner for technical assistance; V. Rybin for helpful discussions on technical points; E. Nielsen and B.Sonnichsen for the supply of rhodamine-labelled early endosomes; P. Scheiffele, B. Sonnichsen and members of the laboratory for discussions and critical reading of the manuscript; and K. Ashman and M. Wilm form providing mass spectroscopy data. S.C. is supported by an EU TMR fellowship. H.M.M. is recipient of an Alexander vonHumboldt Stiftung. This work was supported by the Max Planck Gesellschaft, and by grants from the Human Frontier Science Program, EU TMR and Biomed (to M.Z.). This work is dedicated to the memory of Thomas Kreis.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Marino Zerial.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Christoforidis, S., McBride, H., Burgoyne, R. et al. The Rab5 effector EEA1 is a core component of endosome docking. Nature 397, 621–625 (1999).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


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