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The Rab5 effector EEA1 is a core component of endosome docking

Nature volume 397, pages 621–625 (1999)Cite this article

Abstract

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.

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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.

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Acknowledgements

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.

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Authors and Affiliations

  1. European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, and Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse, 01307, Dresden, Germany

    Savvas Christoforidis, Heidi M. McBride & Marino Zerial

  2. The Physiological Laboratory, University of Liverpool, Crown Street, L69 3BX, Liverpool, UK

    Robert D. Burgoyne

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  1. Savvas Christoforidis
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Correspondence to Marino Zerial.

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Christoforidis, S., McBride, H., Burgoyne, R. et al. The Rab5 effector EEA1 is a core component of endosome docking. Nature 397, 621–625 (1999). https://doi.org/10.1038/17618

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