Reconstitution of Rab- and SNARE-dependent membrane fusion by synthetic endosomes


Rab GTPases and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are evolutionarily conserved essential components of the eukaryotic intracellular transport system. Although pairing of cognate SNAREs is sufficient to fuse membranes in vitro, a complete reconstitution of the Rab–SNARE machinery has never been achieved. Here we report the reconstitution of the early endosomal canine Rab5 GTPase, its key regulators and effectors together with SNAREs into proteoliposomes using a set of 17 recombinant human proteins. These vesicles behave like minimal ‘synthetic’ endosomes, fusing with purified early endosomes or with each other in vitro. Membrane fusion measured by content-mixing and morphological assays requires the cooperativity between Rab5 effectors and cognate SNAREs which, together, form a more efficient ‘core machinery’ than SNAREs alone. In reconstituting a fusion mechanism dependent on both a Rab GTPase and SNAREs, our work shows that the two machineries act coordinately to increase the specificity and efficiency of the membrane tethering and fusion process.

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Figure 1: Rab5 effectors and SNARE priming factors can substitute cytosol in the homotypic fusion between early endosomes in vitro.
Figure 2: Recruitment of Rab5, EEA1 and rabenosyn-5 on proteoliposomes.
Figure 3: The Rab5 machinery and SNAREs cooperatively promote membrane fusion.
Figure 4: Molecular requirements for membrane fusion.
Figure 5: Electron microscopic analysis of proteoliposomes stained with uranyl acetate (a–d) or ammonium molybdenum (e).


  1. 1

    Pfeffer, S. R. Transport-vesicle targeting: tethers before SNAREs. Nature Cell Biol. 1, E17–E22 (1999)

    CAS  Article  Google Scholar 

  2. 2

    Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature Rev. Mol. Cell Biol. 2, 107–117 (2001)

    CAS  Article  Google Scholar 

  3. 3

    Grosshans, B. L., Ortiz, D. & Novick, P. Rabs and their effectors: achieving specificity in membrane traffic. Proc. Natl Acad. Sci. USA 103, 11821–11827 (2006)

    CAS  Article  ADS  Google Scholar 

  4. 4

    Jahn, R. & Scheller, R. H. SNAREs—engines for membrane fusion. Nature Rev. Mol. Cell Biol. 7, 631–643 (2006)

    CAS  Article  Google Scholar 

  5. 5

    McNew, J. A. et al. Compartmental specificity of cellular membrane fusion encoded in SNARE proteins. Nature 407, 153–159 (2000)

    CAS  Article  ADS  Google Scholar 

  6. 6

    Rothman, J. E. & Sollner, T. H. Throttles and dampers: controlling the engine of membrane fusion. Science 276, 1212–1213 (1997)

    CAS  Article  Google Scholar 

  7. 7

    Cai, H., Reinisch, K. & Ferro-Novick, S. Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. Dev. Cell 12, 671–682 (2007)

    CAS  Article  Google Scholar 

  8. 8

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

    CAS  Article  ADS  Google Scholar 

  9. 9

    Wang, L., Merz, A. J., Collins, K. M. & Wickner, W. Hierarchy of protein assembly at the vertex ring domain for yeast vacuole docking and fusion. J. Cell Biol. 160, 365–374 (2003)

    CAS  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    Wickner, W. & Schekman, R. Membrane fusion. Nature Struct. Mol. Biol. 15, 658–664 (2008)

    CAS  Article  Google Scholar 

  12. 12

    Zimmerberg, J. & Gawrisch, K. The physical chemistry of biological membranes. Nature Chem. Biol. 2, 564–567 (2006)

    CAS  Article  Google Scholar 

  13. 13

    Schuette, C. G. et al. Determinants of liposome fusion mediated by synaptic SNARE proteins. Proc. Natl Acad. Sci. USA 101, 2858–2863 (2004)

    CAS  Article  ADS  Google Scholar 

  14. 14

    Fix, M. et al. Imaging single membrane fusion events mediated by SNARE proteins. Proc. Natl Acad. Sci. USA 101, 7311–7316 (2004)

    CAS  Article  ADS  Google Scholar 

  15. 15

    Liu, T. et al. SNARE-driven, 25-millisecond vesicle fusion in vitro . Biophys. J. 89, 2458–2472 (2005)

    CAS  Article  ADS  Google Scholar 

  16. 16

    Parlati, F. et al. Rapid and efficient fusion of phospholipid vesicles by the alpha-helical core of a SNARE complex in the absence of an N-terminal regulatory domain. Proc. Natl Acad. Sci. USA 96, 12565–12570 (1999)

    CAS  Article  ADS  Google Scholar 

  17. 17

    Pobbati, A. V., Stein, A. & Fasshauer, D. N- to C-terminal SNARE complex assembly promotes rapid membrane fusion. Science 313, 673–676 (2006)

    CAS  Article  ADS  Google Scholar 

  18. 18

    Brandhorst, D. et al. Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity. Proc. Natl Acad. Sci. USA 103, 2701–2706 (2006)

    CAS  Article  ADS  Google Scholar 

  19. 19

    Sonnichsen, B. et al. Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J. Cell Biol. 149, 901–914 (2000)

    CAS  Article  Google Scholar 

  20. 20

    Barbero, P., Bittova, L. & Pfeffer, S. R. Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells. J. Cell Biol. 156, 511–518 (2002)

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

    Christoforidis, S. et al. Phosphatidylinositol-3-OH kinases are Rab5 effectors. Nature Cell Biol. 1, 249–252 (1999)

    CAS  Article  Google Scholar 

  23. 23

    Shin, H. W. et al. An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway. J. Cell Biol. 170, 607–618 (2005)

    CAS  Article  Google Scholar 

  24. 24

    Nielsen, E. et al. Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain. J. Cell Biol. 151, 601–612 (2000)

    CAS  Article  Google Scholar 

  25. 25

    Schnatwinkel, C. et al. The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms. PLoS Biol. 2, 1363–1380 (2004)

    CAS  Article  Google Scholar 

  26. 26

    McBride, H. M. et al. Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13. Cell 98, 377–386 (1999)

    CAS  Article  Google Scholar 

  27. 27

    Hoepfner, S. et al. Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B. Cell 121, 437–450 (2005)

    CAS  Article  Google Scholar 

  28. 28

    Del Conte-Zerial, P. et al. Membrane identity and GTPase cascades regulated by toggle and cut-out switches. Mol. Syst. Biol. 4, 206 (2008)

    Article  Google Scholar 

  29. 29

    Zwilling, D. et al. Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes with multiple topologies. EMBO J. 26, 9–18 (2007)

    CAS  Article  Google Scholar 

  30. 30

    Bhalla, A., Chicka, M. C., Tucker, W. C. & Chapman, E. R. Ca2+-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion. Nature Struct. Mol. Biol. 13, 323–330 (2006)

    CAS  Article  Google Scholar 

  31. 31

    Dennison, S. M., Bowen, M. E., Brunger, A. T. & Lentz, B. R. Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion. Biophys. J. 90, 1661–1675 (2006)

    CAS  Article  ADS  Google Scholar 

  32. 32

    Starai, V. J., Jun, Y. & Wickner, W. Excess vacuolar SNAREs drive lysis and Rab bypass fusion. Proc. Natl Acad. Sci. USA 104, 13551–13558 (2007)

    CAS  Article  ADS  Google Scholar 

  33. 33

    Ullrich, O., Horiuchi, H., Bucci, C. & Zerial, M. Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange. Nature 368, 157–160 (1994)

    CAS  Article  ADS  Google Scholar 

  34. 34

    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)

    CAS  Article  Google Scholar 

  35. 35

    Rybin, V. et al. GTPase activity of Rab5 acts as a timer for endocytic membrane fusion. Nature 383, 266–269 (1996)

    CAS  Article  ADS  Google Scholar 

  36. 36

    Sivars, U., Aivazian, D. & Pfeffer, S. R. Yip3 catalyses the dissociation of endosomal Rab–GDI complexes. Nature 425, 856–859 (2003)

    CAS  Article  ADS  Google Scholar 

  37. 37

    Simonsen, A., Gaullier, J. M., D'Arrigo, A. & Stenmark, H. The Rab5 effector EEA1 interacts directly with syntaxin-6. J. Biol. Chem. 274, 28857–28860 (1999)

    CAS  Article  Google Scholar 

  38. 38

    Ungermann, C., Price, A. & Wickner, W. A new role for a SNARE protein as a regulator of the Ypt7/Rab-dependent stage of docking. Proc. Natl Acad. Sci. USA 97, 8889–8891 (2000)

    CAS  Article  ADS  Google Scholar 

  39. 39

    Mima, J. et al. Reconstituted membrane fusion requires regulatory lipids, SNAREs and synergistic SNARE chaperones. EMBO J. 27, 2031–2042 (2008)

    CAS  Article  Google Scholar 

  40. 40

    Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006)

    CAS  Article  Google Scholar 

  41. 41

    Weber, T. et al. SNAREpins are functionally resistant to disruption by NSF and alphaSNAP. J. Cell Biol. 149, 1063–1072 (2000)

    CAS  Article  Google Scholar 

  42. 42

    Collins, K. M. & Wickner, W. T. Trans-SNARE complex assembly and yeast vacuole membrane fusion. Proc. Natl Acad. Sci. USA 104, 8755–8760 (2007)

    CAS  Article  ADS  Google Scholar 

  43. 43

    Kobayashi, T. et al. A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 392, 193–197 (1998)

    CAS  Article  ADS  Google Scholar 

  44. 44

    Pfeffer, S. & Aivazian, D. Targeting Rab GTPases to distinct membrane compartments. Nature Rev. Mol. Cell Biol. 5, 886–896 (2004)

    CAS  Article  Google Scholar 

  45. 45

    Dulubova, I. et al. Munc18–1 binds directly to the neuronal SNARE complex. Proc. Natl Acad. Sci. USA 104, 2697–2702 (2007)

    CAS  Article  ADS  Google Scholar 

  46. 46

    Shen, J. et al. Selective activation of cognate SNAREpins by Sec1/Munc18 proteins. Cell 128, 183–195 (2007)

    CAS  Article  Google Scholar 

  47. 47

    Martens, S., Kozlov, M. M. & McMahon, H. T. How synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007)

    CAS  Article  ADS  Google Scholar 

  48. 48

    Rink, J., Ghigo, E., Kalaidzidis, Y. & Zerial, M. Rab conversion as a mechanism of progression from early to late endosomes. Cell 122, 735–749 (2005)

    CAS  Article  Google Scholar 

  49. 49

    Peplowska, K. et al. The CORVET tethering complex interacts with the yeast Rab5 homolog Vps21 and is involved in endo-lysosomal biogenesis. Dev. Cell 12, 739–750 (2007)

    CAS  Article  Google Scholar 

  50. 50

    Bartlett, G. R. Colorimetric assay methods for free and phosphorylated glyceric acids. J. Biol. Chem. 234, 469–471 (1959)

    CAS  PubMed  Google Scholar 

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We are grateful to K. Simons and B. Hoflack for discussions, to C. Stroupe and W. Wickner for sharing unpublished information, and to G. Marsne, I. Baines, W. Huttner, K. Simons, C. Stroupe and W. Wickner for critical reading of the manuscript. We acknowledge support by the systems biology network HepatoSys of the German Ministry for Education and Research (BMBF, grant 0313082J), the EU Integrated Project EndoTrack, the DFG and the Max Planck Society (including the Max Planck Partner Group grant to M.Z. and M.M.). T.O. was supported by The Nakatomi Foundation.

Author Contributions M.M. conducted the initial studies and tested the recombinant proteins in endosome fusion and the membrane recruitment of Rab5 and its effectors on proteoliposomes, and B.L. further developed such a proteoliposome system. D.D. and A.R. established several of the protocols of purification of recombinant proteins. T.O. completed the development of these procedures and conducted all biochemical experiments on membrane fusion reported in this study. Ü.C. performed the electron microscopy analysis, Y.K. did the statistical analysis and the mathematical model of membrane fusion, and M.Z. conceived and directed the project and wrote the manuscript with the help of T.O., M.M. and Y.K.

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Correspondence to Marino Zerial.

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Ohya, T., Miaczynska, M., Coskun, Ü. et al. Reconstitution of Rab- and SNARE-dependent membrane fusion by synthetic endosomes. Nature 459, 1091–1097 (2009).

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