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

Abstract

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).

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Acknowledgements

We are grateful to K. Simons and B. Hoflack for discussions, to C. Stroupe and W. Wickner for sharing unpublished information, and to G. Marsne, I. Baines, W. Huttner, K. Simons, C. Stroupe and W. Wickner for critical reading of the manuscript.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). https://doi.org/10.1038/nature08107

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