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

Ohya, Takeshi
Miaczynska, Marta
Coskun, Ünal
Lommer, Barbara
Runge, Anja
Drechsel, David
Kalaidzidis, Yannis
Zerial, Marino

¹Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01309, Dresden, Germany
²A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia
³Present address: International Institute of Molecular and Cell Biology (IIMCB), Ks. Trojdena 4, 02-109 Warsaw, Poland.

Correspondence and requests for materials should be addressed to M.Z. ([email protected]).

Received 13 February 2009; accepted 05 May 2009; Published online 20 May 2009

We are grateful to K. Simons and B. Hoflack for discussions, to C. Stroupe and W. Wickner for sharing unpublished information, and to G. Marsne, I. Baines, W. Huttner, K. Simons, C. Stroupe and W. Wickner for critical reading of the manuscript.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.

Nature 459(7250):p 1091-1097, June 25, 2009. | DOI: 10.1038/nature08107

Rab GTPases and SNAREs (solubleN-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 membranesin 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 otherin 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.