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Nature. 2009 Jun 25;459(7250):1091-7. doi: 10.1038/nature08107. Epub 2009 May 20.

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

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1
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01309, Dresden, Germany.

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.

PMID:
19458617
DOI:
10.1038/nature08107
[Indexed for MEDLINE]

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