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. Membrane fusion reconstituted with Rab and SNARE Eukaryotic cells contain membrane-bound organelles that can undergo fusion with each other in a regulated manner. The widely held view is that fusion is mediated by SNARE (soluble N -ethylmaleimide-sensitive factor attachment receptor) proteins. Although SNAREs are capable of fusing vesicles with high specificity, fusion reactions with native SNAREs alone are inefficient and require high concentrations of proteins. In this study, Ohya et al . reconstitute endosome fusion using the Rab machinery along with SNARE proteins. This is the first report of the reconstitution of membrane fusion efficiently and under physiological conditions and also highlights the functional relationship between Rab and SNARE proteins. Eukaryotic cells contain membrane-bound organelles that can undergo fusion with each other in a regulated manner; this is thought to be mediated by SNAREs (soluble N -ethylmaleimide-sensitive factor attachment protein receptors). Here, the complete reconstitution of endosome fusion using Rab5 and SNARE proteins highlights the functional relationship between these two machineries.