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.
