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.

Profile of Thomas Südhof, James Rothman, and Randy Schekman, 2013 Nobel Laureates in Physiology or Medicine

“…SNARE function is tightly coupled to bound proteins such as Munc13 and Munc18 for synaptic fusion and by Sec1/ Munc18 homologs and large tethering complexes for many other intracellular SNARE-dependent membrane fusion events. Reconstituted reactions reflecting more of the complete physiological complexity, regulation, and kinetics have appeared (14)(15)(16)(17). The neuronal fusion reconstitution is strictly dependent on Munc18-1and Munc13 (17), in full accord with Südhof's group's findings.…”

The p97 ATPase associates with EEA1 to regulate the size of early endosomes

“…Endocytic vesicle docking and fusion require cooperation between many soluble and membrane components. Among them, the early endosome-associated autoantigen 1 (EEA1) is an essential component that www.cell-research.com | Cell Research Harish N Ramanathan and Yihong Ye 347 npg dynamically distributes between an endosome membrane-associated and a soluble cytosolic form [17][18][19][20]. Membrane association of EEA1 is regulated by the Rab5 GTPase, a master regulator of endocytic trafficking [21][22][23].…”

Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

“…The lipid mixing that we measure therefore represents authentic membrane fusion. Biochemical reconstitution of Rab5-dependent membrane fusion using purified components has also recently been reported (31).…”