An endosomal tether undergoes an entropic collapse to bring vesicles together.

An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) alone. Here we address the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA1 (refs 6, 7) recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harbouring Rab5. Surprisingly, structural analysis reveals that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers, we confirm that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measure its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induce prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle towards the target membrane to initiate docking and fusion.