An endosomal tether undergoes an entropic collapse to bring vesicles together

A new endosomal tethering mechanism involving a mechanochemical cycle of the dimeric coiled-coil protein EEA1 regulated by Rab5:GTP binding and GTP hydrolysis. Flexible conformation for EEA1 tether protein Within cells, Rab GTPase proteins recruit tethering effectors that allow transport vesicles to dock and fuse with their target membranes. This raises the question of how the long, rod-like tethering molecules such as the protein EEA1 can capture vesicles, yet allow the reduction of the distance between the membranes for fusion. Marino Zerial and colleagues address this question by reconstituting an endosomal tethering machinery consisting of EEA1, membranes and vesicles carrying Rab5. They find that, in a new role, Rab5:GTP induces a change in EEA1 conformation from extended to flexible, allowing it to bend. In addition, they show that membrane tethering by EEA1:Rab5:GTP generates a force that also helps to bring two membranes 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 1 , 2 , 3 , 4 . Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs (soluble N -ethylmaleimide-sensitive factor attachment protein receptors) alone 5 , 6 , 7 . 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.