An endosomal tether undergoes an entropic collapse to bring vesicles together

Abstract

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 effectors1–4. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs alone5,6,7. Here, we addressed 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 EEA16,7 recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harboring Rab5. Surprisingly, structural analysis revealed that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers we confirmed that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measured its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induced 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 toward the target membrane to initiate docking and fusion.

Multivalent Rab interactions determine tether-mediated membrane fusion

“…, 2008; Cheung et al. , 2015; Murray et al. , 2016), or cell membranes and cytosol to identify components (Cao et al.…”

The endosomal trafficking factors CORVET and ESCRT suppress plasma membrane residence of the renal outer medullary potassium channel (ROMK)

“…Four of these subunits, Vps11, Vps16, Vps18, and Vps33, are also components of the HOPS complex found on Rab7-positive vesicles (56). Recent structural studies have shown that these tethering complexes adopt a disordered and highly flexible conformation that coalesces into a tightly ordered structure upon Rab GTPase binding (83)(84)(85). Both of the Rab5-binding subunits that are unique to CORVET, Vps8 and Vps3, were identified in this study as negative regulators of ROMK plasma membrane residence in yeast.…”

Single-molecule force spectroscopy of protein-membrane interactions

“…High-resolution OTs achieve extremely high spatiotemporal resolution (~0.3 nm, 20 ms) in a range of force that can reversibly unfold a biomolecule or is generated by molecular motors (Abbondanzieri et al, 2005;Neupane et al, 2016;Moffitt et al, 2006;Zhang et al, 2013;Gao et al, 2012;Cecconi et al, 2005;Bustamante et al, 2004). Recently, OTs have been used to test membrane binding of the vesicle tethering complex EEA1 (Murray et al, 2016). However, to our knowledge, OTs have not been applied to measure both the energy and the detailed kinetics of protein-membrane interactions.…”