title: 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 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.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: David H
authors.initials: DH
authors.lastname: Murray
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.|Biotechnology Center, Technical University Dresden, Tatzberg 47/49, 01307 Dresden, Germany.|Max Planck Institute for the Physics of Complex Systems, Nöthnitzerstraße 38, 01187 Dresden, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Jahnel
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Janelle
authors.initials: J
authors.lastname: Lauer
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.|Biotechnology Center, Technical University Dresden, Tatzberg 47/49, 01307 Dresden, Germany.
authors.forename: Mario J
authors.initials: MJ
authors.lastname: Avellaneda
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Brouilly
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Alice
authors.initials: A
authors.lastname: Cezanne
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Hernán
authors.initials: H
authors.lastname: Morales-Navarrete
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.|Biotechnology Center, Technical University Dresden, Tatzberg 47/49, 01307 Dresden, Germany.
authors.forename: Enrico D
authors.initials: ED
authors.lastname: Perini
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia 4072, Australia.
authors.forename: Charles
authors.initials: C
authors.lastname: Ferguson
authors.affiliation: Department of Protein Evolution, Max-Planck-Institute for Developmental Biology, 72076 Tübingen, Germany.
authors.forename: Andrei N
authors.initials: AN
authors.lastname: Lupas
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Yannis
authors.initials: Y
authors.lastname: Kalaidzidis
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia 4072, Australia.|Centre for Microscopy and Microanalysis, The University of Queensland, St Lucia 4072, Australia.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Parton
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.|Biotechnology Center, Technical University Dresden, Tatzberg 47/49, 01307 Dresden, Germany.|Max Planck Institute for the Physics of Complex Systems, Nöthnitzerstraße 38, 01187 Dresden, Germany.
authors.forename: Stephan W
authors.initials: SW
authors.lastname: Grill
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Marino
authors.initials: M
authors.lastname: Zerial
mesh_terms: D000494:Allosteric Regulation; D011992:Endosomes / Q000378:metabolism*; D019277:Entropy; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008561:Membrane Fusion; D052898:Optical Tweezers; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D018583:Pliability; D011485:Protein Binding; D011487:Protein Conformation; D050600:SNARE Proteins / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D018129:Phosphatidylinositol Phosphates; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; C055525:phosphatidylinositol 3-phosphate; D006160:Guanosine Triphosphate; D020696:rab5 GTP-Binding Proteins