title: Pulling force generated by interacting SNAREs facilitates membrane hemifusion.
abstract: In biological systems, membrane fusion is mediated by specialized proteins. Although soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) provide the minimal molecular machinery required to drive membrane fusion, the precise mechanism for SNARE-mediated fusion remains to be established. Here, we used atomic force microscope (AFM) spectroscopy to determine whether the pulling force generated by interacting SNAREs is directly coupled to membrane fusion. The mechanical strength of the SNARE binding interaction was determined by single molecule force measurements. It was revealed that the forced unbinding of the SNARE complex formed between opposing (trans) bilayers involves two activation barriers; where the steep inner barrier governs the transition from the bound to an intermediate state and the outer barrier governs the transition between the intermediate and the unbound state. Moreover, truncation of either SNAP-25 or VAMP 2 reduced the slope of the inner barrier significantly and, consequently, reduced the pulling strength of the SNARE complex; thus, suggesting that the inner barrier determines the binding strength of the SNARE complex. In parallel, AFM compression force measurements revealed that truncated SNAREs were less efficient than native SNAREs in facilitating hemifusion of the apposed bilayers. Together, these findings reveal a mechanism by which a pulling force generated by interacting trans-SNAREs reduces the slope of the hemifusion barrier and, subsequently, facilitates hemifusion and makes the membranes more prone to fusion.
authors.affiliation: University of Miami Miller School of Medicine, Physiology & Biophysics Department, 1600 NW 10th Ave., Miami, FL 33136, USA.
authors.forename: Midhat H
authors.initials: MH
authors.lastname: Abdulreda
authors.forename: Akhil
authors.initials: A
authors.lastname: Bhalla
authors.forename: Felix
authors.initials: F
authors.lastname: Rico
authors.forename: Per-Olof
authors.initials: PO
authors.lastname: Berggren
authors.forename: Edwin R
authors.initials: ER
authors.lastname: Chapman
authors.forename: Vincent T
authors.initials: VT
authors.lastname: Moy
mesh_terms: D002462:Cell Membrane / Q000502:physiology*; D008051:Lipid Bilayers / Q000378:metabolism*; D008561:Membrane Fusion / Q000502:physiology*; D050600:SNARE Proteins / Q000502:physiology*; D050825:Synaptosomal-Associated Protein 25 / Q000502:physiology; D013816:Thermodynamics; D050988:Vesicle-Associated Membrane Protein 2 / Q000502:physiology
chemical_list: D008051:Lipid Bilayers; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050988:Vesicle-Associated Membrane Protein 2