Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion

Abstract

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.

Differential diffusional properties in loose and tight docking prior to membrane fusion

“…Vesicles carrying the AA syb mutants showed the fastest diffusion, followed by those containing the Δ84 mutant whereas vesicles with WT protein were the slowest. These data show that the different states uncovered previously by electron microscopy (17) indeed represent physical differences in the nature of membrane attachment between the two vesicles. As discussed above, the loosely docked state is probably governed exclusively by trans-interactions between individual SNAREs whereas in the tightly docked state the membranes are firmly attached, forming a disk that is probably rigid (6,17).…”

“…These data show that the different states uncovered previously by electron microscopy (17) indeed represent physical differences in the nature of membrane attachment between the two vesicles. As discussed above, the loosely docked state is probably governed exclusively by trans-interactions between individual SNAREs whereas in the tightly docked state the membranes are firmly attached, forming a disk that is probably rigid (6,17). Using the experimentally obtained diffusion data, we have therefore carried out simulations in order to evaluate whether the data are compatible with these models.…”

“…Under physiological conditions, the lifetime of these intermediate states is very short. Indeed, only recently docking intermediates preceding hemifusion in SNARE-mediated fusion have been captured by cryo-electron microscopy (6,17,18). Deletion of a single amino acid in the SNARE synaptobrevin (Δ84 syb) results in vesicles that are arrested in a tightly docked (but not hemifused) state in which the membranes are separated by less than one nanometer.…”

“…Deletion of a single amino acid in the SNARE synaptobrevin (Δ84 syb) results in vesicles that are arrested in a tightly docked (but not hemifused) state in which the membranes are separated by less than one nanometer. In contrast, substitution of two amino acids more distal from the transmembrane domain (I45A,M46A; referred to as AA syb) uncovered a loosely docked state where the membranes are separated by a larger gap, only connected by reversible SNARE interactions (17). Finally, extended hemifusion diaphragms were observed when the wild type (WT) syb protein was used (6).…”

“…This technique has been shown to reach microsecond temporal resolution and nm precision in detecting and tracking individual nanoparticles via the interference of their scattered light with a reference beam (22,23, and see Methods). Using iSCAT, we could investigate the diffusion of unlabeled vesicles arrested at a loosely or tightly docked intermediate state using synaptobrevin point mutants as previously described (17). Modeling of the diffusion data allowed for approximating the number of SNARE complexes involved in vesicle docking (loose docking) as well as the size of the interaction interface (tight docking).…”