Interactions between synaptic vesicle fusion proteins explored by atomic force microscopy

Abstract

Measuring the biophysical properties of macromolecular complexes at work is a major challenge of modern biology. The protein complex composed of vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa, and syntaxin 1 [soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex] is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. To better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces and dissociation kinetics of the SNAREs and led us to propose a sequence of interactions. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein neuronal Sec1 injected into the atomic force microscope chamber prevented the complex formation. Finally, we measured the effect of tetanus toxin protease on the SNARE complex and its activity by on-line registration during tetanus toxin injection. These experiments provide a basis for the functional study of protein microdomains and also suggest opportunities for sensitive screening of drugs that can modulate protein-protein interactions. E xocytosis is the process in which an intracellular membranebound vesicle fuses with the plasma membrane, leading to the release of the vesicle content into the extracellular space. One of the most extensively studied forms of exocytosis is the chemical synaptic transmission used by nervous system cells to communicate. A better understanding of this mechanism is a key to understanding synaptic transmission and synaptic plasticity, learning, and memory.The current hypothesis suggests soluble N-ethyl-maleimidesensitive factor attachment protein receptor (SNARE) proteins are mediators of membrane fusion (1-4). In this model, vesicleassociated membrane protein 2 (VAMP 2) is located on the vesicle membrane whereas syntaxin 1 and synaptosomalassociated protein of 25 kDa (SNAP-25) reside in the target membrane (Fig. 1A). Before the fusion process, these proteins assemble into a highly thermostable ternary complex (5-7), which consists of a coiled-coil bundle of four ␣-helices (one from syntaxin 1, one from VAMP 2, and two from SNAP-25) bound in a parallel alignment (8-10). Whether the SNARE complex triggers fusion directly or indirectly is still controversial (11). The ''zipper'' model proposes that the complex zips from the membrane distal amino termini to the proximal carboxyl termini, thus bringing the vesicle and the membrane in a close configuration that might induce fusion (4, 10). The sequence of steps leading to fusion is still not clear. The SNARE complex could exist in a partially zipped conformation (12) and complete zip...

SNARE-mediated membrane fusion trajectories derived from force-clamp experiments

“…Also, the number of SNARE complexes formed is reduced to one to three SNARE complexes. We cannot entirely rule out that extraction of SNAREs upon retraction occurs, but the forces agree well with previous reports and extraction followed by unfolding would not depend that strongly on the presence of calcium or on point mutations as observed here (26,27). Besides, constant fusion efficiency over 1,000 force-distance cycles was found, which speaks against extraction of SNAREs from the bilayer.…”

“…S1). We found discrete detachment events corresponding to one to five SNARE molecules each with a force maximum of 200 ± 37 pN upon separation of nonfused bilayers, in agreement with reported rupture forces for SNARE assemblies (26,27).…”

Evidence for structural and functional diversity among SDS-resistant SNARE complexes in neuroendocrine cells

“…Such structures have been largely neglected in previous studies on the function of the SNARE fusion machinery and should be looked at carefully in future work in the field. This notion is supported by recent evidence that oligomeric SNARE structures form at fusion sites (Cho et al, 2002) and by mathematical modelling, which suggests that cooperation of at least three SNARE complexes is needed to mediate membrane fusion (Hua and Scheller, 2001;Yersin et al, 2003). The complex-230 observed in PC12 cells might represent such cooperative SNARE structure.…”

Tension-induced fusion of bilayer membranes and vesicles

“…In fact, distinguishing between the different models for the action of fusion proteins 4,5 is a compelling target for further DPD simulation studies. It is possible to measure the work performed during the fusion process in a DPD simulation, as has recently been done in coarse-grained molecular dynamics simulations 9 , and to relate this to experimental estimates of the forces exerted by fusion proteins 23 . It should also be possible to calibrate the DPD parameters by extending a method that has recently been applied to coarsegrained molecular dynamics simulations 21 .…”