How Could SNARE Proteins Open a Fusion Pore?

Abstract

The SNARE (Soluble NSF Attachment protein REceptor) complex, which in mammalian neurosecretory cells is composed of the proteins synaptobrevin 2 (also called VAMP2), syntaxin, and SNAP-25, plays a key role in vesicle fusion.In this review, we discuss the hypothesis that, in neurosecretory cells, fusion pore formation is directly accomplished by a conformational change in the SNARE complex via movement of the transmembrane domains.Qinghua Fang 1,2 and Manfred Lindau (49) has three components. The v-SNARE synaptobrevin 2 (syb2 also called VAMP2) is a 116-amino acid protein anchored in the vesicle membrane by a single transmembrane (TM) domain. Syntaxin (stx) is correspondingly anchored in the plasma membrane via a single TM helix. The third component, SNAP-25, has lipid anchors in the plasma membrane. SNAP-25 and stx are called t-SNAREs, being in the target membrane for fusion of secretory vesicles. The key importance of these proteins in the fusion mechanism has been demonstrated by the finding that proteolytic cleavage of the SNARE proteins by specific neurotoxins results in strong inhibition of transmitter release in neurons as well as in chromaffin cells (42,44). One example for the medical relevance of the SNARE complex is the BoTox treatment, which exerts its function through inhibition of transmitter release by specific cleavage of the SNARE protein SNAP-25. The cytosolic so-called SNARE domains of these three proteins form a coiled coil, which incorporates one helix each from syb2 and stx and two helices (named SN1 and SN2) from SNAP-25. The coiled coil structure of the SNARE domains has been solved by X-ray crystallography several years ago (57). Based on this structure, the configuration shown in FIGURE 1A was proposed for a trans configuration, in which a SNARE complex links the vesicle membrane and plasma membrane. The arrows indicate the cleavage sites for the neurotoxins mentioned above. More recently, a crystal structure of the SNARE complex, including the syb2 and stx TM helices, was solved (56), which shows helical extension from the SNARE domains through the linkers into the TM domains (FIGURE 1B). This structure is thought to resemble a post-fusion state in which all components of the SNARE complex are in a cis configuration, located in the same fused membrane.Synaptic vesicles contain ϳ70 copies of syb2 (59), but it is unknown and controversial how many of these copies are acting together in the formation of a fusion pore (43). Interestingly, neurosecretory vesicles in PC 12 cells recruit t-SNARE clusters containing a similar number of stx and SNAP-25 molecules (26). It has been shown that in reconstituted systems a single SNARE complex is sufficient to promote lipid vesicle fusion (50, 62). However, at least three SNARE complexes appear to be required for fast membrane fusion kinetics (8,17,41,50). In cultured hippocampal neurons, two copies of syb2 were found to be sufficient for fusion, but the experiments were performed with low time resolution (52). The Triggering MechanismTransmitter rel...

Exocytotic fusion pores are composed of both lipids and proteins

“…The latter possibility could arise if a few residues in the TMDs of syb2 and syx 1A, near the interface of the membranes destined to fuse, were to physically interact with each other to hold pores open longer 40 . It should also be noted that SNAREs might oligo-merize 45-47 , and at high copy numbers they could potentially form largely or purely proteinaceous channels 11-13 . Transient fusion pores, formed by the reversible assembly of SNAREs into oligomers, is an appealing idea, because different stoichiometries of SNAREs might determine the size of the pore, which can range from 0.5 nm for small vesicles in the posterior pituitary to 2 nm in beige mouse mast cells 48,49 .…”

“…Two distinct hypotheses have been proposed 11-13 . In the lipid-stalk-fusion hypothesis, the outer leaflets of the membranes destined to fuse merge, initially forming a hemifusion stalk that resolves to a hemifusion diaphragm; the fusion pore then forms in the diaphragm and is purely lipidic 14 .…”

The Multifaceted Role of SNARE Proteins in Membrane Fusion

“…The formation of the fusion pore was suggested to be initiated by the movement of the sybII TMD uncharged C-terminus into the membrane interior, induced by the pulling force resulting from SNARE complex zippering, as revealed by coarse-grained simulations (Lindau et al, 2012) (see Figure 5). Addition of charged residues to the C-terminus of SNARE TMD inhibited exocytosis in chromaffin and PC 12 cells (Ngatchou et al, 2010; Wehland et al, 2016), suggesting a mechanism in which the movement of the C-terminus initiates the fusion pore formation by rearranging the bilayer structure in distal leaflets (Fang and Lindau, 2014). …”

Functional anatomy and physiology of gastric secretion

“…Proteins involved in translocation and fusion of H,K-ATPase include myosin, actin, Rab GTPases, ezrin, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The SNARE complex is composed of synaptobrevin 2 [also termed vesicle-associated membrane protein-2 (VAMP2)], syntaxin, and synaptosomal-associated protein, 25 kDa (SNAP-25) [34].…”