Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca2+-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle’s outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion.DOI: http://dx.doi.org/10.7554/eLife.17571.001
ComplexinII (CpxII) inhibits non-synchronized vesicle fusion, but the underlying mechanisms have remained unclear. Here, we provide evidence that the far C-terminal domain (CTD) of CpxII interferes with SNARE assembly, thereby arresting tonic exocytosis. Acute infusion of a CTD-derived peptide into mouse chromaffin cells enhances synchronous release by diminishing premature vesicle fusion like full-length CpxII, indicating a direct, inhibitory function of the CTD that sets the magnitude of the primed vesicle pool. We describe a high degree of structural similarity between the CpxII CTD and the SNAP25-SN1 domain (C-terminal half) and show that the CTD peptide lowers the rate of SDS-resistant SNARE complex formation in vitro. Moreover, corresponding CpxII:SNAP25 chimeras do restore complexin’s function and even ‘superclamp’ tonic secretion. Collectively, these results support a so far unrecognized clamping mechanism wherein the CpxII C-terminus hinders spontaneous SNARE complex assembly, enabling the build-up of a release-ready pool of vesicles for synchronized Ca2+-triggered exocytosis.
Vesicle fusion is mediated by assembly of SNARE proteins between opposing membranes. While previous work suggested an active role of SNARE transmembrane domains (TMDs) in promoting membrane merger (Dhara et al., 2016), the underlying mechanism remained elusive. Here, we show that naturally-occurring v-SNARE TMD variants differentially regulate fusion pore dynamics in mouse chromaffin cells, indicating TMD flexibility as a mechanistic determinant that facilitates transmitter release from differentially-sized vesicles. Membrane curvature-promoting phospholipids like lysophosphatidylcholine or oleic acid profoundly alter pore expansion and fully rescue the decelerated fusion kinetics of TMD-rigidifying VAMP2 mutants. Thus, v-SNARE TMDs and phospholipids cooperate in supporting membrane curvature at the fusion pore neck. Oppositely, slowing of pore kinetics by the SNARE-regulator complexin-2 withstands the curvature-driven speeding of fusion, indicating that pore evolution is tightly coupled to progressive SNARE complex formation. Collectively, TMD-mediated support of membrane curvature and SNARE force-generated membrane bending promote fusion pore formation and expansion.
Hindering premature vesicular fusion is key to build up a molecularly primed ready releasable pool. Complexin-II (CpxII), a 16 KD cytosolic protein, inhibits non-synchronous vesicle fusion at release sites, and by that accumulates a ready releasable pool of primed vesicles that are released in synchrony upon immediate elevation of intracellular calcium. Functionally, CpxII is equipped with four independent yet synergetic domains. A central helix mediates the CpxII-SNARE (Nethylmaleimide-sensitive factor (NSF) attachment protein receptors) complex interaction, while an accessory helix is important for the stability of the central helix and for reducing premature fusion. The upstream N-terminal domain facilitates exocytosis, whereas the downstream C-terminal domain (CTD) inhibits premature fusion of vesicles. The molecular mechanisms how the CTD blocks asynchronous release are still unclear. With the help of high resolution membrane capacitance measurements coupled with calcium imaging, carbon fiber amperometry, and biochemical assays, we show that infusion of CpxII far CTD -derived peptide into mouse chromaffin cells is able to boost the synchronous release by reducing the premature vesicle fusion. A similar phenotype is also observed by over-expressing the full length WT protein. Using in vitro binding assays, we show that the CTD interacts with SNARE proteins. Moreover, we found that the CTD peptide lowers the rate of SDS resistant SNARE complex formation. We also show that the CTD shares a high degree of similarity to the SNARE protein SNAP25-SN1 domain. Furthermore chimeras of CpxII:SNAP25-SN1 fully restore CpxII function, and even ''superclamp'' tonic secretion. Collectively, these results provide evidence for a clamping mechanism in which the CTD of CpxII prevents the spontaneous assembly of SNARE complexes, enabling the build-up of a ready releasable pool for synchronised Ca 2þ -triggered exocytosis.
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