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Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are membrane-associated trafficking proteins that confer identity to lipid membranes and facilitate membrane fusion. These functions are achieved through the complexing of Q-SNAREs with a specific cognate target R-SNARE, leading to the fusion of their associated membranes. These SNARE complexes then dissociate so that the Q-SNAREs and R-SNAREs can repeat this cycle. Whilst the basic function of SNAREs has been long appreciated, it is becoming increasingly clear that the cell can control the localisation and function of SNARE proteins through posttranslational modifications (PTMs), such as phosphorylation and ubiquitylation. Whilst numerous proteomic methods have shown that SNARE proteins are subject to these modifications, little is known about how these modifications regulate SNARE function. However, it is clear that these PTMs provide cells with an incredible functional plasticity; SNARE PTMs enable cells to respond to an ever-changing extracellular environment through the rerouting of membrane traffic. In this Review, we summarise key findings regarding SNARE regulation by PTMs and discuss how these modifications reprogramme membrane trafficking pathways., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

In eukaryotes, most intracellular membrane fusion reactions are mediated by the interaction of SNARE proteins that are present in both fusing membranes. However, the minimal number of SNARE complexes needed for membrane fusion is not known. Here we show unambiguously that one SNARE complex is sufficient for membrane fusion. We performed controlled in vitro Förster resonance energy transfer (FRET) experiments and found that liposomes bearing only a single SNARE molecule are still capable of fusion with other liposomes or with purified synaptic vesicles. Furthermore, we demonstrated that multiple SNARE complexes do not act cooperatively, showing that synergy between several SNARE complexes is not needed for membrane fusion. Our findings shed new light on the mechanism of SNARE-mediated membrane fusion and call for a revision of current views of fusion events such as the fast release of neurotransmitters.

Synaptotagmin-1 triggers Ca2+-sensitive, rapid neurotransmitter release by promoting interactions between SNARE proteins on synaptic vesicles and the plasma membrane. How synaptotagmin-1 promotes this interaction is unclear, and the massive increase in membrane fusion efficiency of Ca2+-bound synaptotagmin-1 has not been reproduced in vitro. However, previous experiments have been performed at relatively high salt concentrations, screening potentially important electrostatic interactions. Using functional reconstitution in liposomes, we show here that at low ionic strength SNARE-mediated membrane fusion becomes strictly dependent on both Ca2+ and synaptotagmin-1. Under these conditions, synaptotagmin-1 functions as a distance regulator that tethers the liposomes too far from the plasma membrane for SNARE nucleation in the absence of Ca2+, but while bringing the liposomes close enough for membrane fusion in the presence of Ca2+. These results may explain how the relatively weak electrostatic interactions between synaptotagmin-1 and membranes substantially accelerate fusion. [ABSTRACT FROM AUTHOR]

SNARE proteins mediate membrane fusion between synaptic vesicles and the plasma membrane. A minimized peptide SNARE model system with reduced complexity was introduced combining the native SNARE transmembrane (TMD) and linker domains with artificial coiled-coil forming peptides. Specific membrane fusion initiated by coiled-coil recognition was shown by lipid and content mixing vesicle assays. [ABSTRACT FROM AUTHOR]

SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Fo? rster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood- derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution. [ABSTRACT FROM AUTHOR]