Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes through pairing of vesicleassociated v-SNAREs with complementary t-SNAREs on target membranes. However, the number of SNARE complexes required for fusion is unknown, and there is controversy about whether additional proteins are required to explain the fast fusion which can occur in cells. Here we show that single vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNAREs syntaxin-SNAP25. Fusion rates decreased dramatically when the number of externally oriented v-SNAREs per vesicle was reduced below 5-10, directly establishing this as the minimum number required for rapid fusion. Docking-to-fusion delay time distributions were consistent with a requirement that 5-11 t-SNAREs be recruited to achieve fusion, closely matching the v-SNARE requirement.lipid bilayer | membrane fusion | SNARE mechanisms | supported bilayer T rafficking of proteins in the cell-as well as secretion of physiological mediators such as hormones and neurotransmitters-depends on intracellular membrane fusion. With few exceptions, intracellular fusion reactions are driven by pairing of vesicle-associated v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with cognate t-SNAREs on the target membrane, resulting in a four-helix bundle (SNAREpin) that brings bilayers into close proximity (1-3). In cells, the action of SNAREs is regulated by auxiliary proteins, some of which, such as the members of the Sec1/Munc18-like (SM) family, are universally required components of the eukaryotic fusion machinery (4). Whether SNAREs alone can catalyze fusion at physiologically meaningful rates in the absence of modulating proteins or peptides (5-9) remains controversial. In addition, it is unknown how many SNAREpins are required to produce fusion. Here, using an in vitro assay that can resolve single docking and fusion events, we show 5-10 SNAREpins mediate fast fusion in the absence of any auxiliary proteins.Reconstituted fusion assays have played a key role in elucidating mechanisms of SNARE-mediated membrane fusion (1, 2, 5, 6, 10, 11). SNARE proteins reconstituted into small unilamellar vesicles (SUVs) fused bilayers in a bulk fusion assay, albeit with slow kinetics (1, 2). More recently, single SUVs containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin were shown to fuse rapidly with planar, supported bilayers (SBLs) containing the synaptic/exocytic t-SNAREs syntaxin 1-SNAP25, with single fusion events occurring in ∼10-100 ms (7, 9) to seconds (8, 12). However, the SNAP25 subunit of the t-SNARE was not required (8, 9), or an artificial peptide was needed (7), raising questions about the physiological relevance of these results. These, and other studies of SNARE-mediated membrane fusion, used lipid bilayers where the active fusion catalysts were the only proteins present. By contrast, natural intracellular membranes are popula...
Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.
Syntaxin-1A and SNAP25 form the receptor component of the SNARE complex, which has been shown to be the minimal machinery for membrane fusion. In vivo studies have revealed that syntaxin-1A exists in cholesterol-dependent clusters that are distinct from lipid rafts. Additionally, SNARE-mediated membrane fusion has been shown to be stimulated by regulatory lipids, such as phosphatidy- linositol 4,5-bisphosphate (PI-4,5-P2). An appreciation of the lipid-protein interactions which define syntaxin clustering dynamics is essential to understand the membrane role in organization of SNARE-mediated membrane fusion. We determined that syntaxin exists in cholesterol-dependent clusters, from which it may be released by as little as 1-5 mole percent PI-4,5-P2 by in vitro fluorescence assays. Lipid-protein fluorescence resonance energy transfer reveals that the phosphoinositide interaction is direct and mediated by electrostatics. To investigate the dynamics of clustering, single-molecule fluorescence quenching microscopy was developed. The observation of syntaxins in single vesicles allows the step-wise statistical analysis of discrete syntaxin-syntaxin interactions, and determination of their dependence on concentration and membrane composition. These in vitro results help explain the mechanisms of dynamic clustering of syntaxin in cell membranes, and the activation of fusion by PI-4,5-P2. Moreover, they suggest a working model for cell membrane regulation of syntaxin clustering.
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