Membrane fusion is the cell’s delivery process, enabling its many compartments to receive cargo and machinery for cell growth and intercellular communication. The overall activation energy of the process must be large enough to prevent frequent and nonspecific spontaneous fusion events, yet must be low enough to allow it to be overcome upon demand by specific fusion proteins [such as soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs)]. Remarkably, to the best of our knowledge, the activation energy for spontaneous bilayer fusion has never been measured. Multiple models have been developed and refined to estimate the overall activation energy and its component parts, and they span a very broad range from 20 kBT to 150 kBT, depending on the assumptions. In this study, using a bulk lipid-mixing assay at various temperatures, we report that the activation energy of complete membrane fusion is at the lowest range of these theoretical values. Typical lipid vesicles were found to slowly and spontaneously fully fuse with activation energies of ∼30 kBT. Our data demonstrate that the merging of membranes is not nearly as energy consuming as anticipated by many models and is ideally positioned to minimize spontaneous fusion while enabling rapid, SNARE-dependent fusion upon demand.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes are the core molecular machinery of membrane fusion, a fundamental process that drives inter- and intracellular communication and trafficking. One of the questions that remains controversial has been whether and how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure rings to template uniform-sized small unilamellar vesicles containing predetermined maximal number of externally facing SNAREs to study the membrane-fusion process. We also incorporated lipid-conjugated complementary ssDNA as tethers into vesicle and target membranes, which enabled bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. With this platform, we confirmed at the single event level that, after docking of the templated-SUVs to supported lipid bilayers (SBL), one to two pairs of SNAREs are sufficient to drive fast lipid mixing. Modularity and programmability of this platform makes it readily amenable to studying more complicated systems where auxiliary proteins are involved.

Here we introduce ApoE-based nanolipoprotein particle (NLP)—a soluble, discoidal bilayer mimetic of ∼23 nm in diameter, as fusion partners to study the dynamics of fusion pores induced by SNARE proteins. Using in vitro lipid mixing and content release assays, we report that NLPs reconstituted with synaptic v-SNARE VAMP2 (vNLP) fuse with liposomes containing the cognate t-SNARE (Syntaxin1/SNAP25) partner, with the resulting fusion pore opening directly to the external buffer. Efflux of encapsulated fluorescent dextrans of different sizes show that unlike the smaller nanodiscs, these larger NLPs accommodate the expansion of the fusion pore to at least ∼9 nm, and dithionite quenching of fluorescent lipid introduced in vNLP confirms that the NLP fusion pores are short-lived and eventually reseal. The NLPs also have capacity to accommodate larger number of proteins and using vNLPs with defined number of VAMP2 protein, including physiologically relevant copy numbers, we find that 3–4 copies of VAMP2 (minimum 2 per face) are required to keep a nascent fusion pore open, and the SNARE proteins act cooperatively to dilate the nascent fusion pore.

Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state.

The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Here we report an unexpected circular arrangement (ring) of SYT’s cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18–43 nm, corresponding to 11–26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide–sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded.

Two classes of proteins that bind to each other and to Golgi membranes have been implicated in the adhesion of Golgi cisternae to each other to form their characteristic stacks: Golgi reassembly and stacking proteins 55 and 65 (GRASP55 and GRASP65) and Golgin of 45 kDa and Golgi matrix protein of 130 kDa. We report here that efficient stacking occurs in the absence of GRASP65/55 when either Golgin is overexpressed, as judged by quantitative electron microscopy. The Golgi stacks in these GRASP-deficient HeLa cells were normal both in morphology and in anterograde cargo transport. This suggests the simple hypothesis that the total amount of adhesive energy gluing cisternae dictates Golgi cisternal stacking, irrespective of which molecules mediate the adhesive process. In support of this hypothesis, we show that adding artificial adhesive energy between cisternae and mitochondria by dimerizing rapamycin-binding domain and FK506-binding protein domains that are attached to cisternal adhesive proteins allows mitochondria to invade the stack and even replace Golgi cisternae within a few hours. These results indicate that although Golgi stacking is a highly complicated process involving a large number of adhesive and regulatory proteins, the overriding principle of a Golgi stack assembly is likely to be quite simple. From this simplified perspective, we propose a model, based on cisternal adhesion and cisternal maturation as the two core principles, illustrating how the most ancient form of Golgi stacking might have occurred using only weak cisternal adhesive processes because of the differential between the rate of influx and outflux of membrane transport through the Golgi.

Complexin (Cpx) is a SNARE-binding protein that regulates neurotransmission by clamping spontaneous synaptic vesicle fusion in the absence of Ca2+ influx while promoting evoked release in response to an action potential. Previous studies indicated Cpx may cross-link multiple SNARE complexes via a trans interaction to function as a fusion clamp. During Ca2+ influx, Cpx is predicted to undergo a conformational switch and collapse onto a single SNARE complex in a cis-binding mode to activate vesicle release. To test this model in vivo, we performed structure–function studies of the Cpx protein in Drosophila. Using genetic rescue approaches with cpx mutants that disrupt SNARE cross-linking, we find that manipulations that are predicted to block formation of the trans SNARE array disrupt the clamping function of Cpx. Unexpectedly, these same mutants rescue action potential-triggered release, indicating trans–SNARE cross-linking by Cpx is not a prerequisite for triggering evoked fusion. In contrast, mutations that impair Cpx-mediated cis–SNARE interactions that are necessary for transition from an open to closed conformation fail to rescue evoked release defects in cpx mutants, although they clamp spontaneous release normally. Our in vivo genetic manipulations support several predictions made by the Cpx cross-linking model, but unexpected results suggest additional mechanisms are likely to exist that regulate Cpx’s effects on SNARE-mediated fusion. Our findings also indicate that the inhibitory and activating functions of Cpx are genetically separable, and can be mapped to distinct molecular mechanisms that differentially regulate the SNARE fusion machinery.

Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes through pairing of vesicle-associated 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.

Cortical endoplasmic reticulum (cER) is a permanent feature of yeast cells but occurs transiently in most animal cell types. Ist2p is a transmembrane protein that permanently localizes to the cER in yeast. When Ist2 is expressed in mammalian cells, it induces abundant cER containing Ist2. Ist2 cytoplasmic C-terminal peptide is necessary and sufficient to induce cER. This peptide sequence resembles classic coat protein complex I (COPI) coatomer protein-binding KKXX signals, and indeed the dimerized peptide binds COPI in vitro. Controlled dimerization of this peptide induces cER in cells. RNA interference experiments confirm that coatomer is required for cER induction in vivo, as are microtubules and the microtubule plus-end binding protein EB1. We suggest that Ist2 dimerization triggers coatomer binding and clustering of this protein into domains that traffic at the microtubule growing plus-end to generate the cER beneath the plasma membrane. Sequences similar to the Ist2 lysine-rich tail are found in mammalian STIM proteins that reversibly induce the formation of cER under calcium control.

Abstract

Membrane fusion between vesicles and target membranes involves the zippering of a four-helix bundle generated by constituent helices derived from target– and vesicle–soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs). In neurons, the protein complexin clamps otherwise spontaneous fusion by SNARE proteins, allowing neurotransmitters and other mediators to be secreted when and where they are needed as this clamp is released. The membrane-proximal accessory helix of complexin is necessary for clamping, but its mechanism of action is unknown. Here, we present experiments using a reconstituted fusion system that suggest a simple model in which the complexin accessory helix forms an alternative four-helix bundle with the target-SNARE near the membrane, preventing the vesicle-SNARE from completing its zippering.

Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE)-mediated lipid mixing can be efficiently recapitulated in vitro by the incorporation of purified vesicle membrane (-v) SNARE and target membrane (t-) SNARE proteins into separate liposome populations. Despite the strong correlation between the observed activities in this system and the known SNARE physiology, some recent works have suggested that SNARE-mediated lipid mixing may be limited to circumstances where membrane defects arise from artifactual reconstitution conditions (such as nonphysiological high-protein concentrations or unrealistically small liposome populations). Here, we show that the previously published strategies used to reconstitute SNAREs into liposomes do not significantly affect either the physical parameters of the proteoliposomes or the ability of SNAREs to drive lipid mixing in vitro. The surface density of SNARE proteins turns out to be the most critical parameter, which controls both the rate and the extent of SNARE-mediated liposome fusion. In addition, the specific activity of the t-SNARE complex is significantly influenced by expression and reconstitution protocols, such that we only observe optimal lipid mixing when the t-SNARE proteins are coexpressed before purification.