Factor Attachment Protein Receptor Research Articles

Roles of the fission yeast UNC-13/Munc13 protein Ync13 in late stages of cytokinesis

Cytokinesis is a complicated yet conserved step of the cell-division cycle that requires the coordination of multiple proteins and cellular processes. Here we describe a previously uncharacterized protein, Ync13, and its roles during fission yeast cytokinesis. Ync13 is a member of the UNC-13/Munc13 protein family, whose animal homologues are essential priming factors for soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex assembly during exocytosis in various cell types, but no roles in cytokinesis have been reported. We find that Ync13 binds to lipids in vitro and dynamically localizes to the plasma membrane at cell tips during interphase and at the division site during cytokinesis. Deletion of Ync13 leads to defective septation and exocytosis, uneven distribution of cell-wall enzymes and components of cell-wall integrity pathway along the division site and massive cell lysis during cell separation. Interestingly, loss of Ync13 compromises endocytic site selection at the division plane. Collectively, we find that Ync13 has a novel function as an UNC-13/Munc13 protein in coordinating exocytosis, endocytosis, and cell-wall integrity during fission yeast cytokinesis.

Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis

Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca2+). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca2+-dependent release has been unclear. Here, we report that the Ca2+-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca2+-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca2+-stimulated release. We propose a parsimonious model whereby Ca2+-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca2+ Our data further suggest Ca2+-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery.

Emerging evidence for the modulation of exocytosis by signalling lipids.

Membrane fusion is a key event in exocytosis of neurotransmitters and hormones stored in intracellular vesicles. In this process, soluble N‐ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins are essential components of the exocytotic molecular machinery, while lipids have been seen traditionally as structural elements. However, the so‐called signalling lipids, such as sphingosine and arachidonic acid, interact with SNAREs and directly modulate the frequency and mode of fusion events. Interestingly, recent work has proved that the sphingosine analogue FTY‐720, used in the treatment of multiple sclerosis, mimics the effects of signalling lipids. In the present Review, we discuss recent investigations suggesting that endogenous signalling lipids and synthetic analogues can modulate important physiological aspects of secretion, such as quantal release, vesicle recruitment into active sites, vesicle transport and even organelle fusion in the cytosol. Therefore, these compounds are far from being merely structural components of cellular membranes.

Mechanism of neurotransmitter release coming into focus.

Research for three decades and major recent advances have provided crucial insights into how neurotransmitters are released by Ca2+ -triggered synaptic vesicle exocytosis, leading to reconstitution of basic steps that underlie Ca2+ -dependent membrane fusion and yielding a model that assigns defined functions for central components of the release machinery. The soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) syntaxin-1, SNAP-25, and synaptobrevin-2 form a tight SNARE complex that brings the vesicle and plasma membranes together and is key for membrane fusion. N-ethyl maleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) disassemble the SNARE complex to recycle the SNAREs for another round of fusion. Munc18-1 and Munc13-1 orchestrate SNARE complex formation in an NSF-SNAP-resistant manner by a mechanism whereby Munc18-1 binds to synaptobrevin and to a self-inhibited "closed" conformation of syntaxin-1, thus forming a template to assemble the SNARE complex, and Munc13-1 facilitates assembly by bridging the vesicle and plasma membranes and catalyzing opening of syntaxin-1. Synaptotagmin-1 functions as the major Ca2+ sensor that triggers release by binding to membrane phospholipids and to the SNAREs, in a tight interplay with complexins that accelerates membrane fusion. Many of these proteins act as both inhibitors and activators of exocytosis, which is critical for the exquisite regulation of neurotransmitter release. It is still unclear how the actions of these various proteins and multiple other components that control release are integrated and, in particular, how they induce membrane fusion, but it can be expected that these fundamental questions can be answered in the near future, building on the extensive knowledge already available.

Light Chain Diversity among the Botulinum Neurotoxins.

Botulinum neurotoxins (BoNT) are produced by several species of clostridium. There are seven immunologically unique BoNT serotypes (A–G). The Centers for Disease Control classifies BoNTs as ‘Category A’ select agents and are the most lethal protein toxins for humans. Recently, BoNT-like proteins have also been identified in several non-clostridia. BoNTs are di-chain proteins comprised of an N-terminal zinc metalloprotease Light Chain (LC) and a C-terminal Heavy Chain (HC) which includes the translocation and receptor binding domains. The two chains are held together by a disulfide bond. The LC cleaves Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The cleavage of SNAREs inhibits the fusion of synaptic vesicles to the cell membrane and the subsequent release of acetylcholine, which results in flaccid paralysis. The LC controls the catalytic properties and the duration of BoNT action. This review discusses the mechanism for LC catalysis, LC translocation, and the basis for the duration of LC action. Understanding these properties of the LC may expand the applications of BoNT as human therapies.

Endosomal and Phagosomal SNAREs.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein family is of vital importance for organelle communication. The complexing of cognate SNARE members present in both the donor and target organellar membranes drives the membrane fusion required for intracellular transport. In the endocytic route, SNARE proteins mediate trafficking between endosomes and phagosomes with other endosomes, lysosomes, the Golgi apparatus, the plasma membrane, and the endoplasmic reticulum. The goal of this review is to provide an overview of the SNAREs involved in endosomal and phagosomal trafficking. Of the 38 SNAREs present in humans, 30 have been identified at endosomes and/or phagosomes. Many of these SNAREs are targeted by viruses and intracellular pathogens, which thereby reroute intracellular transport for gaining access to nutrients, preventing their degradation, and avoiding their detection by the immune system. A fascinating picture is emerging of a complex transport network with multiple SNAREs being involved in consecutive trafficking routes.

Oligomerisation of Synaptobrevin-2 Studied by Native Mass Spectrometry and Chemical Cross-Linking

Synaptobrevin-2 is a key player in signal transmission in neurons. It forms, together with SNAP25 and Syntaxin-1A, the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and mediates exocytosis of synaptic vesicles with the pre-synaptic membrane. While Synaptobrevin-2 is part of a four-helix bundle in this SNARE complex, it is natively unstructured in the absence of lipids or other SNARE proteins. Partially folded segments, presumably SNARE complex formation intermediates, as well as formation of Synaptobrevin-2 dimers and oligomers, were identified in previous studies. Here, we employ three Synaptobrevin-2 variants—the full-length protein Syb(1-116), the soluble, cytosolic variant Syb(1-96) as well as a shorter version Syb(49-96) containing structured segments but omitting a trigger site for SNARE complex formation—to study oligomerisation in the absence of interaction partners or when incorporated into the lipid bilayer of liposomes. Combining native mass spectrometry with chemical cross-linking, we find that the truncated versions show increased oligomerisation. Our findings from both techniques agree well and confirm the presence of oligomers in solution while membrane-bound Synaptobrevin-2 is mostly monomeric. Using ion mobility mass spectrometry, we could further show that lower charge states of Syb(49-96) oligomers, which most likely represent solution structures, follow an isotropic growth curve suggesting that they are intrinsically disordered. From a technical point of view, we show that the combination of native ion mobility mass spectrometry with chemical cross-linking is well-suited for the analysis of protein homo-oligomers.Graphical ᅟ

Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.

Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17.

Macroautophagy is an evolutionarily conserved catabolic mechanism that delivers intracellular constituents to lysosomes using autophagosomes. To achieve degradation, lysosomes must fuse with closed autophagosomes. We previously reported that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (STX) 17 translocates to autophagosomes to mediate fusion with lysosomes. In this study, we report an additional mechanism. We found that autophagosome-lysosome fusion is retained to some extent even in STX17 knockout (KO) HeLa cells. By screening other human SNAREs, we identified YKT6 as a novel autophagosomal SNARE protein. Depletion of YKT6 inhibited autophagosome-lysosome fusion partially in wild-type and completely in STX17 KO cells, suggesting that YKT6 and STX17 are independently required for fusion. YKT6 formed a SNARE complex with SNAP29 and lysosomal STX7, both of which are required for autophagosomal fusion. Recruitment of YKT6 to autophagosomes depends on its N-terminal longin domain but not on the C-terminal palmitoylation and farnesylation that are essential for its Golgi localization. These findings suggest that two independent SNARE complexes mediate autophagosome-lysosome fusion.

Molecular Mechanisms of Fast Neurotransmitter Release.

This review summarizes current knowledge of synaptic proteins that are central to synaptic vesicle fusion in presynaptic active zones, including SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), synaptotagmin, complexin, Munc18 (mammalian uncoordinated-18), and Munc13 (mammalian uncoordinated-13), and highlights recent insights in the cooperation of these proteins for neurotransmitter release. Structural and functional studies of the synaptic fusion machinery suggest new molecular models of synaptic vesicle priming and Ca2+-triggered fusion. These studies will be a stepping-stone toward answering the question of how the synaptic vesicle fusion machinery achieves such high speed and sensitivity.

Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor-mediated phagosome formation and maturation in macrophages.

SNAP-23 is a plasma membrane-localized soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNARE) involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation-specific antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established macrophage lines overexpressing the nonphosphorylatable S95A or the phosphomimicking S95D mutation. The efficiency of phagosome formation and maturation was severely reduced in SNAP-23-S95D–overexpressing cells. To examine whether phosphorylation at Ser95 affected SNAP-23 structure, we constructed intramolecular Förster resonance energy transfer (FRET) probes of SNAP-23 designed to evaluate the approximation of the N termini of the two SNARE motifs. Interestingly, a high FRET efficiency was detected on the membrane when the S95D probe was used, indicating that phosphorylation at Ser95 caused a dynamic structural shift to the closed form. Coexpression of IκB kinase (IKK) 2 enhanced the FRET efficiency of the wild-type probe on the phagosome membrane. Furthermore, the enhanced phagosomal FRET signal in interferon-γ–activated macrophages was largely dependent on IKK2, and this kinase mediated a delay in phagosome-lysosome fusion. These results suggested that SNAP-23 phosphorylation at Ser95 played an important role in the regulation of SNARE-dependent membrane fusion during FcR-mediated phagocytosis.

Activation of disease resistance against Botryosphaeria dothidea by downregulating the expression of MdSYP121 in apple

In plants, the vesicle fusion process plays a vital role in pathogen defence. However, the importance of the vesicle fusion process in apple ring rot has not been studied. Here, we isolated and characterised the apple syntaxin gene MdSYP121. Silencing the MdSYP121 gene in transgenic apple calli increased tolerance to Botryosphaeria dothidea infection; this increased tolerance was correlated with salicylic acid (SA) synthesis-related and signalling-related gene transcription. In contrast, overexpressing MdSYP121 in apple calli resulted in the opposite phenotypes. In addition, the results of RNA sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) assays suggested that MdSYP121 plays an important role in responses to oxidation–reduction reactions. Silencing MdSYP121 in apple calli enhanced the expression levels of reactive oxygen species (ROS)-related genes and the activity of ROS-related enzymes. The enhanced defence response status in MdSYP121-RNAi lines suggests that syntaxins are involved in the defence response to B. dothidea. More importantly, we showed that MdSYP121 forms a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex with MdSNAP33, and the complex may participate in regulating resistance to B. dothidea. In conclusion, by regulating the interaction of SA pathway and oxidation–reduction process, MdSYP121 can influence the pathogen infection process in apple.

Ca2+-Triggered Synaptic Vesicle Fusion Initiated by Release of Inhibition.

Recent structural and functional studies of the synaptic vesicle fusion machinery suggest an inhibited tripartite complex consisting of neuronal soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), synaptotagmin, and complexin prior to Ca2+-triggered synaptic vesicle fusion. We speculate that Ca2+-triggered fusion commences with the release of inhibition by Ca2+ binding to synaptotagmin C2 domains. Subsequently, fusion is assisted by SNARE complex zippering and by active membrane remodeling properties of synaptotagmin. This additional, inhibitory role of synaptotagmin may be a general principle since other recent studies suggest that Ca2+ binding to extended synaptotagmin C2 domains enables lipid transport by releasing an inhibited state of the system, and that Munc13 may nominally be in an inhibited state, which is released upon Ca2+ binding to one of its C2 domains.

Exploiting Botulinum Neurotoxins for the Study of Brain Physiology and Pathology.

Botulinum neurotoxins are metalloproteases that specifically cleave N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in synaptic terminals, resulting in a potent inhibition of vesicle fusion and transmitter release. The family comprises different serotypes (BoNT/A to BoNT/G). The natural target of these toxins is represented by the neuromuscular junction, where BoNTs block acetylcholine release. In this review, we describe the actions of botulinum toxins after direct delivery to the central nervous system (CNS), where BoNTs block exocytosis of several transmitters, with near-complete silencing of neural networks. The use of clostridial neurotoxins in the CNS has allowed us to investigate specifically the role of synaptic activity in different physiological and pathological processes. The silencing properties of BoNTs can be exploited for therapeutic purposes, for example to counteract pathological hyperactivity and seizures in epileptogenic brain foci, or to investigate the role of activity in degenerative diseases like prion disease. Altogether, clostridial neurotoxins and their derivatives hold promise as powerful tools for both the basic understanding of brain function and the dissection and treatment of activity-dependent pathogenic pathways.

High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis.

In vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of fusion entails an accurate in vitro reproduction of the process. To date, supported bilayers have been commonly used to mimic the planar membranes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that induce membrane fusion usually have limited fluidity when embedded in supported bilayers. This alters the kinetics and prevents correct reconstitution of the overall fusion process. Also, observing content release across the membrane is hindered by the lack of a second aqueous compartment. Recently, a step toward resolving these issues was achieved by using membranes spread on holey substrates. The mobility of proteins was preserved but vesicles were prone to bind to the substrate when reaching the edge of the hole, preventing the observation of many fusion events over the suspended membrane. Building on this recent advance, we designed a method for the formation of pore-spanning lipid bilayers containing t-SNARE proteins on Si/SiO2 holey chips, allowing the observation of many individual vesicle fusion events by both lipid mixing and content release. With this setup, proteins embedded in the suspended membrane bounced back when they reached the edge of the hole which ensured vesicles did not bind to the substrate. We observed SNARE-dependent membrane fusion with the freestanding bilayer of about 500 vesicles. The time between vesicle docking and fusion is ∼1 s. We also present a new multimodal open-source software, Fusion Analyzer Software, which is required for fast data analysis.

Doc2b Protects β-Cells Against Inflammatory Damage and Enhances Function.

Loss of functional β-cell mass is an early feature of type 1 diabetes. To release insulin, β-cells require soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, as well as SNARE complex regulatory proteins like double C2 domain-containing protein β (Doc2b). We hypothesized that Doc2b deficiency or overabundance may confer susceptibility or protection, respectively, to the functional β-cell mass. Indeed, Doc2b+/- knockout mice show an unusually severe response to multiple-low-dose streptozotocin (MLD-STZ), resulting in more apoptotic β-cells and a smaller β-cell mass. In addition, inducible β-cell-specific Doc2b-overexpressing transgenic (βDoc2b-dTg) mice show improved glucose tolerance and resist MLD-STZ-induced disruption of glucose tolerance, fasting hyperglycemia, β-cell apoptosis, and loss of β-cell mass. Mechanistically, Doc2b enrichment enhances glucose-stimulated insulin secretion (GSIS) and SNARE activation and prevents the appearance of apoptotic markers in response to cytokine stress and thapsigargin. Furthermore, expression of a peptide containing the Doc2b tandem C2A and C2B domains is sufficient to confer the beneficial effects of Doc2b enrichment on GSIS, SNARE activation, and apoptosis. These studies demonstrate that Doc2b enrichment in the β-cell protects against diabetogenic and proapoptotic stress. Furthermore, they identify a Doc2b peptide that confers the beneficial effects of Doc2b and may be a therapeutic candidate for protecting functional β-cell mass.

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation.

Vesicle-associated V-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans-SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

Involvement of SNARE complex in the hippocampus and prefrontal cortex of offspring with depression induced by prenatal stress

BackgroundPrenatal stress (PS) exposure can cause depression-like behavior in offspring, and maladaptive responses including physiological and neurobiological changes. Glutamate neurotransmission is implicated in effects of PS and in antidepressant mechanisms; however, the mechanisms underlying its involvement remain unclear. In the synapse, the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for vesicular docking and neurotransmitter release. MethodsTo explore effects of PS on the SNARE complex, pregnant rats were assigned to a control or PS group. Both male and female offspring in each group were used in this study. PS rats were exposed to restraint stress three times daily for 45 min on days 14–20 of pregnancy. ResultsIn the PS offspring, the expression of the SNARE protein SNAP-25, vesicle-associated membrane protein (VAMP)-2, and Syntaxin 1a was significantly increased in the hippocampus and prefrontal cortex. These observations were associated with increased levels of proteins that chaperone SNARE complex formation, including Munc-18, α-synuclein, CSPα, complexin1, and complexin2. Immunoblotting of hippocampal and prefrontal cortex homogenates revealed significantly increased SNARE complex formation. vGluT1 protein expression was also significantly increased in the offspring. Additionally, PS was associated with increased mRNA expression of VAMP1, VAMP2, SNAP25, Syntaxin1a, and Syntaxin1b in the hippocampus and prefrontal cortex. Increased monomeric SNARE proteins, SNARE complex formation, vesicle-associated proteins, and vGluT1 may explain the increase in glutamate and its downstream excitotoxicity. ConclusionsThese results support the hypothesis that glutamate release and vesicular glutamate transporters play a role in PS-induced depression-like behavior of rat offspring.

Modulation of Synaptic transmission: Quantitative analysis of Gβγ specificity to adrenergic α2a receptor and SNARE.

Modulation of neurotransmitter exocytosis by activated Gi/o coupled G‐protein coupled receptors (GPCRs) is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. One of the known modulation mechanisms is Gβγ interaction with soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE). There are 5 Gβ and 12 Gγ subunits, but specific Gβγs activated by a given GPCR and the specificity to effectors in vivo are not known. Presynaptic α2a‐adrenergic receptors (α2aARs) in both adrenergic (auto α2aARs) and non‐adrenergic neurons (hetero α2aARs) inhibit neurotransmitter release and affect various physiological functions such as anesthetic sparing and working memory enhancement. With a quantitative MRM proteomic analysis of neuronal Gβ and Gγ subunits to detect neuronal Gβ and Gγ subunits, several mouse models including transgenic Flag‐α2aARs, knock‐in HA‐α2aARs, and other biochemical techniques such as co‐immunoprecipitation, we investigate the specificity of Gβ and Gγ subunits to α2aARs in both adrenergic(auto α2aARs) and non‐adrenergic neurons (hetero α2aARs), and SNARE in presence of epinephrine. Gβ2, Gγ2, Gγ3, and Gγ4 preferentially interact with activated auto α2aARs while Gβ4 and Gγ12 preferentially interact with activated hetero α2aARs. We also detect a subset of Gβ and Gγ subunits interacting with SNARE upon α2aARs activation. Further understanding of Gβγ specificity on its downstream signaling, especially Gβγ‐SNARE interaction, offers new insights into the normal functioning of the brain and lead to an identification of potential pathophysiological states in which the Gβγ‐SNARE interaction may be dysregulated. These studies yield additional insights into Gi/o‐coupled GPCR‐mediated regulation of exocytosis.Support or Funding InformationThis work was supported by the National Institutes of Health (EY10291, MH101679, DK109204, and T32 GM07628).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SNARE complex assembly and disassembly

A fundamental hallmark of eukaryotic cells is their compartmentalization into functionally distinct organelles, including those of the secretory and endocytic pathways. Transport of cargo between these compartments and to/from the cell surface is mediated by membrane-bound vesicles and tubules. Delivery of cargo is facilitated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated membrane fusion of vesicles with their target compartments. Vesicles contain a variety of cargos, including lipids, membrane proteins, signaling molecules, biosynthetic and hydrolytic enzymes, and the trafficking machinery itself. Proper function of membrane trafficking is required for cellular growth, division, movement, and cell-cell communication. Defects in these processes have been implicated in a variety of human diseases, such as cancer, diabetes, neurodegenerative disorders, ciliopathies, and infections. The elucidation of the mechanisms of SNARE assembly and disassembly is key to understanding how membrane fusion is regulated throughout eukaryotes. Here, we introduce the SNARE proteins, their structures and functions in eukaryotic cells, and discuss recent breakthroughs in elucidating the regulation of SNARE assembly and disassembly through the use of high-resolution structural biology and biophysical techniques.