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ABSTRACT: Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex provides mechanical thrust for membrane fusion, but its molecular mechanism is still unclear. Here using magnetic tweezers, we observe mechanical responses of a single neuronal SNARE complex under constant pulling force. Single SNARE complexes may be unzipped with 34 pN force. When rezipping is induced by lowering the force to 11 pN, only a partially assembled state results, with the C-terminal half of the SNARE complex remaining disassembled. Reassembly of the C-terminal half occurs only when the force is further lowered below 11 pN. Thus, mechanical hysteresis, characterized by the unzipping and rezipping cycle of a single SNARE complex, produces the partially assembled state. In this metastable state, unzipping toward the N-terminus is suppressed while zippering toward the C-terminus is initiated as a steep function of force. This ensures the directionality of SNARE-complex formation, making the SNARE complex a robust force-generating machine.
Nature Communications 04/2013; 4:1705. · 7.40 Impact Factor
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ABSTRACT: Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity.
Proceedings of the National Academy of Sciences 03/2013; 110(10):4087-92. · 9.68 Impact Factor
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ABSTRACT: Fusion pore formation and expansion, crucial steps for neurotransmitter release and vesicle recycling in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicle fusion, have not been well studied in vitro due to the lack of a reliable content-mixing fusion assay. Using methods detecting the intervesicular mixing of small and large cargoes at a single-vesicle level, we found that the neuronal SNARE complexes have the capacity to drive membrane hemifusion. However, efficient fusion pore formation and expansion require synaptotagmin 1 and Ca(2+). Real-time measurements show that pore expansion detected by content mixing of large DNA cargoes occurs much slower than initial pore formation that transmits small cargoes. Slow pore expansion perhaps provides a time window for vesicles to escape the full collapse fusion pathway via alternative mechanisms such as kiss-and-run. The results also show that complexin 1 stimulates pore expansion significantly, which could put bias between two pathways of vesicle recycling.
Proceedings of the National Academy of Sciences 01/2013; · 9.68 Impact Factor
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Yoosoo Yang,
Jung-Mi Oh,
Paul Heo,
Jae Yoon Shin,
Byoungjae Kong,
Jonghyeok Shin,
Ji-Chun Lee,
Jeong Su Oh,
Kye Won Park,
Chung Hwan Lee, Yeon-Kyun Shin,
Dae-Hyuk Kweon
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ABSTRACT: Anti-allergic effects of dietary polyphenols were extensively studied in numerous allergic disease models, but the molecular mechanisms of anti-allergic effects by polyphenols remain poorly understood. Here, we show that the release of granular cargo molecules, contained in distinct subsets of granules of mast cells, is specifically mediated by two sets of SNARE (soluble N-ethyl-maleimide-sensitive factor attachment protein receptors) proteins, and that various polyphenols differentially inhibit the formation of those SNARE complexes. Expression analysis of RBL-2H3 cells for 11 SNARE protein genes and lipid mixing assay of 24 possible combinations of reconstituted SNARE proteins indicated that the only two active SNARE complexes involved in mast cell degranulation are syntaxin 4/SNAP-23/VAMP2 and syntaxin 4/SNAP-23/VAMP8. Various polyphenols selectively or commonly interfered with ternary complex formation of these 2 SNARE complexes, thereby stopping membrane fusion between granules and plasma membrane. This led to the differential effect of polyphenols on degranulation of 3 distinct subsets of granules. These results implicate the possibility that formation of a variety of SNARE complexes in numerous cell types is controlled by polyphenols which, in turn, might regulate corresponding membrane trafficking.
Biochemical Journal 12/2012; · 4.90 Impact Factor
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ABSTRACT: SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d.
Nature Protocol 05/2012; 7(5):921-34. · 8.36 Impact Factor
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Chang Hwa Jung,
Yoo Soo Yang,
Jun-Seob Kim, Yeon-Kyun Shin,
Jae Sung Hwang,
Eui Dong Son,
Hong Hwa Lee,
Koo Min Chung,
Jung Mi Oh,
Jong Hwa Lee,
Dae-Hyuk Kweon
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ABSTRACT: Neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins mediate membrane fusion between synaptic
vesicle and presynaptic membrane, resulting in neurotransmitter release. SNARE proteins are specific substrates of botulinum
neurotoxins (BoNT) which are now widely used for therapeutic and cosmetic purposes. While BoNT blocks neuroexocytosis by cleaving
SNAREs, inhibiting SNARE assembly process might exert the same effect on neurotransmission. In the present study, some extracts
of 100 plants reduced neurotransmitter release by inhibiting SNARE complex formation in neuronal cells. The extracts effectively
paralyzed muscle of rat phrenic nerve-hemidiaphragm preparation. Our results raise the possibility that SNARE folding inhibitors
from natural resources might replace some special BoNT application fields.
Biotechnology Letters 04/2012; 31(3):361-369. · 1.68 Impact Factor
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ABSTRACT: Synaptotagmin-1 (Syt1) is a major Ca(2+) sensor for synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Syt1 utilizes its diverse interactions with target membrane (t-) SNARE, SNAREpin, and phospholipids, to regulate vesicle fusion. To dissect the functions of Syt1, we apply a single-molecule technique, alternating-laser excitation (ALEX), which is capable of sorting out subpopulations of fusion intermediates and measuring their kinetics in solution. The results show that Syt1 undergoes at least three distinct steps prior to lipid mixing. First, without Ca(2+), Syt1 mediates vesicle docking by directly binding to t-SNARE/phosphatidylinositol 4,5-biphosphate (PIP(2)) complex and increases the docking rate by 10(3) times. Second, synaptobrevin-2 binding to t-SNARE displaces Syt1 from SNAREpin. Third, with Ca(2+), Syt1 rebinds to SNAREpin, which again requires PIP(2). Thus without Ca(2+), Syt1 may bring vesicles to the plasma membrane in proximity via binding to t-SNARE/PIP(2) to help SNAREpin formation and then, upon Ca(2+) influx, it may rebind to SNAREpin, which may trigger synchronous fusion. The results show that ALEX is a powerful method to dissect multiple kinetic steps in the vesicle fusion pathway.
The EMBO Journal 03/2012; 31(9):2144-55. · 9.20 Impact Factor
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ABSTRACT: Syt1 (synaptotagmin 1) is a major Ca2+ sensor for synaptic vesicle fusion. Although Syt1 is known to bind to SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and to the membrane, the mechanism by which Syt1 regulates vesicle fusion is controversial. In the present study we used in vitro lipid-mixing assays to investigate the Ca2+-dependent Syt1 function in proteoliposome fusion. To study the role of acidic lipids, the concentration of negatively charged DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) in the vesicle was varied. Syt1 stimulated lipid mixing by 3-10-fold without Ca2+. However, with Ca2+ there was an additional 2-5-fold enhancement. This Ca2+-dependent stimulation was observed only when there was excess PS (phosphatidylserine) on the t-SNARE (target SNARE) side. If there was equal or more PS on the v-SNARE (vesicule SNARE) side the Ca2+-dependent stimulation was not observed. We found that Ca2+ at a concentration between 10 and 50 μM was sufficient to give rise to the maximal enhancement. The single-vesicle-fusion assay indicates that the Ca2+-dependent enhancement was mainly on docking, whereas its effect on lipid mixing was small. Thus for Syt1 to function as a Ca2+ sensor, a charge asymmetry appears to be important and this may play a role in steering Syt1 to productively trans bind to the plasma membrane.
Biochemical Journal 01/2012; 443(1):223-9. · 4.90 Impact Factor
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Current Topics in Membranes 01/2011; 68:161-84. · 0.59 Impact Factor
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Yoosoo Yang,
Jae Yoon Shin,
Jung-Mi Oh,
Chang Hwa Jung,
Yunha Hwang,
Sehyun Kim,
Jun-Seob Kim,
Kee-Jung Yoon,
Ji-Young Ryu,
Jaeil Shin,
Jae Sung Hwang,
Tae-Young Yoon, Yeon-Kyun Shin,
Dae-Hyuk Kweon
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ABSTRACT: Neuronal SNARE proteins mediate neurotransmitter release at the synapse by facilitating the fusion of vesicles to the presynaptic plasma membrane. Cognate v-SNAREs and t-SNAREs from the vesicle and the plasma membrane, respectively, zip up and bring about the apposition of two membranes attached at the C-terminal ends. Here, we demonstrate that SNARE zippering can be modulated in the midways by wedging with small hydrophobic molecules. Myricetin, which intercalated into the hydrophobic inner core near the middle of the SNARE complex, stopped SNARE zippering in motion and accumulated the trans-complex, where the N-terminal region of v-SNARE VAMP2 is in the coiled coil with the frayed C-terminal region. Delphinidin and cyanidin inhibited N-terminal nucleation of SNARE zippering. Neuronal SNARE complex in PC12 cells showed the same pattern of vulnerability to small hydrophobic molecules. We propose that the half-zipped trans-SNARE complex is a crucial intermediate waiting for a calcium trigger that leads to fusion pore opening.
Proceedings of the National Academy of Sciences 12/2010; 107(51):22145-50. · 9.68 Impact Factor
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ABSTRACT: The in vitro studies of membrane fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have primarily been conducted by following the mixing of lipids. However, the formation of a fusion pore and its expansion has been difficult to detect directly because of the leakiness of proteoliposomes, vesicle aggregation and rupture that often complicate the interpretation of ensemble fusion experiments. Fusion pore expansion is an essential step for full-collapse fusion and for recycling of fusion mechanisms. Here, we demonstrate a method to detect the inter-vesicular mixing of large cargoes at the single-molecule and -vesicle level. The change in fluorescence resonance energy transfer signal when a DNA hairpin encapsulated in a surface-tethered vesicle encounters a complementary DNA strand from another vesicle indicates content mixing. We found that the yeast SNARE complex alone without any accessory proteins can expand the fusion pore large enough to transmit ~11 kDa cargoes.
Nature Communications 08/2010; 1:54. · 7.40 Impact Factor
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Yeon-Kyun Shin
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ABSTRACT: The functionally important extracellular loop is not resolved in the crystal structures of a putative bacterial Mg(2+) channel CorA. In this issue, Dalmas et al. use EPR to determine a structural model for this conserved loop, providing new insight into the ion selectivity.
Structure 07/2010; 18(7):759-60. · 6.35 Impact Factor
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ABSTRACT: In neurons, synaptotagmin 1 (Syt1) is thought to mediate the fusion of synaptic vesicles with the plasma membrane when presynaptic Ca2+ levels rise. However, in vitro reconstitution experiments have failed to recapitulate key characteristics of Ca2+-triggered membrane fusion. Using an in vitro single-vesicle fusion assay, we found that membrane-anchored Syt1 enhanced Ca2+ sensitivity and fusion speed. This stimulatory activity of membrane-anchored Syt1 dropped as the Ca2+ level rose beyond physiological levels. Thus, Syt1 requires the membrane anchor to stimulate vesicle fusion at physiological Ca2+ levels and may function as a dynamic presynaptic Ca2+ sensor to control the probability of neurotransmitter release.
Science 05/2010; 328(5979):760-3. · 31.20 Impact Factor
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ABSTRACT: Munc18, an essential regulatory protein for intracellular membrane fusion mediated by SNAREs, is known for stabilizing the closed conformation of syntaxin through the interaction with the N-terminal Habc domain (amino acids 28-146) of syntaxin. In addition, Munc18 accelerates membrane fusion and its interaction with SNARE core and the N-peptide (amino acids 1-24) of syntaxin is thought to be necessary for this function. Using the recently developed fluorescence resonance energy transfer assay to detect the fusion between two individual vesicles harboring cognate SNARE proteins, we studied the effect of Munc18 on the fusion induced by neuronal SNARE proteins by following the mixing of lipid molecules between the two vesicles. We found that Munc18-1 stimulates neuronal SNARE-mediated fusion not only with full-length syntaxin 1A but also with a truncated syntaxin 1A that is missing both the Habc domain and the N-peptide. The electron paramagnetic resonance analysis indicates that the SNARE core/Munc18 interaction is responsible for this stimulatory function and the membrane plays a role for establishing this interaction.
ACS Chemical Neuroscience 03/2010; 1(3):168-174. · 3.68 Impact Factor
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ABSTRACT: The calcium-triggered neurotransmitter release requires three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: synaptobrevin 2 (or vesicle-associated membrane protein 2) on the synaptic vesicle and syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa) at the presynaptic plasma membrane. This minimal fusion machinery is believed to drive fusion of the vesicle to the presynaptic membrane. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a major regulator of synaptic vesicle exocytosis. Stimulatory and inhibitory effects of complexin have both been reported, suggesting the duality of its function. To shed light on the molecular basis of the complexin's dual function, we have performed an EPR investigation of the complexin-SNARE quaternary complex. We found that the accessory alpha-helix (amino acids 27-48) by itself has the capacity to replace the C-terminus of the SNARE motif of vesicle-associated membrane protein 2 in the four-helix bundle and makes the SNARE complex weaker when the N-terminal region of complexin I (amino acids 1-26) is removed. However, the accessory alpha-helix remains detached from the SNARE core when the N-terminal region of complexin I is present. Thus, our data show the possibility that the balance between the activities of the accessory alpha-helix and the N-terminal domain might determine the final outcome of the complexin function, either stimulatory or inhibitory.
Journal of Molecular Biology 12/2009; 396(3):602-9. · 4.00 Impact Factor
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Youngdae Yoon,
Jiansong Tong,
Park Joo Lee,
Alexandra Albanese,
Nitin Bhardwaj,
Morten Källberg,
Michelle A Digman,
Hui Lu,
Enrico Gratton, Yeon-Kyun Shin,
Wonhwa Cho
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ABSTRACT: The mechanisms by which cytosolic proteins reversibly bind the membrane and induce the curvature for membrane trafficking and remodeling remain elusive. The epsin N-terminal homology (ENTH) domain has potent vesicle tubulation activity despite a lack of intrinsic molecular curvature. EPR revealed that the N-terminal alpha-helix penetrates the phosphatidylinositol 4,5-bisphosphate-containing membrane at a unique oblique angle and concomitantly interacts closely with helices from neighboring molecules in an antiparallel orientation. The quantitative fluorescence microscopy showed that the formation of highly ordered ENTH domain complexes beyond a critical size is essential for its vesicle tubulation activity. The mutations that interfere with the formation of large ENTH domain complexes abrogated the vesicle tubulation activity. Furthermore, the same mutations in the intact epsin 1 abolished its endocytic activity in mammalian cells. Collectively, these results show that the ENTH domain facilitates the cellular membrane budding and fission by a novel mechanism that is distinct from that proposed for BAR domains.
Journal of Biological Chemistry 11/2009; 285(1):531-40. · 4.77 Impact Factor
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ABSTRACT: Artificial particulate systems such as lipid vesicles are found in a variety of biomedical applications such as drug delivery and targeting. More versatile layers of control would be added if liposomes could be glued together on demand while stabilized against fusion. Here, we present a two-component molecular glue composed of a protein and calcium ions, with each component specialized for fast and specific binding to negatively charged lipid membranes. Upon mixing the two components, the high affinity binding of this glue starts to tightly bridge two lipid vesicles on a subsecond scale. Furthermore, highly charged liposomes are beneficial in preventing spontaneous fusion before applying the molecular glue.
Langmuir 08/2009; 25(13):7177-80. · 4.19 Impact Factor
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ABSTRACT: Conversion of the normal soluble form of prion protein, PrP (PrP(C)), to proteinase K-resistant form (PrP(Sc)) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from alpha-helix to beta-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111-135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.
Molecules and Cells 07/2009; 27(6):673-80. · 2.18 Impact Factor
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ABSTRACT: Cholesterol is a major component of biological membranes and is known to affect vesicle fusion. However, the mechanism by which cholesterol modulates SNARE-dependent intracellular fusion is not well understood. Using the fluorescence assay and dye-labeled SNAREs and the fluorescent lipids, we dissected cholesterol effects on individual fusion steps including SNARE complex formation, hemifusion, pore formation, and pore dilation. At physiological high concentrations, cholesterol stimulated hemifusion as much as 30-fold, but its stimulatory effect diminished to 10-fold and three-fold for subsequent pore formation and pore expansion at 40 mol %, respectively. The results show that cholesterol serves as a strong stimulator for hemifusion but acts as mild stimulators for pore opening and expansion. Strong stimulation of hemifusion and mild stimulation of pore formation are consistent with the fusion model based on the intrinsic negative curvature of cholesterol. However, even a milder effect of cholesterol on pore expansion is contradictory to such a simple curvature-based prediction. Thus, we speculate that cholesterol also affects the conformation of the transmembrane domains of SNAREs, which modulates the fusion kinetics.
Biophysical Journal 04/2009; 96(5):1839-46. · 3.65 Impact Factor
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ABSTRACT: Neurotransmitter release at the synapse requires membrane fusion. The SNARE complex, composed of the plasma membrane t-SNAREs syntaxin 1A and SNAP-25 and the vesicle v-SNARE synaptobrevin, mediates the fusion of 2 membranes. Synaptic vesicles contain unusually high cholesterol, but the exact role of cholesterol in fusion is not known. In this study, cholesterol was found to stimulate SNARE-mediated lipid mixing of proteoliposomes by a factor of 5 at a physiological concentration. Surprisingly, however, the stimulatory effect was more pronounced when cholesterol was on the v-SNARE side than when it was on the t-SNARE side. Site-directed spin labeling and both continuous wave (CW) and pulsed EPR revealed that cholesterol induces a conformational change of the v-SNARE transmembrane domain (TMD) from an open scissors-like dimer to a parallel dimer. When the TMD was forced to form a parallel dimer by the disulfide bond, the rate was stimulated 2.3-fold even without cholesterol, supporting the relevance of the open-to-closed conformational change to the fusion activity. The open scissors-like conformation may be unfavorable for fusion and cholesterol may relieve this inhibitory factor.
Proceedings of the National Academy of Sciences 03/2009; 106(13):5141-6. · 9.68 Impact Factor
