SNARE Complex Zero Layer Residues Are Not Critical forN-Ethylmaleimide-sensitive Factor-mediated Disassembly

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25–50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and35S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 ± 2% of control and reduces the rate of H+ secretion by 77 ± 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.

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Gβγ Interferes with Ca2+-Dependent Binding of Synaptotagmin to the Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor (SNARE) Complex

Molecular Pharmacology ◽

10.1124/mol.107.039446 ◽

2007 ◽

Vol 72 (5) ◽

pp. 1210-1219 ◽

Cited By ~ 54

Author(s):

Eun-Ja Yoon ◽

Tatyana Gerachshenko ◽

Bryan D. Spiegelberg ◽

Simon Alford ◽

Heidi E. Hamm

Keyword(s):

Snare Complex ◽

Attachment Protein ◽

Protein Receptor ◽

Sensitive Factor ◽

Factor Attachment Protein Receptor

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Syntaxin 16’s Newly Deciphered Roles in Autophagy

Cells ◽

10.3390/cells8121655 ◽

2019 ◽

Vol 8 (12) ◽

pp. 1655 ◽

Cited By ~ 3

Author(s):

Bor Luen Tang

Keyword(s):

Membrane Trafficking ◽

Functional Redundancy ◽

Transport Processes ◽

Snare Complex ◽

Dual Roles ◽

Trans Golgi Network ◽

Protein Receptor ◽

Sensitive Factor ◽

Golgi Network

Syntaxin 16, a Qa-SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor), is involved in a number of membrane-trafficking activities, particularly transport processes at the trans-Golgi network (TGN). Recent works have now implicated syntaxin 16 in the autophagy process. In fact, syntaxin 16 appears to have dual roles, firstly in facilitating the transport of ATG9a-containing vesicles to growing autophagosomes, and secondly in autolysosome formation. The former involves a putative SNARE complex between syntaxin 16, VAMP7 and SNAP-47. The latter occurs via syntaxin 16’s recruitment by Atg8/LC3/GABARAP family proteins to autophagosomes and endo-lysosomes, where syntaxin 16 may act in a manner that bears functional redundancy with the canonical autophagosome Qa-SNARE syntaxin 17. Here, I discuss these recent findings and speculate on the mechanistic aspects of syntaxin 16’s newly found role in autophagy.

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VAMP-8 segregates mast cell–preformed mediator exocytosis from cytokine trafficking pathways

Blood ◽

10.1182/blood-2007-07-103309 ◽

2008 ◽

Vol 111 (7) ◽

pp. 3665-3674 ◽

Cited By ~ 110

Author(s):

Neeraj Tiwari ◽

Cheng-Chun Wang ◽

Cristiana Brochetta ◽

Gou Ke ◽

Francesca Vita ◽

...

Keyword(s):

Mast Cells ◽

Inflammatory Responses ◽

Secretory Granules ◽

Snare Complex ◽

Protein Receptor ◽

Sensitive Factor ◽

Syntaxin 4 ◽

Deficient Mice ◽

Snare Complex Formation ◽

Vesicular Compartment

Abstract Inflammatory responses by mast cells are characterized by massive exocytosis of prestored granular mediators followed by cytokine/chemokine release. The vesicular trafficking mechanisms involved remain poorly understood. Vesicular-associated membrane protein-8 (VAMP-8), a member of the soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) family of fusion proteins initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. Here we show that in bone marrow–derived mast cells (BMMCs) VAMP-8 partially colocalized with secretory granules and redistributed upon stimulation. This was associated with increased SNARE complex formation with the target t-SNAREs, SNAP-23 and syntaxin-4. VAMP-8–deficient BMMCs exhibited a markedly reduced degranulation response after IgE+ antigen-, thapsigargin-, or ionomycin-induced stimulation. VAMP-8–deficient mice also showed reduced plasma histamine levels in passive systemic anaphylaxis experiments, while cytokine/chemokine release was not affected. Unprocessed TNF accumulated at the plasma membrane where it colocalized with a VAMP-3–positive vesicular compartment but not with VAMP-8. The findings demonstrate that VAMP-8 segregates secretory lysosomal granule exocytosis in mast cells from cytokine/chemokine molecular trafficking pathways.

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PTP1B DephosphorylatesN-Ethylmaleimide-sensitive Factor and Elicits SNARE Complex Disassembly during Human Sperm Exocytosis

Journal of Biological Chemistry ◽

10.1074/jbc.m807614200 ◽

2009 ◽

Vol 284 (16) ◽

pp. 10491-10503 ◽

Cited By ~ 35

Author(s):

Valeria E. P. Zarelli ◽

Maria C. Ruete ◽

Carlos M. Roggero ◽

Luis S. Mayorga ◽

Claudia N. Tomes

Keyword(s):

Human Sperm ◽

Snare Complex ◽

Sensitive Factor

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SNARE complexes of different composition jointly mediate membrane fusion in Arabidopsis cytokinesis

Molecular Biology of the Cell ◽

10.1091/mbc.e13-02-0074 ◽

2013 ◽

Vol 24 (10) ◽

pp. 1593-1601 ◽

Cited By ~ 55

Author(s):

Farid El Kasmi ◽

Cornelia Krause ◽

Ulrike Hiller ◽

York-Dieter Stierhof ◽

Ulrike Mayer ◽

...

Keyword(s):

Membrane Fusion ◽

Membrane Vesicles ◽

Snare Complex ◽

The Other ◽

Division Plane ◽

Daughter Cells ◽

Protein Receptor ◽

Sensitive Factor ◽

Membrane Type ◽

Cell Division Plane

Membrane fusion is mediated by soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. Although membrane fusion is required for separating daughter cells in eukaryotic cytokinesis, the SNARE complexes involved are not known. In plants, membrane vesicles targeted to the cell division plane fuse with one another to form the partitioning membrane, progressing from the center to the periphery of the cell. In Arabidopsis, the cytokinesis-specific Qa-SNARE KNOLLE interacts with two other Q-SNAREs, SNAP33 and novel plant-specific SNARE 11 (NPSN11), whose roles in cytokinesis are not clear. Here we show by coimmunoprecipitation that KNOLLE forms two SNARE complexes that differ in composition. One complex is modeled on the trimeric plasma membrane type of SNARE complex and includes, in addition to KNOLLE, the promiscuous Qb,c-SNARE SNAP33 and the R-SNARE vesicle-associated membrane protein (VAMP) 721,722, also involved in innate immunity. In contrast, the other KNOLLE-containing complex is tetrameric and includes Qb-SNARE NPSN11, Qc-SNARE SYP71, and VAMP721,722. Elimination of only one or the other type of KNOLLE complex by mutation, including the double mutant npsn11 syp71, causes a mild or no cytokinesis defect. In contrast, the two double mutants snap33 npsn11 and snap33 syp71 eliminate both types of KNOLLE complexes and display knolle-like cytokinesis defects. Thus the two distinct types of KNOLLE complexes appear to jointly mediate membrane fusion in Arabidopsis cytokinesis.

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Alternative Splicing of SNAP-25 Regulates Secretion through Nonconservative Substitutions in the SNARE Domain

Molecular Biology of the Cell ◽

10.1091/mbc.e05-07-0595 ◽

2005 ◽

Vol 16 (12) ◽

pp. 5675-5685 ◽

Cited By ~ 43

Author(s):

Gábor Nagy ◽

Ira Milosevic ◽

Dirk Fasshauer ◽

E. Matthias Müller ◽

Bert L. de Groot ◽

...

Keyword(s):

Mammalian Cells ◽

Chromaffin Cells ◽

Snare Complex ◽

Functional Difference ◽

Protein Receptor ◽

Accessory Factor ◽

Sensitive Factor ◽

Alternatively Spliced ◽

Secretory Phenotype ◽

Different Levels

The essential membrane fusion apparatus in mammalian cells, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consists of four α-helices formed by three proteins: SNAP-25, syntaxin 1, and synaptobrevin 2. SNAP-25 contributes two helices to the complex and is targeted to the plasma membrane by palmitoylation of four cysteines in the linker region. It is alternatively spliced into two forms, SNAP-25a and SNAP-25b, differing by nine amino acids substitutions. When expressed in chromaffin cells from SNAP-25 null mice, the isoforms support different levels of secretion. Here, we investigated the basis of that different secretory phenotype. We found that two nonconservative substitutions in the N-terminal SNARE domain and not the different localization of one palmitoylated cysteine cause the functional difference between the isoforms. Biochemical and molecular dynamic simulation experiments revealed that the two substitutions do not regulate secretion by affecting the property of SNARE complex itself, but rather make the SNAP-25b-containing SNARE complex more available for the interaction with accessory factor(s).

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Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1506409112 ◽

2015 ◽

Vol 112 (18) ◽

pp. E2290-E2297 ◽

Cited By ~ 32

Author(s):

Michael Zick ◽

Amy Orr ◽

Matthew L. Schwartz ◽

Alexey J. Merz ◽

William T. Wickner

Keyword(s):

Lipid Bilayer ◽

Energy Barrier ◽

Fatty Acyl ◽

Snare Complex ◽

Fusion Reactions ◽

Attachment Protein ◽

Limited Ability ◽

Acyl Composition ◽

Sensitive Factor ◽

Stable Association

Sec17 [soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17L291A,L292A did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17F21S,M22S, with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17F21S,M22S, interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion.

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Epilepsy-causing STX1B mutations translate altered protein functions into distinct phenotypes in mouse neurons

Brain ◽

10.1093/brain/awaa151 ◽

2020 ◽

Vol 143 (7) ◽

pp. 2119-2138 ◽

Cited By ~ 4

Author(s):

Gülçin Vardar ◽

Fabian Gerth ◽

Xiao Jakob Schmitt ◽

Pia Rautenstrauch ◽

Thorsten Trimbuch ◽

...

Keyword(s):

Snare Complex ◽

Epileptic Encephalopathies ◽

Unfolded Protein ◽

Readily Releasable Pool ◽

Protein Receptor ◽

Vesicular Release ◽

Sensitive Factor ◽

Protein Functions ◽

Syntaxin 1B ◽

Snare Complex Formation

Abstract Syntaxin 1B (STX1B) is a core component of the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that is critical for the exocytosis of synaptic vesicles in the presynapse. SNARE-mediated vesicle fusion is assisted by Munc18-1, which recruits STX1B in the auto-inhibited conformation, while Munc13 catalyses the fast and efficient pairing of helices during SNARE complex formation. Mutations within the STX1B gene are associated with epilepsy. Here we analysed three STX1B mutations by biochemical and electrophysiological means. These three paradigmatic mutations cause epilepsy syndromes of different severity, from benign fever-associated seizures in childhood to severe epileptic encephalopathies. An insertion/deletion (K45/RMCIE, L46M) mutation (STX1BInDel), causing mild epilepsy and located in the early helical Habc domain, leads to an unfolded protein unable to sustain neurotransmission. STX1BG226R, causing epileptic encephalopathies, strongly compromises the interaction with Munc18-1 and reduces expression of both proteins, the size of the readily releasable pool of vesicles, and Ca2+-triggered neurotransmitter release when expressed in STX1-null neurons. The mutation STX1BV216E, also causing epileptic encephalopathies, only slightly diminishes Munc18-1 and Munc13 interactions, but leads to enhanced fusogenicity and increased vesicular release probability, also in STX1-null neurons. Even though the synaptic output remained unchanged in excitatory hippocampal STX1B+/− neurons exogenously expressing STX1B mutants, the manifestation of clear and distinct molecular disease mechanisms by these mutants suggest that certain forms of epilepsies can be conceptualized by assigning mutations to structurally sensitive regions of the STX1B−Munc18-1 interface, translating into distinct neurophysiological phenotypes.

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Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1

Molecular Biology of the Cell ◽

10.1091/mbc.e11-08-0670 ◽

2012 ◽

Vol 23 (2) ◽

pp. 337-346 ◽

Cited By ~ 67

Author(s):

Francesca Morgera ◽

Margaret R. Sallah ◽

Michelle L. Dubuke ◽

Pallavi Gandhi ◽

Daniel N. Brewer ◽

...

Keyword(s):

Membrane Fusion ◽

Secretory Pathway ◽

Secretory Vesicle ◽

Snare Complex ◽

Attachment Protein ◽

Protein Receptor ◽

Sensitive Factor ◽

Membrane Bound ◽

Sm Protein ◽

Snare Complex Formation

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function—it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6–Sec1 interaction is exclusive of Sec6–Sec9 but compatible with Sec6–exocyst assembly. In contrast, the Sec6–exocyst interaction is incompatible with Sec6–Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6–exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.

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