scholarly journals A common mechanism for the regulation of vesicular SNAREs on phospholipid membranes

2004 ◽  
Vol 377 (3) ◽  
pp. 781-785 ◽  
Author(s):  
Kuang HU ◽  
Colin RICKMAN ◽  
Joe CARROLL ◽  
Bazbek DAVLETOV
Keyword(s):  
Membrane Fusion ◽  
Snare Complex ◽  
Common Mechanism ◽  
Cell Physiology ◽  
Phospholipid Membranes ◽  
Intracellular Traffic ◽  
Protein Receptor ◽  
Liposomal Membranes ◽  
Eukaryotic Organisms ◽  
Protein Attachment

The SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) family of proteins is essential for membrane fusion in intracellular traffic in eukaryotic organisms. v-SNAREs (vesicular SNAREs) must engage target SNAREs in the opposing membrane to form the fusogenic SNARE complex. Temporal and spatial control of membrane fusion is important for many aspects of cell physiology and may involve the regulation of the SNAREs resident on intracellular membranes. Here we show that the v-SNARE synaptobrevin 2, also known as VAMP (vesicle-associated membrane protein) 2, is restricted from forming the SNARE complex in chromaffin granules from adrenal medullae to the same degree as in brain-purified synaptic vesicles. Our analysis indicates that the previously reported synaptophysin–synaptobrevin interaction is not likely to be involved in regulation of the v-SNARE. Indeed, the restriction can be reproduced for two distinct v-SNARE homologues, synaptobrevin 2 and cellubrevin/VAMP3, by reconstituting them in pure liposomal membranes. Overall, our data uncover a common mechanism for the control of SNARE engagement where intact phospholipid membranes rather than proteins down-regulate vesicular SNAREs in different cellular organelles.

S-nitrosylation of syntaxin 1 at Cys145 is a regulatory switch controlling Munc18-1 binding

2008 ◽  
Vol 413 (3) ◽  
pp. 479-491 ◽  
Author(s):  
Zoë J. Palmer ◽  
Rory R. Duncan ◽  
James R. Johnson ◽  
Lu-Yun Lian ◽  
Luciane V. Mello ◽  
...  
Keyword(s):  
Release Kinetics ◽  
Cell Types ◽  
Snare Complex ◽  
Syntaxin 1A ◽  
Dynamic Simulations ◽  
Closed Conformation ◽  
Quantal Size ◽  
Protein Receptor ◽  
Protein Attachment

Exocytosis is regulated by NO in many cell types, including neurons. In the present study we show that syntaxin 1a is a substrate for S-nitrosylation and that NO disrupts the binding of Munc18-1 to the closed conformation of syntaxin 1a in vitro. In contrast, NO does not inhibit SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein] receptor} complex formation or binding of Munc18-1 to the SNARE complex. Cys145 of syntaxin 1a is the target of NO, as a non-nitrosylatable C145S mutant is resistant to NO and novel nitrosomimetic Cys145 mutants mimic the effect of NO on Munc18-1 binding in vitro. Furthermore, expression of nitrosomimetic syntaxin 1a in living cells affects Munc18-1 localization and alters exocytosis release kinetics and quantal size. Molecular dynamic simulations suggest that NO regulates the syntaxin–Munc18 interaction by local rearrangement of the syntaxin linker and H3c regions. Thus S-nitrosylation of Cys145 may be a molecular switch to disrupt Munc18-1 binding to the closed conformation of syntaxin 1a, thereby facilitating its engagement with the membrane fusion machinery.

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

2013 ◽  
Vol 24 (10) ◽  
pp. 1593-1601 ◽  
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.

scholarly journals Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

IUCrJ ◽  
2014 ◽  
Vol 1 (6) ◽  
pp. 505-513 ◽  
Author(s):  
Asma Rehman ◽  
Julia K. Archbold ◽  
Shu-Hong Hu ◽  
Suzanne J. Norwood ◽  
Brett M. Collins ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Blood Glucose Control ◽  
Vital Role ◽  
Snare Complex ◽  
Snare Proteins ◽  
Regulatory Role ◽  
Sm Proteins ◽  
Protein Receptor

Membrane fusion is essential for human health, playing a vital role in processes as diverse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the solubleN-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for example through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins.

Sec22b-dependent assembly of endoplasmic reticulum Q-SNARE proteins

2008 ◽  
Vol 410 (1) ◽  
pp. 93-100 ◽  
Author(s):  
Takehiro Aoki ◽  
Masaki Kojima ◽  
Katsuko Tani ◽  
Mitsuo Tagaya
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Cd Spectroscopy ◽  
Snare Proteins ◽  
Peripheral Membrane Proteins ◽  
Protein Receptor ◽  
Snare Motif ◽  
Α Helix ◽  
Protein Attachment

SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins involved in membrane fusion usually contain a conserved α-helix (SNARE motif) that is flanked by a C-terminal transmembrane domain. They can be classified into Q-SNARE and R-SNARE based on the structural property of their motifs. Assembly of four SNARE motifs (Qa, b, c and R) is supposed to trigger membrane fusion. We have previously shown that ER (endoplasmic reticulum)-localized syntaxin 18 (Qa) forms a complex with BNIP1 (Qb), p31/Use1 (Qc), Sec22b (R) and several peripheral membrane proteins. In the present study, we examined the interaction of syntaxin 18 with other SNAREs using pulldown assays and CD spectroscopy. We found that the association of syntaxin 18 with Sec22b induces an increase in α-helicity of their SNARE motifs, which results in the formation of high-affinity binding sites for BNIP1 and p31. This R-SNARE-dependent Q-SNARE assembly is quite different from the assembly mechanisms of SNAREs localized in organelles other than the ER. The implication of the mechanism of ER SNARE assembly is discussed in the context of the physiological roles of the syntaxin 18 complex.

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

2012 ◽  
Vol 23 (2) ◽  
pp. 337-346 ◽  
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.

scholarly journals A Novel Site of Action for α-SNAP in the SNARE Conformational Cycle Controlling Membrane Fusion

2008 ◽  
Vol 19 (3) ◽  
pp. 776-784 ◽  
Author(s):  
Marcin Barszczewski ◽  
John J. Chua ◽  
Alexander Stein ◽  
Ulrike Winter ◽  
Rainer Heintzmann ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Site Of Action ◽  
Snare Motif ◽  
Liposome Fusion

Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of α-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of α-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of α-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of α-SNAP potently inhibits Ca2+-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an α-SNAP mutant defective in NSF activation is used. We conclude that α-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for α-SNAP in the SNARE cycle that drives exocytotic membrane fusion.

scholarly journals HOPS drives vacuole fusion by binding the vacuolar SNARE complex and the Vam7 PX domain via two distinct sites

2011 ◽  
Vol 22 (14) ◽  
pp. 2601-2611 ◽  
Author(s):  
Lukas Krämer ◽  
Christian Ungermann
Keyword(s):  
Membrane Fusion ◽  
Snare Complex ◽  
Endomembrane System ◽  
Attachment Protein ◽  
Vacuole Fusion ◽  
Px Domain ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Membrane Tethering

Membrane fusion within the endomembrane system follows a defined order of events: membrane tethering, mediated by Rabs and tethers, assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes, and lipid bilayer mixing. Here we present evidence that the vacuolar HOPS tethering complex controls fusion through specific interactions with the vacuolar SNARE complex (consisting of Vam3, Vam7, Vti1, and Nyv1) and the N-terminal domains of Vam7 and Vam3. We show that homotypic fusion and protein sorting (HOPS) binds Vam7 via its subunits Vps16 and Vps18. In addition, we observed that Vps16, Vps18, and the Sec1/Munc18 protein Vps33, which is also part of the HOPS complex, bind to the Q-SNARE complex. In agreement with this observation, HOPS-stimulated fusion was inhibited if HOPS was preincubated with the minimal Q-SNARE complex. Importantly, artificial targeting of Vam7 without its PX domain to membranes rescued vacuole morphology in vivo, but resulted in a cytokinesis defect if the N-terminal domain of Vam3 was also removed. Our data thus support a model of HOPS-controlled membrane fusion by recognizing different elements of the SNARE complex.

scholarly journals A VAMP7/Vti1a SNARE complex distinguishes a non-conventional traffic route to the cell surface used by KChIP1 and Kv4 potassium channels

2009 ◽  
Vol 418 (3) ◽  
pp. 529-540 ◽  
Author(s):  
Sarah E. Flowerdew ◽  
Robert D. Burgoyne
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Snare Complex ◽  
K Channel ◽  
Protein Receptor ◽  
Ef Hand ◽  
Neuro2a Cells ◽  
Vesicular Stomatitis ◽  
Interfering Rna ◽  
Protein Attachment

The KChIPs (K+ channel-interacting proteins) are EF hand-containing proteins required for the traffic of channel-forming Kv4 K+ subunits to the plasma membrane. KChIP1 is targeted, through N-terminal myristoylation, to intracellular vesicles that appear to be trafficking intermediates from the ER (endoplasmic reticulum) to the Golgi but differ from those underlying conventional ER–Golgi traffic. To define KChIP1 vesicles and the traffic pathway followed by Kv4/KChIP1 traffic, we examined their relationship to potential SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins mediating the trafficking step. To distinguish Kv4/KChIP1 from conventional constitutive traffic, we compared it to the traffic of the VSVG (vesicular-stomatitis virus G-protein). Expression of KChIP with single or triple EF hand mutations quantitatively inhibited Kv4/KChIP1 traffic to the cell surface but had no effect on VSVG traffic. KChIP1-expressing vesicles co-localized with the SNARE proteins Vti1a and VAMP7 (vesicle-associated membrane protein 7), but not with the components of two other ER–Golgi SNARE complexes. siRNA (small interfering RNA)-mediated knockdown of Vti1a or VAMP7 inhibited Kv4/KChIP1traffic to the plasma membrane in HeLa and Neuro2A cells. Vti1a and VAMP7 siRNA had no effect on VSVG traffic or that of Kv4.2 when stimulated by KChIP2, a KChIP with different intrinsic membrane targeting compared with KChIP1. The present results suggest that a SNARE complex containing VAMP7 and Vti1a defines a novel traffic pathway to the cell surface in both neuronal and non-neuronal cells.

scholarly journals A Second SNARE Role for Exocytic SNAP25 in Endosome Fusion

2006 ◽  
Vol 17 (5) ◽  
pp. 2113-2124 ◽  
Author(s):  
Yoshikatsu Aikawa ◽  
Kara L. Lynch ◽  
Kristin L. Boswell ◽  
Thomas F.J. Martin
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Snare Proteins ◽  
Recycling Endosome ◽  
Protein Receptor ◽  
Interfering Rna

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca2+-dependent secretory vesicle–plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway.

What is the role of SNARE proteins in membrane fusion?

2003 ◽  
Vol 285 (2) ◽  
pp. C237-C249 ◽  
Author(s):  
Joseph G. Duman ◽  
John G. Forte
Keyword(s):  
Subcellular Localization ◽  
Membrane Fusion ◽  
Biological Membrane ◽  
Snare Complex ◽  
Model Systems ◽  
Snare Proteins ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Membrane Contact

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins have been at the fore-front of research on biological membrane fusion for some time. The subcellular localization of SNAREs and their ability to form the so-called SNARE complex may be integral to determining the specificity of intracellular fusion (the SNARE hypothesis) and/or serving as the minimal fusion machinery. Both the SNARE hypothesis and the idea of the minimal fusion machinery have been challenged by a number of experimental observations in various model systems, suggesting that SNAREs may have other functions. Considering recent advances in the SNARE literature, it appears that SNAREs may actually function as part of a complex fusion “machine.” Their role in the machinery could be any one or a combination of roles, including establishing tight membrane contact, formation of a scaffolding on which to build the machine, binding of lipid surfaces, and many others. It is also possible that complexations other than the classic SNARE complex participate in membrane fusion.

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