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.

scholarly journals SNAP25, but Not Syntaxin 1A, Recycles via an ARF6-regulated Pathway in Neuroendocrine Cells

2006 ◽  
Vol 17 (2) ◽  
pp. 711-722 ◽  
Author(s):  
Yoshikatsu Aikawa ◽  
Xiaofeng Xia ◽  
Thomas F.J. Martin
Keyword(s):  
Plasma Membrane ◽  
Cellular Membrane ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Proteins ◽  
Syntaxin 1A ◽  
Recycling Endosome ◽  
Protein Receptor ◽  
Vesicle Exocytosis ◽  
Donor Acceptor

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate cellular membrane fusion events and provide a level of specificity to donor–acceptor membrane interactions. However, the trafficking pathways by which individual SNARE proteins are targeted to specific membrane compartments are not well understood. In neuroendocrine cells, synaptosome-associated protein of 25 kDa (SNAP25) is localized to the plasma membrane where it functions in regulated secretory vesicle exocytosis, but it is also found on intracellular membranes. We identified a dynamic recycling pathway for SNAP25 in PC12 cells through which plasma membrane SNAP25 recycles in ∼3 h. Approximately 20% of the SNAP25 resides in a perinuclear recycling endosome–trans-Golgi network (TGN) compartment from which it recycles back to the plasma membrane. SNAP25 internalization occurs by constitutive, dynamin-independent endocytosis that is distinct from the dynamin-dependent endocytosis that retrieves secretory vesicle constituents after exocytosis. Endocytosis of SNAP25 is regulated by ADP-ribosylation factor (ARF)6 (through phosphatidylinositol bisphosphate synthesis) and is dependent upon F-actin. SNAP25 endosomes, which exclude the plasma membrane SNARE syntaxin 1A, merge with those derived from clathrin-dependent endocytosis containing endosomal syntaxin 13. Our results characterize a robust ARF6-dependent internalization mechanism that maintains an intracellular pool of SNAP25, which is compatible with possible intracellular roles for SNAP25 in neuroendocrine cells.

Synaptotagmin-1: A Multi-Functional Protein that Mediates Vesicle Docking, Priming, and Fusion

2021 ◽  
Vol 22 ◽  
Author(s):  
Najeeb Ullah ◽  
Ezzouhra El Maaiden ◽  
Md. Sahab Uddin ◽  
Ghulam Md Ashraf
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Secretory Vesicles ◽  
Functional Protein ◽  
Vesicle Docking ◽  
Synaptotagmin 1

: The fusion of secretory vesicles with the plasma membrane depends on the assembly of v-SNAREs (VAMP2/synaptobrevin2) and t-SNAREs (SNAP25/syntaxin1) into the SNARE complex. Vesicles go through several upstream steps, referred to as docking and priming, to gain fusion competence. The vesicular protein synaptotagmin-1 (Syt-1) is the principal Ca2+ sensor for fusion in several central nervous system neurons and neuroendocrine cells and part of the docking complex for secretory granules. Syt-1 binds to the acceptor complex such as synaxin1, SNAP-25 on the plasma membrane to facilitate secretory vesicle docking, and upon Ca2+-influx promotes vesicle fusion. This review assesses the role of the Syt-1 protein involved in the secretory vesicle docking, priming, and fusion.

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.

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.

scholarly journals Geranylgeranylated Snares Are Dominant Inhibitors of Membrane Fusion

2000 ◽  
Vol 151 (2) ◽  
pp. 453-466 ◽  
Author(s):  
Eric Grote ◽  
Misuzu Baba ◽  
Yoshinori Ohsumi ◽  
Peter J. Novick
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Secretory Vesicle ◽  
Snare Complex ◽  
Snare Proteins ◽  
Wild Type ◽  
Protein Receptor ◽  
High Level ◽  
Order Complexes ◽  
Mutant Cells

Exocytosis in yeast requires the assembly of the secretory vesicle soluble N-ethylmaleimide–sensitive factor attachment protein receptor (v-SNARE) Sncp and the plasma membrane t-SNAREs Ssop and Sec9p into a SNARE complex. High-level expression of mutant Snc1 or Sso2 proteins that have a COOH-terminal geranylgeranylation signal instead of a transmembrane domain inhibits exocytosis at a stage after vesicle docking. The mutant SNARE proteins are membrane associated, correctly targeted, assemble into SNARE complexes, and do not interfere with the incorporation of wild-type SNARE proteins into complexes. Mutant SNARE complexes recruit GFP-Sec1p to sites of exocytosis and can be disassembled by the Sec18p ATPase. Heterotrimeric SNARE complexes assembled from both wild-type and mutant SNAREs are present in heterogeneous higher-order complexes containing Sec1p that sediment at greater than 20S. Based on a structural analogy between geranylgeranylated SNAREs and the GPI-HA mutant influenza virus fusion protein, we propose that the mutant SNAREs are fusion proteins unable to catalyze fusion of the distal leaflets of the secretory vesicle and plasma membrane. In support of this model, the inverted cone–shaped lipid lysophosphatidylcholine rescues secretion from SNARE mutant cells.

scholarly journals Ordering the Final Events in Yeast Exocytosis

2000 ◽  
Vol 151 (2) ◽  
pp. 439-452 ◽  
Author(s):  
Eric Grote ◽  
Chavela M. Carr ◽  
Peter J. Novick
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Late Stage ◽  
Binding Protein ◽  
Secretory Vesicle ◽  
Snare Complex ◽  
Wild Type ◽  
Attachment Protein ◽  
Complex Assembly ◽  
Protein Receptor

In yeast, assembly of exocytic soluble N-ethylmaleimide–sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

scholarly journals Insulin and Hypertonicity Recruit GLUT4 to the Plasma Membrane of Muscle Cells by Using N-Ethylmaleimide-sensitive Factor-dependent SNARE Mechanisms but Different v-SNAREs: Role of TI-VAMP

2004 ◽  
Vol 15 (12) ◽  
pp. 5565-5573 ◽  
Author(s):  
Varinder K. Randhawa ◽  
Farah S.L. Thong ◽  
Dawn Y. Lim ◽  
Dailin Li ◽  
Rami R. Garg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Muscle Cells ◽  
Dominant Negative ◽  
Attachment Protein ◽  
Insulin Effect ◽  
Tetanus Neurotoxin ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Interfering Rna

Insulin and hypertonicity each increase the content of GLUT4 glucose transporters at the surface of muscle cells. Insulin enhances GLUT4 exocytosis without diminishing its endocytosis. The insulin but not the hypertonicity response is reduced by tetanus neurotoxin, which cleaves vesicle-associated membrane protein (VAMP)2 and VAMP3, and is rescued upon introducing tetanus neurotoxin-resistant VAMP2. Here, we show that hypertonicity enhances GLUT4 recycling, compounding its previously shown ability to reduce GLUT4 endocytosis. To examine whether the canonical soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) mechanism is required for the plasma membrane fusion of the tetanus neurotoxin-insensitive GLUT4 vesicles, L6 myoblasts stably expressing myc-tagged GLUT4 (GLUT4myc) were transiently transfected with dominant negative N-ethylmaleimide-sensitive factor (NSF) (DN-NSF) or small-interfering RNA to tetanus neurotoxin-insensitive VAMP (TI-VAMP siRNA). Both strategies markedly reduced the basal level of surface GLUT4myc and the surface gain of GLUT4myc in response to hypertonicity. The insulin effect was abolished by DN-NSF, but only partly reduced by TI-VAMP siRNA. We propose that insulin and hypertonicity recruit GLUT4myc from partly overlapping, but distinct sources defined by VAMP2 and TI-VAMP, respectively.

scholarly journals Differential inhibition by botulinum neurotoxin A of cotransmitters released from autonomic vasodilator neurons

2001 ◽  
Vol 281 (5) ◽  
pp. H2124-H2132 ◽  
Author(s):  
Judy L. Morris ◽  
Phillip Jobling ◽  
Ian L. Gibbins
Keyword(s):  
Botulinum Neurotoxin ◽  
Snare Complex ◽  
Snare Proteins ◽  
Botulinum Neurotoxin A ◽  
Uterine Arteries ◽  
Synaptic Vesicle Exocytosis ◽  
Protein Receptor ◽  
Autonomic Neurons ◽  
Vesicle Exocytosis

The role of the soluble NSF attachment protein receptor (SNARE) protein complex in release of multiple cotransmitters from autonomic vasodilator neurons was examined in isolated segments of guinea pig uterine arteries treated with botulinum neurotoxin A (BoNTA; 50 nM). Western blotting of protein extracts from uterine arteries demonstrated partial cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) to a NH2-terminal fragment of ∼24 kDa by BoNTA. BoNTA reduced the amplitude (by 70–80%) of isometric contractions of arteries in response to repeated electrical stimulation of sympathetic axons at 1 or 10 Hz. The amplitude of neurogenic relaxations mediated by neuronal nitric oxide (NO) was not affected by BoNTA, whereas the duration of peptide-mediated neurogenic relaxations to stimulation at 10 Hz was reduced (67% reduction in integrated responses). In contrast, presynaptic cholinergic inhibition of neurogenic relaxations was abolished by BoNTA. These results demonstrate that the SNARE complex has differential involvement in release of cotransmitters from the same autonomic neurons: NO release is not dependant on synaptic vesicle exocytosis, acetylcholine release from small vesicles is highly dependant on the SNARE complex, and neuropeptide release from large vesicles involves SNARE proteins that may interact differently with regulatory factors such as calcium.

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.

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