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.

scholarly journals Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

2013 ◽  
Vol 24 (4) ◽  
pp. 510-520 ◽  
Author(s):  
Matyáš Fendrych ◽  
Lukáš Synek ◽  
Tamara Pečenková ◽  
Edita Janková Drdová ◽  
Juraj Sekereš ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Complex Dynamics ◽  
Active Regions ◽  
Snare Complex ◽  
Epifluorescence Microscopy ◽  
Rab Gtpases ◽  
Exocyst Complex ◽  
Protein Receptor ◽  
Outer Domain

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6–green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.

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 Golgin-160 Is Required for the Golgi Membrane Sorting of the Insulin-responsive Glucose Transporter GLUT4 in Adipocytes

2006 ◽  
Vol 17 (12) ◽  
pp. 5346-5355 ◽  
Author(s):  
Dumaine Williams ◽  
Stuart W. Hicks ◽  
Carolyn E. Machamer ◽  
Jeffrey E. Pessin
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Glucose Transporter ◽  
General Distribution ◽  
Amino Terminal ◽  
Vesicular Stomatitis ◽  
Glucose Transporter Glut4 ◽  
Golgi Network ◽  
Interfering Rna ◽  
Regulated Trafficking

The peripheral Golgi protein golgin-160 is induced during 3T3L1 adipogenesis and is primarily localized to the Golgi cisternae distinct from the trans-Golgi network (TGN) in a general distribution similar to p115. Small interfering RNA (siRNA)-mediated reduction in golgin-160 protein resulted in an increase accumulation of the insulin-responsive amino peptidase (IRAP) and the insulin-regulated glucose transporter (GLUT4) at the plasma membrane concomitant with enhanced glucose uptake in the basal state. The redistribution of GLUT4 was rescued by expression of a siRNA-resistant golgin-160 cDNA. The basal state accumulation of plasma membrane GLUT4 occurred due to an increased rate of exocytosis without any significant effect on the rate of endocytosis. This GLUT4 trafficking to the plasma membrane in the absence of golgin-160 was independent of TGN/Golgi sorting, because it was no longer inhibited by the expression of a dominant-interfering Golgi-localized, γ-ear–containing ARF-binding protein mutant and displayed reduced binding to the lectin wheat germ agglutinin. Moreover, expression of the amino terminal head domain (amino acids 1–393) had no significant effect on the distribution or insulin-regulated trafficking of GLUT4 or IRAP. In contrast, expression of carboxyl α helical region (393–1498) inhibited insulin-stimulated GLUT4 and IRAP translocation, but it had no effect on the sorting of constitutive membrane trafficking proteins, the transferrin receptor, or vesicular stomatitis virus G protein. Together, these data demonstrate that golgin-160 plays an important role in directing insulin-regulated trafficking proteins toward the insulin-responsive compartment in adipocytes.

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 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.

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.

The molecular mechanisms of the mammalian exocyst complex in exocytosis

2006 ◽  
Vol 34 (5) ◽  
pp. 687-690 ◽  
Author(s):  
S. Wang ◽  
S.C. Hsu
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Secretory Vesicles ◽  
Attachment Protein ◽  
Trafficking Pathway ◽  
Exocyst Complex ◽  
Protein Receptor ◽  
Associated Proteins ◽  
Protein Attachment

Exocytosis is a highly ordered vesicle trafficking pathway that targets proteins to the plasma membrane for membrane addition or secretion. Research over the years has discovered many proteins that participate at various stages in the mammalian exocytotic pathway. At the early stage of exocytosis, co-atomer proteins and their respective adaptors and GTPases have been shown to play a role in the sorting and incorporation of proteins into secretory vesicles. At the final stage of exocytosis, SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) and SNARE-associated proteins are believed to mediate the fusion of secretory vesicles at the plasma membrane. There are multiple events that may occur between the budding of secretory vesicles from the Golgi and the fusion of these vesicles at the plasma membrane. The most obvious and best-known event is the transport of secretory vesicles from Golgi to the vicinity of the plasma membrane via microtubules and their associated motors. At the vicinity of the plasma membrane, however, it is not clear how vesicles finally dock and fuse with the plasma membrane. Identification of proteins involved in these events should provide important insights into the mechanisms of this little known stage of the exocytotic pathway. Currently, a protein complex, known as the sec6/8 or the exocyst complex, has been implicated to play a role at this late stage of exocytosis.

scholarly journals Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking

2015 ◽  
Vol 26 (3) ◽  
pp. 530-536 ◽  
Author(s):  
Jessica B. A. Sadler ◽  
Nia J. Bryant ◽  
Gwyn W. Gould
Keyword(s):  
Plasma Membrane ◽  
Snare Complex ◽  
Cell Type ◽  
Attachment Protein ◽  
Systematic Analysis ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Factor Attachment Protein Receptor

The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.

scholarly journals Transport of the membrane glycoprotein of vesicular stomatitis virus to the cell surface in two stages by clathrin-coated vesicles.

1980 ◽  
Vol 86 (1) ◽  
pp. 162-171 ◽  
Author(s):  
J E Rothman ◽  
H Bursztyn-Pettegrew ◽  
R E Fine
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Coated Vesicles ◽  
Transmembrane Glycoprotein ◽  
Vesicular Stomatitis ◽  
Two Stages

The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Pulse-chase experiments suggest that G is transported to the cell surface in two successive waves of clathrin-coated vesicles. The oligosaccharides of G protein carried in the early wave are of the "high-mannose" (G1) form, whereas the oligosaccharides in the second, later wave are of the mature "complex" (G2) form. the early wave is therefore proposed to correspond to transport of G in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, and the succeeding wave to transport from the Golgi apparatus to the plasma membrane. The G1- and G2-containing coated vesicles appear to be structurally distinct, as judged by their differential precipitation by anticoated vesicle serum.

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