The mesoscale organization of syntaxin 1A and SNAP25 is determined by SNARE-SNARE-interactions

SNARE proteins have been described as the effectors of fusion events in the secretory pathway more than two decades ago. The strong interactions between SNARE-domains are clearly important in membrane fusion, but it is unclear whether they are involved in any other cellular processes. Here, we analyzed two classical SNARE proteins, syntaxin 1A and SNAP25. Although they are supposed to be engaged in tight complexes, we surprisingly find them largely segregated in the plasma membrane. Syntaxin 1A only occupies a small fraction of the plasma membrane area. Yet, we find it is able to redistribute the far more abundant SNAP25 on the mesoscale by gathering crowds of SNAP25 molecules onto syntaxin-clusters in a SNARE-domain dependent manner. Our data suggests that SNARE-domain interactions are not only involved in driving membrane fusion on the nanoscale, but also play an important role in controlling the general organization of proteins on the mesoscale. Further, we propose this mechanisms preserves active syntaxin 1A-SNAP25 complexes at the plasma membrane.

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Mechanisms of CFTR regulation by syntaxin 1A and PKA

Journal of Cell Science ◽

10.1242/jcs.115.4.783 ◽

2002 ◽

Vol 115 (4) ◽

pp. 783-791 ◽

Cited By ~ 3

Author(s):

Steven Y. Chang ◽

Anke Di ◽

Anjaparavanda P. Naren ◽

H. Clive Palfrey ◽

Kevin L. Kirk ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Interactions ◽

Membrane Trafficking ◽

Anion Channel ◽

Cell Types ◽

Snare Proteins ◽

Snare Protein ◽

Membrane Turnover ◽

Open Probability ◽

Syntaxin 1A

Activation of the chloride selective anion channel CFTR is stimulated by cAMP-dependent phosphorylation and is regulated by the target membrane t-SNARE syntaxin 1A. The mechanism by which SNARE proteins modulate CFTR in secretory epithelia is controversial. In addition, controversy exists as to whether PKA activates CFTR-mediated Cl- currents (ICFTR) by increasing the number of channels in the plasma membrane and/or by stimulating membrane-resident channels. SNARE proteins play a well known role in exocytosis and have recently been implicated in the regulation of ion channels; therefore this investigation sought to resolve two related issues:(a) is PKA activation or SNARE protein modulation of CFTR linked to changes in membrane turnover and (b) does syntaxin 1A modulate CFTR via direct effects on the gating of channels residing in the plasma membrane versus alterations in membrane traffic. Our data demonstrate that syntaxin 1A inhibits CFTR as a result of direct protein-protein interactions that decrease channel open probability (Po) and serves as a model for other SNARE protein-ion channel interactions. We also show that PKA activation can enhance membrane trafficking in some epithelial cell types, and this is independent from CFTR activation or syntaxin 1A association.

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MUNC13 REGULATES SECRETION AT THE GOLGI IN A LOCALIZATION-DEPENDENT MANNER

Clinical & Investigative Medicine ◽

10.25011/cim.v31i4.4805 ◽

2008 ◽

Vol 31 (4) ◽

pp. 11

Author(s):

Neil M Goldenberg ◽

Mel Silverman

Keyword(s):

Plasma Membrane ◽

Secretory Pathway ◽

Phorbol Esters ◽

Cell Structure ◽

Eukaryotic Cell ◽

Temperature Sensitive ◽

Dependent Manner ◽

C1 Domain ◽

Surface Labelling ◽

And Function

Background: Constitutive secretion is critical for the maintenance of eukaryotic cell structure and function. Our lab has shown that Rab34 is required for secretion at the Golgi^1, and that the C1 domain-containing protein, Munc13, is an effector of Rab34^2. Current studies seek to elucidate potential roles for Munc13in secretion at the Golgi. Methods: Using a temperature-sensitive mutant of the Vesicular Stomatitis G-protein fused to GFP (VSVG-GFP) to monitor secretion, we examinedthe role of Munc13 in secretion in HeLa cells. Cells transfected with VSVG-GFP were treated with Munc13, amutant lacking the C1 domain (C1-less), and the phorbol esters TPA andPDBu. The rate of VSVG-GFP secretion was monitored using surface labelling of plasmalemmal VSVG-GFP and spinning disc confocal microscopy. Results: TPA treatment resulted in an increase in the rate of VSVG-GFP appearance at the plasma membrane. Co-transfection of either Munc13 or C1-less alone also resulted in an increased rate of VSVG-GFP transport. Transfection of Munc13 plus TPA treatment resulted in amarked decrease in the rate of VSVG-GFP transport. Since TPA treatment relocalizes Munc13 to the plasma membrane, this result suggests that the availability of Munc13 in the cytosol is required for its effect on VSVG-GFP secretion. Conclusions: Munc13 over-expression increases the rate of VSVG-GFP secretion to the plasma membrane. Sequestration of Munc13 at the plasma membrane with TPA abrogates thiseffect, and reduces the rate of VSVG-GFP secretion. We propose that Munc13 effects VSVG-GFP secretion via its interaction with Rab34 at the Golgi. References: 1. Goldenberg, NM, S. Grinstein, M. Silverman. Golgi-bound Rab34 is a Novel Member ofthe Secretory Pathway. Mol BiolCell. 18(12):4762-4771 (2007). 2. Speight, P, M. Silverman.Diacylglycerol-Activated Hmunc13 Serves as an Effector of the GTPaseRab34. Traffic.6(10):858-865 (2005).

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A Second SNARE Role for Exocytic SNAP25 in Endosome Fusion

Molecular Biology of the Cell ◽

10.1091/mbc.e06-01-0074 ◽

2006 ◽

Vol 17 (5) ◽

pp. 2113-2124 ◽

Cited By ~ 34

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.

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Mechanochemical feedback and control of endocytosis and membrane tension

10.1101/201509 ◽

2017 ◽

Author(s):

Joseph Jose Thottacherry ◽

Anita Joanna Kosmalska ◽

Alberto Elosegui-Artola ◽

Susav Pradhan ◽

Sumit Sharma ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Trafficking ◽

Feedback Loop ◽

Negative Feedback Loop ◽

Dependent Manner ◽

Homeostatic Regulation ◽

Membrane Tension ◽

Cellular Processes ◽

Tightly Coupled ◽

And Control

AbstractPlasma membrane tension is an important factor that regulates many key cellular processes. Membrane trafficking is tightly coupled to membrane tension and can modulate the latter by addition or removal of the membrane. However, the cellular pathway(s) involved in these processes are poorly understood. Here we find that, among a number of endocytic processes operating simultaneously at the cell surface, a dynamin and clathrin-independent pathway, the CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon reduction of tension. On the other hand, inhibition of the CG pathway results in lower membrane tension, while up regulation significantly enhances membrane tension. We find that vinculin, a well-studied mechanotransducer, mediates the tension-dependent regulation of the CG pathway. Vinculin negatively regulates a key CG pathway regulator, GBF1, at the plasma membrane in a tension dependent manner. Thus, the CG pathway operates in a negative feedback loop with membrane tension which leads to a homeostatic regulation of membrane tension.

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Analysis of Sec22p in Endoplasmic Reticulum/Golgi Transport Reveals Cellular Redundancy in SNARE Protein Function

Molecular Biology of the Cell ◽

10.1091/mbc.e02-04-0204 ◽

2002 ◽

Vol 13 (9) ◽

pp. 3314-3324 ◽

Cited By ~ 84

Author(s):

Yiting Liu ◽

Charles Barlowe

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Fusion ◽

Protein Function ◽

Secretory Pathway ◽

Snare Proteins ◽

Snare Protein ◽

Intracellular Membrane ◽

Protein Receptor ◽

Intracellular Membrane Fusion

Membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form heteromeric complexes that are required for intracellular membrane fusion and are proposed to encode compartmental specificity. In yeast, the R-SNARE protein Sec22p acts in transport between the endoplasmic reticulum (ER) and Golgi compartments but is not essential for cell growth. Other SNARE proteins that function in association with Sec22p (i.e., Sed5p, Bos1p, and Bet1p) are essential, leading us to question how transport through the early secretory pathway is sustained in the absence of Sec22p. In wild-type strains, we show that Sec22p is directly required for fusion of ER-derived vesicles with Golgi acceptor membranes. Insec22Δ strains, Ykt6p, a related R-SNARE protein that operates in later stages of the secretory pathway, is up-regulated and functionally substitutes for Sec22p. In vivo combination of thesec22Δ mutation with a conditionalykt6-1 allele results in lethality, consistent with a redundant mechanism. Our data indicate that the requirements for specific SNARE proteins in intracellular membrane fusion are less stringent than appreciated and suggest that combinatorial mechanisms using both upstream-targeting elements and SNARE proteins are required to maintain an essential level of compartmental organization.

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Nd6p, a Novel Protein with RCC1-Like Domains Involved in Exocytosis in Paramecium tetraurelia

Eukaryotic Cell ◽

10.1128/ec.4.12.2129-2139.2005 ◽

2005 ◽

Vol 4 (12) ◽

pp. 2129-2139 ◽

Cited By ~ 7

Author(s):

Delphine Gogendeau ◽

Anne-Marie Keller ◽

Akira Yanagi ◽

Jean Cohen ◽

France Koll

Keyword(s):

Plasma Membrane ◽

Membrane Fusion ◽

Secretory Pathway ◽

Small Gtpase ◽

Paramecium Tetraurelia ◽

Acid Protein ◽

Nuclear Envelope Assembly ◽

Regulated Secretory Pathway ◽

Dense Core Granules ◽

Intracellular Membrane Fusion

ABSTRACT In Paramecium tetraurelia, the regulated secretory pathway of dense core granules called trichocysts can be altered by mutation and genetically studied. Seventeen nondischarge (ND) genes controlling exocytosis have already been identified by a genetic approach. The site of action of the studied mutations is one of the three compartments, the cytosol, trichocyst, or plasma membrane. The only ND genes cloned to date correspond to mutants affected in the cytosol or in the trichocyst compartment. In this work, we investigated a representative of the third compartment, the plasma membrane, by cloning the ND6 gene. This gene encodes a 1,925-amino-acid protein containing two domains homologous to the regulator of chromosome condensation 1 (RCC1). In parallel, 10 new alleles of the ND6 gene were isolated. Nine of the 12 available mutations mapped in the RCC1-like domains, showing their importance for the Nd6 protein (Nd6p) function. The RCC1 protein is well known for its guanine exchange factor activity towards the small GTPase Ran but also for its involvement in membrane fusion during nuclear envelope assembly. Other proteins with RCC1-like domains are also involved in intracellular membrane fusion, but none has been described yet as involved in exocytosis. The case of Nd6p is thus the first report of such a protein with a documented role in exocytosis.

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Lethal giant larvae proteins interact with the exocyst complex and are involved in polarized exocytosis

The Journal of Cell Biology ◽

10.1083/jcb.200502055 ◽

2005 ◽

Vol 170 (2) ◽

pp. 273-283 ◽

Cited By ~ 72

Author(s):

Xiaoyu Zhang ◽

Puyue Wang ◽

Akanksha Gangar ◽

Jian Zhang ◽

Patrick Brennwald ◽

...

Keyword(s):

Plasma Membrane ◽

Epithelial Cell ◽

Membrane Fusion ◽

Cell Polarity ◽

Rho Gtpases ◽

Critical Role ◽

Cell Polarization ◽

Snare Proteins ◽

Exocyst Complex ◽

Lethal Giant Larvae

The tumor suppressor lethal giant larvae (Lgl) plays a critical role in epithelial cell polarization. However, the molecular mechanism by which Lgl carries out its functions is unclear. In this study, we report that the yeast Lgl proteins Sro7p and Sro77p directly interact with Exo84p, which is a component of the exocyst complex that is essential for targeting vesicles to specific sites of the plasma membrane for exocytosis, and that this interaction is important for post-Golgi secretion. Genetic analyses demonstrate a molecular pathway from Rab and Rho GTPases through the exocyst and Lgl to SNAREs, which mediate membrane fusion. We also found that overexpression of Lgl and t-SNARE proteins not only improves exocytosis but also rescues polarity defects in exocyst mutants. We propose that, although Lgl is broadly distributed in the cells, its localized interaction with the exocyst and kinetic activation are important for the establishment and reenforcement of cell polarity.

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Synthesis of fusogenic lipids through activation of phospholipase D1 by GTPases and the kinase RSK2 is required for calcium-regulated exocytosis in neuroendocrine cells

Biochemical Society Transactions ◽

10.1042/bst0380167 ◽

2010 ◽

Vol 38 (1) ◽

pp. 167-171 ◽

Cited By ~ 13

Author(s):

Nicolas Vitale

Keyword(s):

Plasma Membrane ◽

Membrane Fusion ◽

Neuroendocrine Cells ◽

Dependent Manner ◽

Regulatory Pathways ◽

Calcium Dependent ◽

Protein Receptors ◽

Phospholipase D1 ◽

Fusion Site ◽

Ribosomal S6 Kinase

Exocytosis of hormones occurs through the fusion of large dense-core secretory vesicles with the plasma membrane. This highly regulated process involves key proteins such as SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) and also specific lipids at the site of membrane fusion. Among the different lipids required for exocytosis, our recent observations have highlighted the crucial role of PA (phosphatidic acid) in the late stages of membrane fusion in various exocytotic events. An RNAi (RNA interference) strategy coupled with the detection of PA in living cells has pointed to plasma membrane-associated PLD1 (phospholipase D1) as the main producer of PA in response to secretagogue stimulation. We have identified several GTPases which regulate the activation level of PLD1 in neuroendocrine cells. Finally, RSK2 (ribosomal S6 kinase 2) appears to phosphorylate and regulate the activity of PLD1 in a calcium-dependent manner. Altogether our results have unravelled a complex set of regulatory pathways controlling the synthesis of fusogenic lipids at the secretory granule fusion site by PLD1.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e05-05-0382 ◽

2006 ◽

Vol 17 (2) ◽

pp. 711-722 ◽

Cited By ~ 23

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.

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Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01043-16 ◽

2016 ◽

Vol 60 (11) ◽

pp. 6532-6539 ◽

Cited By ~ 134

Author(s):

Mizuki Yamamoto ◽

Shutoku Matsuyama ◽

Xiao Li ◽

Makoto Takeda ◽

Yasushi Kawaguchi ◽

...

Keyword(s):

Plasma Membrane ◽

Middle East ◽

Membrane Fusion ◽

Serine Protease ◽

Potent Inhibitor ◽

Middle East Respiratory Syndrome ◽

Target Cells ◽

Dependent Manner ◽

S Protein

ABSTRACTMiddle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressingRenillaluciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infectionin vitro. Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.

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