The proteins of exocytosis: lessons from the sperm model

2015 ◽  
Vol 465 (3) ◽  
pp. 359-370 ◽  
Author(s):  
Claudia Nora Tomes
Keyword(s):  
Plasma Membrane ◽  
Second Messengers ◽  
Human Sperm ◽  
Secretory Vesicle ◽  
Snare Complex ◽  
Calcium Signal ◽  
Rab Gtpases ◽  
Attachment Protein ◽  
Protein Receptors ◽  
Signalling Cascade

Exocytosis is a highly regulated process that consists of multiple functionally, kinetically and/or morphologically definable stages such as recruitment, targeting, tethering and docking of secretory vesicles with the plasma membrane, priming of the fusion machinery and calcium-triggered membrane fusion. After fusion, the membrane around the secretory vesicle is incorporated into the plasma membrane and the granule releases its contents. The proteins involved in these processes belong to several highly conserved families: Rab GTPases, SNAREs (soluble NSF-attachment protein receptors), α-SNAP (α-NSF attachment protein), NSF (N-ethylmaleimide-sensitive factor), Munc13 and -18, complexins and synaptotagmins. In the present article, the molecules of exocytosis are reviewed, using human sperm as a model system. Sperm exocytosis is driven by isoforms of the same proteinaceous fusion machinery mentioned above, with their functions orchestrated in a hierarchically organized and unidirectional signalling cascade. In addition to the universal exocytosis regulator calcium, this cascade includes other second messengers such as diacylglycerol, inositol 1,4,5-trisphosphate and cAMP, as well as the enzymes that synthesize them and their target proteins. Of special interest is the cAMP-binding protein Epac (exchange protein directly activated by cAMP) due in part to its enzymatic activity towards Rap. The activation of Epac and Rap leads to a highly localized calcium signal which, together with assembly of the SNARE complex, governs the final stages of exocytosis. The source of this releasable calcium is the secretory granule itself.

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 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.

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.

Roles of the cytoplasmic and transmembrane domains of syntaxins in intracellular localization and trafficking

2001 ◽  
Vol 114 (17) ◽  
pp. 3115-3124 ◽  
Author(s):  
Kazuo Kasai ◽  
Kimio Akagawa
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Transmembrane Domains ◽  
Cytoplasmic Domains ◽  
Immunofluorescence Analysis ◽  
Attachment Protein ◽  
Transport Pathways ◽  
Sensitive Factor ◽  
Protein Receptors

Syntaxins are target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) involved in docking and fusion of vesicles in exocytosis and endocytosis. Many syntaxin isoforms have been isolated, and each one displays a distinct intracellular localization pattern. However, the signals that drive the specific intracellular localization of syntaxins are poorly understood. In this study, we used indirect immunofluorescence analysis to examine the localization of syntaxin chimeras, each containing a syntaxin transmembrane domain fused to a cytoplasmic domain derived from a different syntaxin. We show that the cytoplasmic domains of syntaxins 5, 6, 7 and 8 have important effects on intracellular localization. We also demonstrate that the transmembrane domain of syntaxin 5 is sufficient to localize the chimera to the compartment expected for wild-type syntaxin 5. Additionally, we find that syntaxins 6, 7 and 8, but not syntaxin 5, are present at the plasma membrane, and that these syntaxins cycle through the plasma membrane by virtue of their cytoplasmic domains. Finally, we find that di-leucine-based motifs in the cytoplasmic domains of syntaxins 7 and 8 are necessary for their intracellular localization and trafficking via distinct transport pathways. Combined, these results suggest that both the cytoplasmic and the transmembrane domains play important roles in intracellular localization and trafficking of syntaxins.

scholarly journals Munc18-1 binding to the neuronal SNARE complex controls synaptic vesicle priming

2009 ◽  
Vol 184 (5) ◽  
pp. 751-764 ◽  
Author(s):  
Ferenc Deák ◽  
Yi Xu ◽  
Wen-Pin Chang ◽  
Irina Dulubova ◽  
Mikhail Khvotchev ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Tight Binding ◽  
Point Mutations ◽  
Snare Complex ◽  
Attachment Protein ◽  
Macromolecular Assembly ◽  
Closed Conformation ◽  
Protein Receptors ◽  
Primed State ◽  
Synaptic Vesicle Fusion

Munc18-1 and soluble NSF attachment protein receptors (SNAREs) are critical for synaptic vesicle fusion. Munc18-1 binds to the SNARE syntaxin-1 folded into a closed conformation and to SNARE complexes containing open syntaxin-1. Understanding which steps in fusion depend on the latter interaction and whether Munc18-1 competes with other factors such as complexins for SNARE complex binding is critical to elucidate the mechanisms involved. In this study, we show that lentiviral expression of Munc18-1 rescues abrogation of release in Munc18-1 knockout mice. We describe point mutations in Munc18-1 that preserve tight binding to closed syntaxin-1 but markedly disrupt Munc18-1 binding to SNARE complexes containing open syntaxin-1. Lentiviral rescue experiments reveal that such disruption selectively impairs synaptic vesicle priming but not Ca2+-triggered fusion of primed vesicles. We also find that Munc18-1 and complexin-1 bind simultaneously to SNARE complexes. These results suggest that Munc18-1 binding to SNARE complexes mediates synaptic vesicle priming and that the resulting primed state involves a Munc18-1–SNARE–complexin macromolecular assembly that is poised for Ca2+ triggering of fusion.

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 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 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 All three components of the neuronal SNARE complex contribute to secretory vesicle docking

2012 ◽  
Vol 198 (3) ◽  
pp. 323-330 ◽  
Author(s):  
Yao Wu ◽  
Yiwen Gu ◽  
Mary K. Morphew ◽  
Jun Yao ◽  
Felix L. Yeh ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Secretory Vesicle ◽  
Distal Portion ◽  
Snare Complex ◽  
Vesicle Docking ◽  
Large Dense Core Vesicles ◽  
Target Membrane ◽  
Snare Motif ◽  
Clostridial Neurotoxin

Before exocytosis, vesicles must first become docked to the plasma membrane. The SNARE complex was originally hypothesized to mediate both the docking and fusion steps in the secretory pathway, but previous electron microscopy (EM) studies indicated that the vesicular SNARE protein synaptobrevin (syb) was dispensable for docking. In this paper, we studied the function of syb in the docking of large dense-core vesicles (LDCVs) in live PC12 cells using total internal reflection fluorescence microscopy. Cleavage of syb by a clostridial neurotoxin resulted in significant defects in vesicle docking in unfixed cells; these results were confirmed via EM using cells that were prepared using high-pressure freezing. The membrane-distal portion of its SNARE motif was critical for docking, whereas deletion of a membrane-proximal segment had little effect on docking but diminished fusion. Because docking was also inhibited by toxin-mediated cleavage of the target membrane SNAREs syntaxin and SNAP-25, syb might attach LDCVs to the plasma membrane through N-terminal assembly of trans-SNARE pairs.

scholarly journals Munc18a clusters SNARE-bearing liposomes prior to trans-SNARE zippering

2017 ◽  
Vol 474 (19) ◽  
pp. 3339-3354 ◽  
Author(s):  
Matthew Grant Arnold ◽  
Pratikshya Adhikari ◽  
Baobin Kang ◽  
Hao Xu (徐昊)
Keyword(s):  
Snare Complex ◽  
Binary Interaction ◽  
Fusion Reactions ◽  
Sm Proteins ◽  
Attachment Protein ◽  
Direct Role ◽  
Vesicle Docking ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Snare Complex Formation

Sec1–Munc18 (SM) proteins co-operate with SNAREs {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein] receptors} to mediate membrane fusion in eukaryotic cells. Studies of Munc18a/Munc18-1/Stxbp1 in neurotransmission suggest that SM proteins accelerate fusion kinetics primarily by activating the partially zippered trans-SNARE complex. However, accumulating evidence has argued for additional roles for SM proteins in earlier steps in the fusion cascade. Here, we investigate the function of Munc18a in reconstituted exocytic reactions mediated by neuronal and non-neuronal SNAREs. We show that Munc18a plays a direct role in promoting proteoliposome clustering, underlying vesicle docking during exocytosis. In the three different fusion reactions examined, Munc18a-dependent clustering requires an intact N-terminal peptide (N-peptide) motif in syntaxin that mediates the binary interaction between syntaxin and Munc18a. Importantly, clustering is preserved under inhibitory conditions that abolish both trans-SNARE complex formation and lipid mixing, indicating that Munc18a promotes membrane clustering in a step that is independent of trans-SNARE zippering and activation.

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