Soluble N-ethylmaleimide-sensitive Factor (NSF) Attachment Protein Receptor Research Articles

P97 and p47 function in membrane tethering in cooperation with FTCD during mitotic Golgi reassembly.

p97ATPase-mediated membrane fusion is required for the biogenesis of the Golgi complex. p97 and its cofactor p47 function in soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) priming, but the tethering complex for p97/p47-mediated membrane fusion remains unknown. In this study, we identified formiminotransferase cyclodeaminase (FTCD) as a novel p47-binding protein. FTCD mainly localizes to the Golgi complex and binds to either p47 or p97 via its association with their polyglutamate motifs. FTCD functions in p97/p47-mediated Golgi reassembly at mitosis in vivo and in vitro via its binding to p47 and to p97. We also showed that FTCD, p47, and p97 form a big FTCD-p97/p47-FTCD tethering complex. In vivo tethering assay revealed that FTCD that was designed to localize to mitochondria caused mitochondria aggregation at mitosis by forming a complex with endogenous p97 and p47, which support a role for FTCD in tethering biological membranes in cooperation with the p97/p47 complex. Therefore, FTCD is thought to act as a tethering factor by forming the FTCD-p97/p47-FTCD complex in p97/p47-mediated Golgi membrane fusion.

Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A-F Employing Taguchi Design-of-Experiments.

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins’ light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A–F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A–F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.

Detection of Novel Fusion Transcript VTI1A-CFAP46 in Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is now the second-highest cause of cancer death worldwide. Recent studies have discovered a wide range of somatic mutations in HCC. These mutations involve various vital signaling pathways such as: Wnt/β-Catenin, p53, telome­rase reverse transcriptase (TERT), chromatin remodeling, RAS/MAPK signaling, and oxidative stress. However, fusion transcripts have not been broadly explored in HCC. Methods: To identify novel fusion transcripts in HCC, in the first phase of our study, we performed targeted RNA sequencing (in HCC and paired non-HCC tissues) on 6 patients with a diagnosis of HCC undergoing liver transplantation. Results: As a result of these studies, we discovered the novel fusion transcript, VTI1A-CFAP46. In the second phase of our study, we measured the expression of wild-type VTI1A in 21 HCC specimens, which showed that 10 of 21 exhibited upregulation of wild-type VTI1A in their tumors. VTI1A (Vesicle Transport via Interaction with t-SNARE homolog 1A) is a member of the Soluble N-ethylmaleimide-Sensitive Factor (NSF) attachment protein receptor (SNARE) gene family, which is essential for membrane trafficking and function in endocytosis, autophagy, and Golgi transport. Notably, it is known that autophagy is involved in HCC. Conclusions: The link between novel fusion transcript VTI1A-CFAP46 and autophagy as a potential therapeutic target in HCC patients deserves further investigation. Moreover, this study shows that fusion transcripts are worthy of additional exploration in HCC.

Visualization of SNARE-Mediated Hemifusion between Giant Unilamellar Vesicles Arrested by Myricetin.

Neurotransmitters are released within a millisecond after Ca2+ arrives at an active zone. However, the vesicle fusion pathway underlying this synchronous release is yet to be understood. At the center of controversy is whether hemifusion, in which outer leaflets are merged while inner leaflets are still separated, is an on-pathway or off-pathway product of Ca2+-triggered exocytosis. Using the single vesicle fusion assay, we recently demonstrated that hemifusion is an on-pathway intermediate that immediately proceeds to full fusion upon Ca2+ triggering. It has been shown that the flavonoid myricetin arrests soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-mediated vesicle fusion at hemifusion, but that the hemifused vesicles spontaneously convert to full fusion when the myricetin clamp is removed by the enzyme laccase. In the present study, we visualized SNARE-mediated hemifusion between two SNARE-reconstituted giant unilamellar vesicles (GUVs) arrested by myricetin. The large size of the GUVs enabled us to directly image the hemifusion between them. When two merging GUVs were labeled with different fluorescent dyes, GUV pairs showed asymmetric fluorescence intensities depending on the position on the GUV pair consistent with what is expected for hemifusion. The flow of lipids from one vesicle to the other was revealed with fluorescence recovery after photobleaching (FRAP), indicating that the two membranes had hemifused. These results support the hypothesis that hemifusion may be the molecular status that primes Ca2+-triggered millisecond exocytosis. This study represents the first imaging of SNARE-driven hemifusion between GUVs.

Dopamine D1A directly interacts with otoferlin synaptic pathway proteins: Ca2+ and phosphorylation underlie an NSF-to-AP2mu1 molecular switch.

Dopamine receptors regulate exocytosis via protein-protein interactions (PPIs) as well as via adenylyl cyclase transduction pathways. Evidence has been obtained for PPIs in inner ear hair cells coupling D1A to soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-related proteins snapin, otoferlin, N-ethylmaleimide-sensitive factor (NSF), and adaptor-related protein complex 2, mu 1 (AP2mu1), dependent on [Ca2+] and phosphorylation. Specifically, the carboxy terminus of dopamine D1A was found to directly bind t-SNARE-associated protein snapin in teleost and mammalian hair cell models by yeast two-hybrid (Y2H) and pull-down assays, and snapin directly interacts with hair cell calcium-sensor otoferlin. Surface plasmon resonance (SPR) analysis, competitive pull-downs, and co-immunoprecipitation indicated that these interactions were promoted by Ca2+ and occur together. D1A was also found to separately interact with NSF, but with an inverse dependence on Ca2+ Evidence was obtained, for the first time, that otoferlin domains C2A, C2B, C2D, and C2F interact with NSF and AP2mu1, whereas C2C or C2E do not bind to either protein, representing binding characteristics consistent with respective inclusion or omission in individual C2 domains of the tyrosine motif YXXΦ. In competitive pull-down assays, as predicted by KD values from SPR (+Ca2+), C2F pulled down primarily NSF as opposed to AP2mu1. Phosphorylation of AP2mu1 gave rise to a reversal: an increase in binding by C2F to phosphorylated AP2mu1 was accompanied by a decrease in binding to NSF, consistent with a molecular switch for otoferlin from membrane fusion (NSF) to endocytosis (AP2mu1). An increase in phosphorylated AP2mu1 at the base of the cochlear inner hair cell was the observed response elicited by a dopamine D1A agonist, as predicted.

The SNARE Proteins SNAP25 and Synaptobrevin Are Involved in Endocytosis at Hippocampal Synapses

SNAP25, an essential component of the soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) complex that mediates exocytosis, is not considered to play a role in endocytosis, which couples to exocytosis by retrieving a similar amount of exocytosed vesicles. By knocking down SNAP25 and imaging slow endocytosis at a conventional synapse, the rat cultured hippocampal synapse, we found that SNAP25 is involved in slow, clathrin-dependent endocytosis. With similar techniques, we found that not only SNAP25, but also synaptobrevin is involved in slow endocytosis. These results provide the first evidence showing the dual role of SNAP25 and synaptobrevin in both exocytosis and slow endocytosis at conventional synapses. Such a dual role may contribute to mediate the coupling between exocytosis and clathrin-dependent endocytosis at conventional synapses, a mechanism critical for the maintenance of synaptic transmission and the normal structure of nerve terminals.

The Dsl1 Tethering Complex Actively Participates in Soluble NSF (N-Ethylmaleimide-sensitive Factor) Attachment Protein Receptor (SNARE) Complex Assembly at the Endoplasmic Reticulum in Saccharomyces cerevisiae

Intracellular transport is largely dependent on vesicles that bud off from one compartment and fuse with the target compartment. The first contact of an incoming vesicle with the target membrane is mediated by tethering factors. The tethering factor responsible for recruiting Golgi-derived vesicles to the ER is the Dsl1 tethering complex, which is comprised of the essential proteins Dsl1p, Dsl3p, and Tip20p. We investigated the role of the Tip20p subunit at the ER by analyzing two mutants, tip20-5 and tip20-8. Both mutants contained multiple mutations that were scattered throughout the TIP20 sequence. Individual mutations could not reproduce the temperature-sensitive phenotype of tip20-5 and tip20-8, indicating that the overall structure of Tip20p might be altered in the mutants. Using molecular dynamics simulations comparing Tip20p and Tip20-8p revealed that some regions, particularly the N-terminal domain and parts of the stalk region, were more flexible in the mutant protein, consistent with its increased susceptibility to proteolysis. Both Tip20-5p and Tip20-8p mutants prevented proper ER trans-SNARE complex assembly in vitro. Moreover, Tip20p mutant proteins disturbed the interaction between Dsl1p and the coatomer coat complex, indicating that the Dsl1p-coatomer interaction could be stabilized or regulated by Tip20p. We provide evidence for a direct role of the Dsl1 complex, in particular Tip20p, in the formation and stabilization of ER SNARE complexes.

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

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.

Palmitoylation of the synaptic vesicle fusion machinery

The fusion of synaptic vesicles with the pre-synaptic plasma membrane mediates the secretion of neurotransmitters at nerve terminals. This pathway is regulated by an array of protein-protein interactions. Of central importance are the soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP25, which are associated with the pre-synaptic plasma membrane and vesicle-associated membrane protein (VAMP2), a synaptic vesicle SNARE. Syntaxin 1, SNAP25 and VAMP2 interact to form a tight complex bridging the vesicle and plasma membranes, which has been suggested to represent the minimal membrane fusion machinery. Synaptic vesicle fusion is stimulated by a rise in intraterminal Ca2+ levels, and a major Ca2+ sensor for vesicle fusion is synaptotagmin I. Synaptotagmin is likely to couple Ca2+ entry to vesicle fusion via Ca2+-dependent and independent interactions with membrane phospholipids and the SNARE proteins. Intriguingly, syntaxin 1, SNAP25, VAMP2 and synaptotagmin I have all been reported to be modified by palmitoylation in neurons. In this review, we discuss the mechanisms and dynamics of palmitoylation of these proteins and speculate on how palmitoylation might contribute to the regulation of synaptic vesicle fusion.

Identification of the Neuroblastoma-amplified Gene Product as a Component of the Syntaxin 18 Complex Implicated in Golgi-to-Endoplasmic Reticulum Retrograde Transport

Syntaxin 18, a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein implicated in endoplasmic reticulum (ER) membrane fusion, forms a complex with other SNAREs (BNIP1, p31, and Sec22b) and several peripheral membrane components (Sly1, ZW10, and RINT-1). In the present study, we showed that a peripheral membrane protein encoded by the neuroblastoma-amplified gene (NAG) is a subunit of the syntaxin 18 complex. NAG encodes a protein of 2371 amino acids, which exhibits weak similarity to yeast Dsl3p/Sec39p, an 82-kDa component of the complex containing the yeast syntaxin 18 orthologue Ufe1p. Under conditions favoring SNARE complex disassembly, NAG was released from syntaxin 18 but remained in a p31-ZW10-RINT-1 subcomplex. Binding studies showed that the extreme N-terminal region of p31 is responsible for the interaction with NAG and that the N- and the C-terminal regions of NAG interact with p31 and ZW10-RINT-1, respectively. Knockdown of NAG resulted in a reduction in the expression of p31, confirming their intimate relationship. NAG depletion did not substantially affect Golgi morphology and protein export from the ER, but it caused redistribution of Golgi recycling proteins accompanied by a defect in protein glycosylation. These results together suggest that NAG links between p31 and ZW10-RINT-1 and is involved in Golgi-to-ER transport.

Cell vacuolization induced by Helicobacter pylori VacA cytotoxin does not depend on late endosomal SNAREs.

Cellular vacuoles induced by the Helicobacter pylori vacuolating cytotoxin VacA originate from late endosomal compartments. Their biogenesis requires the activity of both rab7 GTPase and the ATPase proton pump. The toxin has been suggested to cause an increased luminal osmotic pressure via its anion-specific channel activity localized on late endosomal compartments after endocytosis. Here, we show that the extensive membrane fusion that takes place in the transition from the small late endosomal compartments to the large vacuoles does not depend on soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins. The process of vacuolization leads to disappearance of the large array of internal membranes of late endosomes. We suggest that most of the vacuole-limiting membrane derives from internal membranes.

Stable SNARE Complex Prior to Evoked Synaptic Vesicle Fusion Revealed by Fluorescence Resonance Energy Transfer

Although it is clear that soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex plays an essential role in synaptic vesicle fusion, the dynamics of SNARE assembly during vesicle fusion remain to be determined. In this report, we employ fluorescence resonance energy transfer technique to study the formation of SNARE complexes. Donor/acceptor pair variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP) are fused with the N termini of SNAP-25 and synaptobrevin, respectively. In vitro assembly of SNARE core complex in the presence of syntaxin shows strong fluorescence resonance energy transfer (FRET) between the CFP-SNAP-25 and YFP-synaptobrevin. Under the same conditions, CFP fused to the C terminus of SNAP-25, and YFP- synaptobrevin have no FRET. Adenovirus-mediated gene transfer is used to express the fusion proteins in PC12 cells and cultured rat cerebellar granule cells. Strong FRET is associated with neurite membranes and vesicular structures in PC12 cells co-expressing CFP-SNAP-25 and YFP-synaptobrevin. In cultured rat cerebellar granule cells, FRET between CFP-SNAP-25 and YFP-synaptobrevin is mostly associated with sites presumed to be synaptic junctions. Neurosecretion in PC12 cells initiated by KCl depolarization leads to an increase in the extent of FRET. These results demonstrate that significant amounts of stable SNARE complex exist prior to evoked synaptic vesicle fusion and that the assembly of SNARE complex occurs during vesicle docking/priming stage. Moreover, it demonstrates that FRET can be used as an effective tool for investigating dynamic SNARE interactions during synaptic vesicle fusion.