Maleimide-sensitive Factor Attachment Protein Receptor Research Articles

Requirements of Qa-SNARE LjSYP132s for Nodulation and Seed Development in Lotus japonicus

SNAREs (soluble N-ethyl maleimide-sensitive factor attachment protein receptors) mediate membrane fusion of vesicle transport in eukaryotic cells. LjSYP132s are the members of Qa-SNAREs in Lotus japonicus. Two isoforms, LjSYP132a and LjSYP132b, are generated by alternative splicing. Immunoblot analysis detected strong expression of LjSYP132s in infected root nodules and seeds by posttranscriptional modification. In either LjSYP132a or LjSYP132b silenced roots (RNAi-LjSYP132a, RNAi-LjSYP132b), the infection thread (IT) was not elongated, suggesting that both LjSYP132a and LjSYP132b have a role in IT progression. The results were consistent with the data of qRT-PCR showing that both genes were expressed at the early stage of infection. However, during the nodulation, only LjSYP132a was induced. LjSYP132s protein was observed in the Mesorhizobium loti-inoculated roots of mutants, nfr1, castor and pollux, suggesting that LjSYP132s can be induced without Nod factor signaling. Accumulation of LjSYP132s in the peribacteroid membrane suggests the function of not only IT formation but also nutrient transport. In contrast, qRT-PCR showed that LjSYP132b was expressed in the seeds. A stable transgenic plant of LjSYP132b, R132b, was produced by RNAi silencing. In the R132b plants, small pods with a few seeds and abnormal tip growth of the pollen tubes were observed, suggesting that LjSYP132b has a role in pollen tube growth and nutrient transport in the plasma membrane of seeds.

Retrograde trafficking and quality control of yeast synaptobrevin, Snc1, are conferred by its transmembrane domain.

Synaptobrevin/vesicle-associated membrane protein 2 (VAMP2) is an essential soluble N-ethyl maleimide–sensitive factor attachment protein receptor (SNARE) protein that has been extensively studied in its role in synaptic vesicle fusion. However, sorting and trafficking of VAMP2 within the endosomal system is not well understood. Here, we use the yeast VAMP2 homologue Snc1 to investigate the pathways and signals required for endocytic trafficking. We identify two genetically distinct retrieval pathways from the endosomal system: a plasma membrane recycling pathway that requires the Rcy1 F-box protein and a retrograde pathway originating from the multivesicular/prevacuole endosome dependent on the Snx4-Atg20 sorting nexin complex. Lysine residues within the transmembrane domain of Snc1 are necessary for presentation of a Snx4-Atg20–dependent sorting signal located within its juxtamembrane region. Mutations of the transmembrane lysine residues ablate retrograde sorting and subject Snc1 to quality control via sorting into the degradative multivesicular endosome pathway. Degradative sorting requires lysine residues in the juxtamembrane region of Snc1 and is mediated by the Rsp5 ubiquitin ligase and its transmembrane adapters, Ear1 and Ssh4, which localize to endosome and vacuole membranes. This study shows that Snc1 is trafficked between the endosomal system and the Golgi apparatus via multiple pathways and provides evidence for protein quality control surveillance of a SNARE protein in the endo-vacuolar system.

SNARE-dependent glutamate release in megakaryocytes

ObjectiveThe identification of signaling pathways involved in megakaryocytopoiesis is essential for development of novel therapeutics to treat hematological disorders. Following our previous findings that megakaryocytes express functional channel-forming N-methyl-D-aspartate-type glutamate receptors, here we aimed to determine the glutamate release capacity in undifferentiated and differentiated megakaryocytes and the role of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) proteins that are known to be associated with vesicular exocytosis.Materials and MethodsUsing the megakaryocytic cell line MEG-01, primary megakaryocytes, and tissue sections of bone marrow, reverse transcription polymerase chain reaction, Western blot analysis, and immunolocalization were employed to detect factors required for vesicular glutamate release. Vesicle recycling was monitored by acridine orange and FM1-43 staining and glutamate release activity was assessed by an enzyme-linked fluorimetric assay. Genetically modified MEG-01 cells, with deletion or overexpression of SNARE and vesicular proteins, were also examined for glutamate release activity.ResultsWe demonstrated that megakaryocytes express numerous proteins required for vesicular glutamate release, including core SNARE proteins, vesicle-associated membrane protein, soluble N-ethyl maleimide-sensitive factor attachment protein−23, and syntaxin, as well as specific glutamate-loading vesicle proteins, VGLUT1 and VGLUT2. Moreover, active vesicle recycling and differentiation-dependent glutamate release were observed in megakaryocytes. Vesicle-associated membrane protein−deficient MEG-01 cells, which are impaired in vesicle recycling, showed a 30% decrease in released glutamate, whereas overexpression of VGLUT1 exhibited up to a 2.2-fold increase in glutamate release.ConclusionThese data show that glutamate release from megakaryocytes occurs in a SNARE-dependent, exocytotic manner and is increased during differentiation, suggesting that manipulation of glutamate signaling could influence megakaryocytopoiesis and, therefore, offer a suitable target for the treatment of thrombosis and other hematological disorders.

Inhibition of degranulation and cytokine production in bone marrow-derived mast cells by hydrolyzed rice bran

We investigated the effects of hydrolyzed rice bran (HRB), an arabinoxylan extracted from rice bran, on mast cell degranulation and cytokine production. HRB was obtained by treating rice bran with an extract obtained from shiitake mushrooms. Bone marrow-derived mast cells (BMMCs) were prepared by culturing bone marrow cells from BALB/c mice in the presence of interleukin-3 and stem cell factor for 4 weeks. BMMCs were pretreated with HRB (0-3 mg/ml) for 30 min and were then antigen activated. Pretreatment of BMMCs with HRB significantly inhibited antigen-induced degranulation and cytokine production (tumor necrosis factor-alpha and interleukin-4) in a dose-dependent manner. HRB also diminished membrane fusion between liposomes in which soluble N-ethyl maleimide-sensitive factor attachment protein receptors were reconstituted. Phosphorylation of RelA and mitogen-activated kinases after antigen stimulation was suppressed by pretreatment of BMMCs with HRB. These findings suggest that HRB may have an anti-inflammatory effect by inhibiting mast cell degranulation and cytokine production.

Single Molecule Study of Disassembly of SNARE Complex by NSF/α−SNAP

In eukaryotic cells, material exchange between distinct organelles within the cytosol is required for many biochemical processes. The phospholipid vesicles containing the materials are transported to the target membrane and merged. All known forms of intracellular membrane fusion or vesicle fusion involve a highly conserved family of proteins termed SNAREs (Soluble N-ethyl maleimide sensitive-factor Attachment Protein Receptors). The assembly and dissociation of SNARE proteins drives membrane fusion. A good example is chemical release in the synapse, which is essential for inter-communication between neurons. Although it has been known that the ATPase activity of NSF (N-ethyl maleimide Sensitive-Factor) is involved in the disassembly of SNARE complex, the molecular details of the reaction are not understood. In order to investigate the molecular mechanism, we applied a fluorescence-based in vitro approach at the single molecule level. SNARE complexes from three neuronal proteins (Syntanxin1a, Snap-25, and Synaptobrevin) were labeled with suitably chosen FRET dye pairs based on the available structural information about the SNARE complex. For the particular labeling pairs, high FRET corresponds to assembled complex, low FRET corresponds to dissociated complex. To test the labels, we used a conventional bulk FRET assay and found a progressive temporal change from high FRET to low FRET states, indicating complex dissociation into each individual protein in a Mg2+, ATP, and α-SNAP dependent manner. Using a single molecule TIRF optical setup, the same reactions were monitored, and likewise show a change in FRET signal corresponding to complex disassembly. We are studying the disassembly reaction for a number of different labeling pairs along the SNARE helical bundle. Single molecule kinetic studies are underway to understand the molecular mechanism of NSF catalyzed SNARE protein disassembly.

Membrane fusion between liposomes containing SNARE proteins involved in mast cell exocytosis

We monitored the membrane fusion of liposomes to determine if the minimal components of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE), which is involved in mast cell exocytosis, have fusogenic activity. Three core components of SNARE were reconstituted into liposomes. Membrane fusion between liposomes containing vesicle associated membrane protein (VAMP) -7 or -8 and liposomes containing synaptosomal-associated protein 23 kDa (SNAP23) and syntaxin-3 or -4 was monitored by fluorescence resonance energy transfer. The combination of SNAP23/syntaxin-3/VAMP-8 showed the most efficient liposome-liposome fusogenic activity. Liposomes with VAMP-7 exhibited poor fusogenic activity regardless of the syntaxin isoform. The core components of SNAP23, syntaxin-3, and VAMP-8 appear to be minimal machinery to induce membrane fusion, while VAMP-7 appears to be unessential for membrane fusion.

Multiple intermediates in SNARE-induced membrane fusion

Membrane fusion in eukaryotic cells is thought to be mediated by a highly conserved family of proteins called SNAREs (soluble N-ethyl maleimide sensitive-factor attachment protein receptors). The vesicle-associated v-SNARE engages with its partner t-SNAREs on the target membrane to form a coiled coil that bridges two membranes and facilitates fusion. As demonstrated by recent findings on the hemifusion state, identifying intermediates of membrane fusion can help unveil the underlying fusion mechanism. Observation of SNARE-driven fusion at the single-liposome level has the potential to dissect and characterize fusion intermediates most directly. Here, we report on the real-time observation of lipid-mixing dynamics in a single fusion event between a pair of SNARE-reconstituted liposomes. The assay reveals multiple intermediate states characterized by discrete values of FRET between membrane-bound fluorophores. Hemifusion, flickering of fusion pores, and kinetic transitions between intermediates, which would be very difficult to detect in ensemble assays, are now identified. The ability to monitor the time course of fusion events between two proteoliposomes should be useful for addressing many important issues in SNARE-mediated membrane fusion.

Complexin II facilitates exocytotic release in mast cells by enhancing Ca2+ sensitivity of the fusion process

Recent studies have shown that soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in exocytotic release in mast cells as in neurotransmitter release. However, the roles of the proteins that regulate the structure and activity of SNARE proteins are poorly understood. Complexin is one such regulatory protein and is involved in neurotransmitter release, although ideas about its role are still controversial. In this study, we investigated the expression and role of complexin in the regulation of exocytotic release (degranulation) in mast cells. We found that complexin II, but not complexin I, is expressed in mast cells. We obtained RBL-2H3 cells that expressed a low level of complexin II and found that antigen-induced degranulation was suppressed in these cells. No significant changes in the Ca2+ response or expression levels of syntaxins and synaptotagmin were observed in knockdown cells. An immunocytochemical study revealed that complexin II was distributed throughout the cytoplasm before antigen stimulation. However, the distribution of complexin II changed dramatically with stimulation and it became localized on the plasma membrane. This change in the intracellular distribution was observed even in the absence of extracellular Ca2+, while exocytotic release was inhibited almost completely under this condition. The degranulation induced by phorbol 12-myristate 13-acetate and A23187 depended on the extracellular Ca2+ concentration, and its sensitivity to Ca2+ was decreased in knockdown cells. These results suggest that complexin II regulates exocytosis positively by translocating to the plasma membrane and enhancing the Ca2+ sensitivity of fusion machinery, although this translocation to the plasma membrane is not sufficient to trigger exocytotic membrane fusion.

Single Molecule Observation of Liposome-Bilayer Fusion Thermally Induced by Soluble N-Ethyl Maleimide Sensitive-Factor Attachment Protein Receptors (SNAREs)

A single molecule fluorescence assay is presented for studying the mechanism of soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs)-mediated liposome fusion to supported lipid bilayers. The three neuronal SNAREs syntaxin-1A, synaptobrevin-II (VAMP), and SNAP-25A were expressed separately, and various dye-labeled combinations of the SNAREs were tested for their ability to dock liposomes and induce fusion. Syntaxin and synaptobrevin in opposing membranes were both necessary and sufficient to dock liposomes to supported bilayers and to induce thermally activated fusion. As little as one SNARE interaction was sufficient for liposome docking. Fusion of docked liposomes with the supported bilayer was monitored by the dequenching of soluble fluorophores entrapped within the liposomes. Fusion was stimulated by illumination with laser light, and the fusion probability was enhanced by raising the ambient temperature from 22 to 37°C, suggesting a thermally activated process. Surprisingly, SNAP-25 had little effect on docking efficiency or the probability of thermally induced fusion. Interprotein fluorescence resonance energy transfer experiments suggest the presence of other conformational states of the syntaxin•synaptobrevin interaction in addition to those observed in the crystal structure of the SNARE complex. Furthermore, although SNARE complexes involved in liposome docking preferentially assemble into a parallel configuration, both parallel and antiparallel configurations were observed.

Taxilin; a novel syntaxin-binding protein that is involved in Ca2+-dependent exocytosis in neuroendocrine cells.

The syntaxin family is a central coordinator and participates in multiple protein-protein interactions in the soluble N-ethyl maleimide-sensitive factor attachment protein receptor machinery, which is involved in intracellular vesicle traffic. However, the molecular mechanism by which the syntaxin family regulates intracellular vesicle transport is not well known. We have identified and purified a novel binding partner of syntaxin-3 from rat lung, and isolated and sequenced the cDNA of its human homologue from a human brain cDNA library. The cDNA had an open reading frame encoding a protein of 546 amino acids with a calculated Mr of 61,890. We tentatively referred to this protein as taxilin. A structural analysis of taxilin revealed the existence of an extraordinarily long coiled-coil domain in its C-terminal half. Syntaxin-1a and -4, as well as syntaxin-3 interacted with taxilin, but syntaxin-7 or -8 did not. Northern blot analysis showed that taxilin was ubiquitously expressed. Over-expression of full-length taxilin inhibited Ca2+-dependent exocytosis in PC12 cells. These results indicate that taxilin is a novel binding partner of several syntaxin family members and suggest that taxilin is involved in Ca2+-dependent exocytosis in neuroendocrine cells.

Interaction of Syntaxin with α-Fodrin, a Major Component of the Submembranous Cytoskeleton

The soluble N-ethyl maleimide-sensitive factor attachment protein receptor machinery is involved in membrane docking and fusion. In this machinery, the syntaxin family is a central coordinator and participates in multiple protein–protein interactions. In this study we have shown that α-fodrin, nonerythroid spectrin, is a new binding partner of the syntaxin family. α-Fodrin bound to syntaxin-1a, -3, and -4, all of which are localized on the plasma membrane. Syntaxin-3 interacted with α-fodrin in dose-dependent and saturable manners but not with α-spectrin, erythroid spectrin. Syntaxin-3 interacted with α-fodrin through its C-terminal coiled-coil region. Binding of Munc18 or SNAP-25 to syntaxin-1a inhibited the interaction of α-fodrin with syntaxin-1a. Available evidence indicates that α-fodrin is implicated in exocytosis, but a precise mode of action of α-fodrin in exocytosis remains unclear. Our results suggest that α-fodrin regulates exocytosis through the interaction with members of the syntaxin family.

SNARE protein trafficking in polarized MDCK cells.

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains. This polarity is generated and maintained by the continuous sorting of apical and basolateral components in the secretory and endocytic pathways. Soluble N-ethyl maleimide-sensitive factor attachment protein receptors (SNARE) proteins of vesicle-associated membrane protein (VAMP) and syntaxin families have been suggested to play a role in the biosynthetic transport to the apical and basolateral plasma membranes of polarized cells, where they likely mediate membrane fusion. To investigate the involvement of SNARE proteins in membrane trafficking to the apical and basolateral plasma membrane in the endocytic pathway we have monitored the recycling of various VAMP and syntaxin molecules between intracellular compartments and the two plasma membrane domains in Madin-Darby canine kidney (MDCK) cells. Here we show that VAMP8/endobrevin cycles through the apical but not through the basolateral plasma membrane. Furthermore, we found that VAMP8 localizes to apical endosomal membranes in nephric tubule epithelium and in MDCK cells. This asymmetry in localization and cycling behavior suggests that VAMP8/endobrevin may play a role in apical endosomal trafficking in polarized epithelium cells.

Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells.

We have investigated the relationships between the apical sorting mechanism using lipid rafts and the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin- Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP protein stimulates the apical transport whereas a SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.

Quantal analysis of synaptic potentials in neurons of the central nervous system.

This review concentrates on the results of quantal analysis at central synapses, particularly where the morphology and the electrophysiology of the same synaptic connection has been investigated. Results from quantal analysis at peripheral synapses are drawn on to help resolve some issues. The major topics covered in this review are the properties of the quantal synaptic potential and the junctional mechanisms involved in its generation