Soluble NSF Attachment Protein Research Articles

A short region upstream of the yeast vacuolar Qa-SNARE heptad-repeats promotes membrane fusion through enhanced SNARE complex assembly.

Whereas SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) heptad-repeats are well studied, SNAREs also have upstream N-domains of indeterminate function. The assembly of yeast vacuolar SNAREs into complexes for fusion can be studied in chemically defined reactions. Complementary proteoliposomes bearing a Rab:GTP and either the vacuolar R-SNARE or one of the three integrally anchored Q-SNAREs were incubated with the tethering/SM protein complex HOPS and the two other soluble SNAREs (lacking a transmembrane anchor) or their SNARE heptad-repeat domains. Fusion required a transmembrane-anchored R-SNARE on one membrane and an anchored Q-SNARE on the other. The N-domain of the Qb-SNARE was completely dispensable for fusion. Whereas fusion can be promoted by very high concentrations of the Qa-SNARE heptad-repeat domain alone, at physiological concentrations the Qa-SNARE heptad-repeat domain alone has almost no fusion activity. The 181-198 region of Qa, immediately upstream of the SNARE heptad-repeat domain, is required for normal fusion activity with HOPS. This region is needed for normal SNARE complex assembly.

Identification of syntaxin 4 as an essential factor for the hepatitis C virus life cycle

Although there is evidence that multivesicular bodies (MVBs) are involved in the release of hepatitis C virus (HCV), many aspects of HCV release are still not fully understood. The amount of α-taxilin that prevents SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) complex formation by binding to free syntaxin 4 is reduced in HCV-positive cells. Therefore, it was analyzed whether the t-SNARE protein syntaxin 4 which mediates vesicles fusion is involved in the HCV life cycle.HCV-positive cells possess an increased amount of syntaxin 4 protein, although the amount of syntaxin 4-specific transcripts is decreased in HCV-positive Huh7.5 cells and in HCV-infected primary human hepatocytes. In HCV-positive cells a significant longer half-life of syntaxin 4 was found that overcompensates for the decreased expression and leads to the elevated level of syntaxin 4.Overexpression of syntaxin 4 reduces the intracellular amount of infectious viral particles by facilitating viral release, while silencing of syntaxin 4 expression using specific siRNAs inhibits the release of HCV particles and so leads to an increase in the intracellular amount of infectious viral particles. This indicates that HCV uses a SNARE-dependent pathway for viral release. Confocal immunofluorescence microscopy revealed a colocalization of syntaxin 4 with a MVB-specific marker, exosomes and HCV core, which suggests a fraction of syntaxin 4 is associated with exosomes loaded with HCV. Altogether, it is assumed that syntaxin 4 is a novel essential cellular factor for the release of HCV.

The soybean GmSNAP18 gene underlies two types of resistance to soybean cyst nematode

Two types of resistant soybean (Glycine max (L.) Merr.) sources are widely used against soybean cyst nematode (SCN, Heterodera glycines Ichinohe). These include Peking-type soybean, whose resistance requires both the rhg1-a and Rhg4 alleles, and PI 88788-type soybean, whose resistance requires only the rhg1-b allele. Multiple copy number of PI 88788-type GmSNAP18, GmAAT, and GmWI12 in one genomic segment simultaneously contribute to rhg1-b resistance. Using an integrated set of genetic and genomic approaches, we demonstrate that the rhg1-a Peking-type GmSNAP18 is sufficient for resistance to SCN in combination with Rhg4. The two SNAPs (soluble NSF attachment proteins) differ by only five amino acids. Our findings suggest that Peking-type GmSNAP18 is performing a different role in SCN resistance than PI 88788-type GmSNAP18. As such, this is an example of a pathogen resistance gene that has evolved to underlie two types of resistance, yet ensure the same function within a single plant species.

Characterization of the Soluble NSF Attachment Protein gene family identifies two members involved in additive resistance to a plant pathogen

Proteins with Tetratricopeptide-repeat (TPR) domains are encoded by large gene families and distributed in all plant lineages. In this study, the Soluble NSF-Attachment Protein (SNAP) subfamily of TPR containing proteins is characterized. In soybean, five members constitute the SNAP gene family: GmSNAP18, GmSNAP11, GmSNAP14, GmSNAP02, and GmSNAP09. Recently, GmSNAP18 has been reported to mediate resistance to soybean cyst nematode (SCN). Using a population of recombinant inbred lines from resistant and susceptible parents, the divergence of the SNAP gene family is analysed over time. Phylogenetic analysis of SNAP genes from 22 diverse plant species showed that SNAPs were distributed in six monophyletic clades corresponding to the major plant lineages. Conservation of the four TPR motifs in all species, including ancestral lineages, supports the hypothesis that SNAPs were duplicated and derived from a common ancestor and unique gene still present in chlorophytic algae. Syntenic analysis of regions harbouring GmSNAP genes in soybean reveals that this family expanded from segmental and tandem duplications following a tetraploidization event. qRT-PCR analysis of GmSNAPs indicates a co-regulation following SCN infection. Finally, genetic analysis demonstrates that GmSNAP11 contributes to an additive resistance to SCN. Thus, GmSNAP11 is identified as a novel minor gene conferring resistance to SCN.

Mechanistic Insights into NSF Mediated SNARE Complex Disassembly

Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with the adaptor protein SNAP (soluble NSF attachment protein), disassembles the SNARE complex, making individual SNAREs available for subsequent rounds of fusion. We determined structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE supercomplex (known as 20S particle) using single-particle cryo-electron microscopy (cryo-EM). We further dissected the mechanism of NSF mediated SNARE complex disassembly using single-molecule fluorescence microscopy based on the cryo-EM structures.

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles.

Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell-like RBL-2H3 cells provide direct evidence that Munc13-4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells.

Protein Conjugation to Nanoparticles by Designer Affinity Tags

For the purpose of proteins bio-conjugation to Gold Nano-Particles (GNPs), we designed and synthesized a polypeptide named NanoLock, derived from SNARE (Soluble NSF Attachment protein REceptor) proteins and able to form a remarkably stable complex with the SNARE protein SNAP25 (Synaptosome Associated Protein of 25 kDa). We also characterized the adsorption of a SNAP25 recombinant fusion to Glutathione S-Transferases (GST) named GST-SNAP25 onto GNPs and found that it forms a stable protein corona surrounding GNPs. Using GST-SNAP25 as an intermediate protein, passively adsorbed on GNPs, we were able to stably bind NanoLock to GNPs.By fusing an arbitrary protein of interest to the affinity tag NanoLock, it would be in principle possible to bind any protein to GST-SNAP25 coated GNPs by simple mixing, in a site-oriented way. Therefore, we propose the pair NanoLock/GST-SNAP25 as a universal tool for the easy bio-conjugation of recombinant proteins to GNPs and possibly other gold surfaces. Further engineered versions of SNAP25, able to bind surfaces other than gold, could be used to decorate with proteins other materials, taking advantage of the same modular approach of the system described here in the case of GNPs.

Identification of lipopolysaccharide-interacting plasma membrane-type proteins in Arabidopsis thaliana

Lipopolysaccharide (LPS) is an amphiphatic bacterial glycoconjugate found on the external membrane of Gram-negative bacteria. This endotoxin is considered as a microbe-associated molecular pattern (MAMP) molecule and has been shown to elicit defense responses in plants. Here, LPS-interacting proteins from Arabidopsis thaliana plasma membrane (PM)-type fractions were captured and identified in order to investigate those involved in LPS perception and linked to triggering of innate immune responses. A novel proteomics-based affinity-capture strategy coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for the enrichment and identification of LPS-interacting proteins. As such, LPS isolated from Burkholderia cepacia (LPSB.cep.) was immobilized on three independent and distinct affinity-based matrices to serve as bait for interacting proteins from A. thaliana leaf and callus tissue. These were resolved by 1D electrophoresis and identified by mass spectrometry. Proteins specifically bound to LPSB.cep. have been implicated in membrane structure (e.g. COBRA-like and tubulin proteins), membrane trafficking and/or transport (e.g. soluble NSF attachment protein receptor (SNARE) proteins, patellin, aquaporin, PM instrinsic proteins (PIP) and H+-ATPase), signal transduction (receptor-like kinases and calcium-dependent protein kinases) as well as defense/stress responses (e.g. hypersensitive-induced response (HIR) proteins, jacalin-like lectin domain-containing protein and myrosinase-binding proteins). The novel affinity-capture strategy for the enrichment of LPS-interacting proteins proved to be effective, especially in the binding of proteins involved in plant defense responses, and can thus be used to elucidate LPS-mediated molecular recognition and disease mechanism(s).

Interactions Between SNAP-25 and Synaptotagmin-1 Are Involved in Vesicle Priming, Clamping Spontaneous and Stimulating Evoked Neurotransmission.

The function of synaptotagmin-1 (syt-1):soluble NSF attachment protein receptor (SNARE) interactions during neurotransmission remains unclear. We mutated SNAP-25 within the recently identified region I and region II of the primary synaptotagmin:SNARE interface. Using in vitro assays and rescue experiments in autaptic neurons, we show that interactions within region II of the primary interface are necessary for synchronized calcium-triggered release, whereas region I is involved in vesicle priming. Spontaneous release was disinhibited by region I mutation and found to correlate with defective complexin (Cpx) clamping in vitro, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Therefore, vesicle priming, clamping spontaneous release, and eliciting evoked release are three different functions of syt-1 that involve different interaction modes with the SNARE complex.

Disease resistance through impairment of α-SNAP–NSF interaction and vesicular trafficking by soybean Rhg1

α-SNAP [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein] and NSF proteins are conserved across eukaryotes and sustain cellular vesicle trafficking by mediating disassembly and reuse of SNARE protein complexes, which facilitate fusion of vesicles to target membranes. However, certain haplotypes of the Rhg1 (resistance to Heterodera glycines 1) locus of soybean possess multiple repeat copies of an α-SNAP gene (Glyma.18G022500) that encodes atypical amino acids at a highly conserved functional site. These Rhg1 loci mediate resistance to soybean cyst nematode (SCN; H. glycines), the most economically damaging pathogen of soybeans worldwide. Rhg1 is widely used in agriculture, but the mechanisms of Rhg1 disease resistance have remained unclear. In the present study, we found that the resistance-type Rhg1 α-SNAP is defective in interaction with NSF. Elevated in planta expression of resistance-type Rhg1 α-SNAPs depleted the abundance of SNARE-recycling 20S complexes, disrupted vesicle trafficking, induced elevated abundance of NSF, and caused cytotoxicity. Soybean, due to ancient genome duplication events, carries other loci that encode canonical (wild-type) α-SNAPs. Expression of these α-SNAPs counteracted the cytotoxicity of resistance-type Rhg1 α-SNAPs. For successful growth and reproduction, SCN dramatically reprograms a set of plant root cells and must sustain this sedentary feeding site for 2-4 weeks. Immunoblots and electron microscopy immunolocalization revealed that resistance-type α-SNAPs specifically hyperaccumulate relative to wild-type α-SNAPs at the nematode feeding site, promoting the demise of this biotrophic interface. The paradigm of disease resistance through a dysfunctional variant of an essential gene may be applicable to other plant-pathogen interactions.

Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells.

Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion.

Prion Aggregates Are Recruited to the Insoluble Protein Deposit (IPOD) via Myosin 2-Based Vesicular Transport

Aggregation of amyloidogenic proteins is associated with several neurodegenerative diseases. Sequestration of misfolded and aggregated proteins into specialized deposition sites may reduce their potentially detrimental properties. Yeast exhibits a distinct deposition site for amyloid aggregates termed “Insoluble PrOtein Deposit (IPOD)”, but nothing is known about the mechanism of substrate recruitment to this site. The IPOD is located directly adjacent to the Phagophore Assembly Site (PAS) where the cell initiates autophagy and the Cytoplasm-to-Vacuole Targeting (CVT) pathway destined for delivery of precursor peptidases to the vacuole. Recruitment of CVT substrates to the PAS was proposed to occur via vesicular transport on Atg9 vesicles and requires an intact actin cytoskeleton and “SNAP (Soluble NSF Attachment Protein) Receptor Proteins (SNARE)” protein function. It is, however, unknown how this vesicular transport machinery is linked to the actin cytoskeleton. We demonstrate that recruitment of model amyloid PrD-GFP and the CVT substrate precursor-aminopeptidase 1 (preApe1) to the IPOD or PAS, respectively, is disturbed after genetic impairment of Myo2-based actin cable transport and SNARE protein function. Rather than accumulating at the respective deposition sites, both substrates reversibly accumulated often together in the same punctate structures. Components of the CVT vesicular transport machinery including Atg8 and Atg9 as well as Myo2 partially co-localized with the joint accumulations. Thus we propose a model where vesicles, loaded with preApe1 or PrD-GFP, are recruited to tropomyosin coated actin cables via the Myo2 motor protein for delivery to the PAS and IPOD, respectively. We discuss that deposition at the IPOD is not an integrated mandatory part of the degradation pathway for amyloid aggregates, but more likely stores excess aggregates until downstream degradation pathways have the capacity to turn them over after liberation by the Hsp104 disaggregation machinery.

Altered T-UCRs expression profile in the spinal cord of mice with neuropathic pain

Spinal cord plays an important role in the transmission and modulation of nociceptive information. Global changes in gene expression in the spinal cord contribute to the induction and maintenance of neuropathic pain. Transcribed Ultraconserved Regions (T-UCRs), a novel class of long noncoding RNAs, can regulate gene expression at both transcriptional and post-transcriptional levels and are related to many human diseases such as cancer, Alzheimer's disease, and heart diseases. In this study, we screened abnormal T-UCRs expression in the spinal cord under spinal nerve ligation (SNL)-induced neuropathic pain condition. Microarray data showed the alternation of T-UCRs at the transcriptional level in the spinal cord 10 days after SNL. Among 78 altered T-UCRs, 23 T-UCRs were upregulated by more than 1.5-fold and 55 ones downregulated by less than 0.5-fold after SNL. Hierarchical cluster analysis of T-UCRs expression profiles showed the opposite expression pattern between SNL and sham-operated mice. The quantitative real-time reverse transcription polymerase chain reaction analysis further confirmed the expression patterns of uc.305, uc.189, uc.46, and uc.217 after SNL. The gene ontology annotation and signaling pathway analysis for the T-UCRs host genes indicated that differentially expressed T-UCRs were involved in several intracellular activities and signaling pathways, including Ephrin receptor activity, soluble NSF attachment protein receptor (SNARE) interactions in vesicular transport pathway, and WNT signaling pathway. Collectively, the current data suggest the possible role of T-UCR in the pathogenesis of neuropathic pain. T-UCRs may serve as a new kind of target for the treatment of neuropathic pain.

Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion

Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state.

Syntaxin-17 delivers PINK1/parkin-dependent mitochondrial vesicles to the endolysosomal system.

Mitochondria are considered autonomous organelles, physically separated from endocytic and biosynthetic pathways. However, recent work uncovered a PINK1/parkin-dependent vesicle transport pathway wherein oxidized or damaged mitochondrial content are selectively delivered to the late endosome/lysosome for degradation, providing evidence that mitochondria are indeed integrated within the endomembrane system. Given that mitochondria have not been shown to use canonical soluble NSF attachment protein receptor (SNARE) machinery for fusion, the mechanism by which mitochondrial-derived vesicles (MDVs) are targeted to the endosomal compartment has remained unclear. In this study, we identify syntaxin-17 as a core mitochondrial SNARE required for the delivery of stress-induced PINK1/parkin-dependent MDVs to the late endosome/lysosome. Syntaxin-17 remains associated with mature MDVs and forms a ternary SNARE complex with SNAP29 and VAMP7 to mediate MDV-endolysosome fusion in a manner dependent on the homotypic fusion and vacuole protein sorting (HOPS) tethering complex. Syntaxin-17 can be traced to the last eukaryotic common ancestor, hinting that the removal of damaged mitochondrial content may represent one of the earliest vesicle transport routes in the cell.

Multiple ER-Golgi SNARE transmembrane domains are dispensable for trafficking but required for SNARE recycling.

The formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes between opposing membranes is an essential prerequisite for fusion between vesicles and their target compartments. The composition and length of a SNARE's transmembrane domain (TMD) is also an indicator for their steady-state distribution in cells. The evolutionary conservation of the SNARE TMD, together with the strict requirement of this feature for membrane fusion in biochemical studies, implies that the TMD represents an essential protein module. Paradoxically, we find that for several essential ER- and Golgi-localized SNAREs, a TMD is unnecessary. Moreover, in the absence of a covalent membrane tether, such SNAREs can still support ER-Golgi vesicle transport and recapitulate established genetic interactions. Transport anomalies appear to be restricted to retrograde trafficking, but these defects are overcome by the attachment of a C-terminal lipid anchor to the SNARE. We conclude that the TMD functions principally to support the recycling of Qb-, Qc-, and R-SNAREs and, in so doing, retrograde transport.

Synaptotagmin-7 Functions to Replenish Insulin Granules for Exocytosis in Human Islet β-Cells.

Synaptotagmin (Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic β-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7’s precise exocytotic actions in β-cells remain unknown. We show that Syt-7 is abundant in human β-cells. Adenovirus–short hairpin RNA knockdown (KD) of Syt-7 in human islets reduced first- and second-phase GSIS attributed to the reduction of exocytosis of predocked and newcomer insulin secretory granules (SGs). Glucose stimulation expectedly induced Syt-7 association in a Ca2+-dependent manner with syntaxin-3 and syntaxin-1A soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes known to mediate exocytosis of newcomer and predocked SGs, respectively. However, Syt-7-KD did not disrupt SNARE complex assembly. Instead, electron microscopy analysis showed that Syt-7-KD reduced the recruitment of SGs to the plasma membrane after glucose-stimulated depletion, which could not be rescued by glucagon-like peptide 1 pretreatment. To assess the possibility that this new action of Syt-7 on SG recruitment may involve calmodulin (CaM), pretreatment of islets with CaM blocker calmidazolium showed effects very similar to those of Syt-7-KD. Syt-7 therefore plays a novel more dominant function in the replenishment of releasable SG pools in human β-cells than its previously purported role in exocytotic fusion per se.

N-Ethylmaleimide Dissociates α7 ACh Receptor from a Complex with NSF and Promotes Its Delivery to the Presynaptic Membrane.

N-Ethylmaleimide (NEM)-sensitive factor (NSF) associates with soluble NSF attachment protein (SNAP), that binds to SNAP receptors (SNAREs) including syntaxin, SNAP25, and synaptobrevin. The complex of NSF/SNAP/SNAREs plays a critical role in the regulation of vesicular traffic. The present study investigated NEM-regulated α7 ACh receptor translocation. NSF associated with β-SNAP and the SNAREs syntaxin 1 and synaptobrevin 2 in the rat hippocampus. NSF also associated with the α7 ACh receptor subunit, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, and the γ-aminobutyric acid A (GABAA) receptor γ2 subunit. NEM, an inhibitor of NSF, significantly dissociated the α7 ACh receptor subunit from a complex with NSF and increased cell surface localization of the receptor subunit, but such effect was not obtained with the GluA1, GluA2 or γ2 subunits. NEM, alternatively, dissociated synaptobrevin 2 from an assembly of NSF/β-SNAP/syntaxin 1/synaptobrevin 2. NEM significantly increased the rate of nicotine-triggered AMPA receptor-mediated miniature excitatory postsynaptic currents, without affecting the amplitude, in rat hippocampal slices. The results of the present study indicate that NEM releases the α7 ACh receptor subunit and synaptobrevin 2 from an assembly of α7 ACh receptor subunit/NSF/β-SNAP/syntaxin 1/synaptobrevin 2, thereby promoting delivery of the α7 ACh receptor subunit to presynaptic membrane.

Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion.

The Munc13 family of exocytosis regulators has multiple Ca(2+)-binding, C2 domains. Here, we probed the mechanism by which Munc13-4 regulates invitro membrane fusion and platelet exocytosis. We show that Munc13-4 enhances invitro soluble NSF attachment protein receptor (SNARE)-dependent, proteoliposome fusion in a Ca(2+)- and phosphatidylserine (PS)-dependent manner that was independent of SNARE concentrations. Munc13-4-SNARE interactions, under the conditions used, were minimal in the absence or presence of Ca(2+). However, Munc13-4 was able to bind and cluster liposomes harbouring PS in response to Ca(2+). Interestingly, Ca(2+)-dependent liposome binding/clustering and enhancement of proteoliposome fusion required both Munc13-4 C2 domains, but only the Ca(2+)-liganding aspartate residues of the C2B domain. Analytical ultracentrifugation (AUC) measurements indicated that, in solution, Munc13-4 was a monomeric prolate ellipsoid with dimensions consistent with a molecule that could bridge two fusing membranes. To address the potential role of Munc13-4 as a tethering protein in platelets, we examined mepacrine-stained, dense granule mobility and secretion in platelets from wild-type and Munc13-4 null (Unc13d(Jinx)) mice. In the absence of Munc13-4, dense granules were highly mobile in both resting and stimulated platelets, and stimulation-dependent granule release was absent. These observations suggest that dense granules are stably docked in resting platelets awaiting stimulation and that Munc13-4 plays a vesicle-stabilizing or tethering role in resting platelets and also in activated platelets in response to Ca(2+). In summary, we show that Munc13-4 conveys Ca(2+) sensitivity to platelet SNARE-mediated membrane fusion and reveal a potential mechanism by which Munc13-4 bridges and stabilizes apposing membranes destined for fusion.

A Fine Balance of Synaptophysin Levels Underlies Efficient Retrieval of Synaptobrevin II to Synaptic Vesicles

Synaptobrevin II (sybII) is a vesicular soluble NSF attachment protein receptor (SNARE) protein that is essential for neurotransmitter release, and thus its correct trafficking to synaptic vesicles (SVs) is critical to render them fusion competent. The SV protein synaptophysin binds to sybII and facilitates its retrieval to SVs during endocytosis. Synaptophysin and sybII are the two most abundant proteins on SVs, being present in a 1:2 ratio. Synaptophysin and sybII are proposed to form a large multimeric complex, and the copy number of the proteins in this complex is also in a 1:2 ratio. We investigated the importance of this ratio between these proteins for the localisation and trafficking of sybII in central neurons. SybII was overexpressed in mouse hippocampal neurons at either 1.6 or 2.15–2.35-fold over endogenous protein levels, in the absence or presence of varying levels of synaptophysin. In the absence of exogenous synaptophysin, exogenous sybII was dispersed along the axon, trapped on the plasma membrane and retrieved slowly during endocytosis. Co-expression of exogenous synaptophysin rescued all of these defects. Importantly, the expression of synaptophysin at nerve terminals in a 1:2 ratio with sybII was sufficient to fully rescue normal sybII trafficking. These results demonstrate that the balance between synaptophysin and sybII levels is critical for the correct targeting of sybII to SVs and suggests that small alterations in synaptophysin levels might affect the localisation of sybII and subsequent presynaptic performance.