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Congenital nystagmus (CN) is an ocular movement disorder manifested as involuntary conjugated binocular oscillation and usually occurs in early infancy. The pathological mechanism underlying CN is still poorly understood. We mapped a novel genetic locus 9q33.1-q34.2 in a larger Chinese family with autosomal dominant CN and identified a variant (c.47A>G/p.His16Arg) of STXBP1 by exome sequencing, which fully co-segregated with the nystagmus phenotype in this family and was absent in 571 healthy unrelated individuals. The STXBP1 encodes syntaxin binding protein 1 (also known as MUNC18-1), which plays a pivotal role in neurotransmitter release. In unc-18 (nematode homolog of MUNC18-1) null Caenorhabditis elegans, we found that the p.His16Arg exhibits a compromised ability to rescue the locomotion defect and aldicarb sensitivity, indicating a functional defect in neurotransmitter release. In addition, we also found an enhanced binding of the p.His16Arg mutant to syntaxin 3B, which is a homolog of syntaxin 1A and specifically located in retinal ribbon synapses. We hypothesize that the variant p.His16Arg of STXBP1 is likely to affect neurotransmitter release in the retina, which may be the underlying etiology of CN in this family. Our results provide a new perspective on understanding the molecular mechanism of CN.

The phylum of Apicomplexa groups medically relevant parasites such as those responsible for malaria and toxoplasmosis. As members of the Alveolata superphylum, these protozoans possess specialized organelles in addition to those found in all members of the eukaryotic kingdom. Vesicular trafficking is the major route of communication between membranous organelles. Neither the molecular mechanism that allows communication between organelles nor the vesicular fusion events that underlie it are completely understood in Apicomplexa. Here, we assessed the function of SEC1/Munc18 and SNARE proteins to identify factors involved in the trafficking of vesicles between these various organelles. We show that SEC1/Munc18 in interaction with SNARE proteins allows targeting of vesicles to the inner membrane complex, prerhoptries, micronemes, apicoplast, and vacuolar compartment from the endoplasmic reticulum, Golgi apparatus, or endosomal-like compartment. These data provide an exciting look at the “ZIP code” of vesicular trafficking in apicomplexans, essential for precise organelle biogenesis, homeostasis, and inheritance., Apicomplexans are obligate intracellular parasites harboring three sets of unique secretory organelles termed micronemes, rhoptries, and dense granules that are dedicated to the establishment of infection in the host cell. Apicomplexans rely on the endolysosomal system to generate the secretory organelles and to ingest and digest host cell proteins. These parasites also possess a metabolically relevant secondary endosymbiotic organelle, the apicoplast, which relies on vesicular trafficking for correct incorporation of nuclear-encoded proteins into the organelle. Here, we demonstrate that the trafficking and destination of vesicles to the unique and specialized parasite compartments depend on SNARE proteins that interact with tethering factors. Specifically, all secreted proteins depend on the function of SLY1 at the Golgi. In addition to a critical role in trafficking of endocytosed host proteins, TgVps45 is implicated in the biogenesis of the inner membrane complex (alveoli) in both Toxoplasma gondii and Plasmodium falciparum, likely acting in a coordinated manner with Stx16 and Stx6. Finally, Stx12 localizes to the endosomal-like compartment and is involved in the trafficking of proteins to the apical secretory organelles rhoptries and micronemes as well as to the apicoplast.

Replenishment of readily releasable synaptic vesicles (SVs) with vesicles in the recycling pool is important for sustained transmitter release during repetitive stimulation. Kinetics of replenishment and available pool size define synaptic performance. However, whether all SVs in the recycling pool are recruited for release with equal probability is unknown. Here, using comprehensive optical imaging for various presynaptic endosomal SNARE proteins in cultured hippocampal neurons, we demonstrate that part of the recycling pool bearing the endosomal Q–SNARE Syntaxin 7 (Stx7) is preferentially mobilized for release during high–frequency repetitive stimulation. Recruitment of the SV pool marked with the Stx7–reporter requires high intra–terminal Ca2+ concentrations and actin polymerization. Furthermore, disruption of Stx7 function by overexpressing the N–terminal domain selectively abolished this pool. Thus, our data indicate that endosomal membrane fusion involving Stx7 is essential for adaptation of synapses to respond high-frequency repetitive stimulation.

Niemann-Pick type C (NPC) is a neurological disorder with no cure. NPC proteins deliver cholesterol from endosomes to other compartments including trans-Golgi network (TGN) and endoplasmic reticulum (ER). Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident ER enzyme that converts cholesterol to cholesteryl esters for storage. Here, we report the surprising finding that in a mutantNpc1mice,Acat1-deficiency delayed the onset of weight loss and declining motor skill, prolonged lifespan, delayed Purkinje neuron death, and improved hepatosplenic pathology. Furthermore, syntaxin 6, a cholesterol-binding t-SNARE normally localized to TGN, is mislocalized in mutant NPC cells. However, upon ACAT1 inhibition this mislocalization is corrected, and increase the level of a few proteins further downstream. Our results imply that ACAT1 inhibition diverts a cholesterol storage pool in a way that replenished the low cholesterol level in NPC-deficient TGN. Taking together, we identify ACAT1 inhibition as a potential therapeutic target for NPC treatment.

Background: Colorectal cancer (CRC) is one of the most common cancers with high mortality worldwide. Uncontrolled growth is an important hallmark of CRC. However, the mechanisms are poorly understood. Methods: Syntaxin 2 (STX2) expression was analyzed in 160 cases of paraffin-embedded CRC tissue by immunohistochemistry, in 10 cases of fresh CRC tissue by western blot, and in 2 public databases. Gain- and loss-of-function analyses were used to investigate the biological function of STX2 in CRC growth. Exosomes isolation, characterization, Co-immunoprecipitation (Co-IP), flow cytometry and fluorescence were conducted to study the molecular mechanism of STX2 in CRC growth. Results: The expression of STX2 was obviously up-regulated in human CRC tissues. Overexpression of STX2 increased the growth of CRC cells in vitro and in vivo. Downregulation of STX2 repressed the growth of CRC. STX2 modulated exosomes secretion of CRC cells which might correlated with Rab8a expression. The secreted exosomes could be ingested by CRC cells, and ultimately promoted the growth of CRC by arresting the tumor cells at S phase. Conclusions: Our data provide evidence that STX2 promotes CRC growth by increasing exosomes secretion of CRC cells; And the modulation of STX2 in exosomes secretion correlates with Rab8a. Thus, our study identified a new mechanism of STX2 in CRC growth and may provide a possible strategy for CRC therapy.

Nonsense-mediated decay (NMD) is a translation-dependent RNA quality control mechanism that occurs in the cytoplasm. However, it is unknown how NMD regulates the stability of RNAs translated at the endoplasmic reticulum (ER). Here, we identify a localized NMD pathway dedicated to ER-translated mRNAs. We previously identified NBAS, a component of the Syntaxin 18 complex involved in Golgi-to-ER trafficking, as a novel NMD factor. Furthermore, we show that NBAS fulfills an independent function in NMD. This ER–NMD pathway requires the interaction of NBAS with the core NMD factor UPF1, which is partially localized at the ER in the proximity of the translocon. NBAS and UPF1 coregulate the stability of ER-associated transcripts, in particular those associated with the cellular stress response. We propose a model where NBAS recruits UPF1 to the membrane of the ER and activates an ER-dedicated NMD pathway, thus providing an ER-protective function by ensuring quality control of ER-translated mRNAs.

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, composed of synaptobrevin, syntaxin, and SNAP25, forms the essential fusion machinery for neurotransmitter release. Recent studies have reported several mutations in the gene encoding SNAP25 as a causative factor for developmental and epileptic encephalopathies of infancy and childhood with diverse clinical manifestations. However, it remains unclear how SNAP25 mutations give rise to these disorders. Here, we show that although structurally clustered mutations in SNAP25 give rise to related synaptic transmission phenotypes, specific alterations in spontaneous neurotransmitter release are a key factor to account for disease heterogeneity. Importantly, we identified a single mutation that augments spontaneous release without altering evoked release, suggesting that aberrant spontaneous release is sufficient to cause disease in humans.

Dedicated chemistries for on-demand capture and release of biomolecules at the solid–liquid interface are required for applications in drug delivery, for the synthesis of switchable surfaces used in analytical devices and for the assembly of next-generation biomaterials with complex architectures and functions. Here we report the engineering of a binary self-assembling polypeptide system for reversible protein capture, immobilisation and controlled thermo-responsive release from a solid surface. The first element of the binary system is a universal protein substrate immobilised on a solid surface. This protein is bio-inspired by the neuronal SNAP25, which is the protein involved in the docking and fusion of synaptic vesicles to the synaptic membrane. The second element is an artificial chimeric protein engineered to include distinct domains from three different proteins: Syntaxin, VAMP and SNAP25. These native proteins constitute the machinery dedicated to vesicle trafficking in eukaryotes. We removed approximately 70% of native protein sequence from these proteins and constructed a protein chimera capable of high affinity interaction and self-assembly with immobilised substrate. The interaction of the two parts of the engineered protein complex is strong but fully-reversible and therefore the chimera can be recombinantly fused as a tag to a protein of interest, to allow spontaneous assembly and stimuli-sensitive release from the surface upon heating at a predetermined temperature. Two thermo-responsive tags are reported: the first presents remarkable thermal stability with melting temperature of the order of 80 °C; the second disassembles at a substantially lower temperature of about 45 °C. The latter is a promising candidate for remote-controlled localised delivery of therapeutic proteins, as physiologically tolerable local increase of temperatures in the 40–45 °C range can be achieved using magnetic fields, infra-red light or focused ultrasound. Importantly, these two novel polypeptides provide a broader blueprint for the engineering of future functional proteins with predictable folding and response to external stimuli.

The domain in SNAP25 that links its two SNARE motifs plays important roles in fusion pore formation, secretion kinetics, and the formation of a dynamic plasma membrane SNAP25/syntaxin “acceptor” complex for the v-SNARE synaptobrevin., Central to the exocytotic release of hormones and neurotransmitters is the interaction of four SNARE motifs in proteins on the secretory granule/synaptic vesicle membrane (synaptobrevin/VAMP, v-SNARE) and on the plasma membrane (syntaxin and SNAP25, t-SNAREs). The interaction is thought to bring the opposing membranes together to enable fusion. An underlying motivation for this Viewpoint is to synthesize from recent diverse studies possible new insights about these events. We focus on a recent paper that demonstrates the importance of the linker region joining the two SNARE motifs of the neuronal t-SNARE SNAP25 for maintaining rates of secretion with roles for distinct segments in speeding fusion pore expansion. Remarkably, lipid-perturbing agents rescue a palmitoylation-deficient mutant whose phenotype includes slow fusion pore expansion, suggesting that protein–protein interactions have a role not only in bringing together the granule or vesicle membrane with the plasma membrane but also in orchestrating protein–lipid interactions leading to the fusion reaction. Unexpectedly, biochemical investigations demonstrate the importance of the C-terminal domain of the linker in the formation of the plasma membrane t-SNARE “acceptor” complex for synaptobrevin2. This insight, together with biophysical and optical studies from other laboratories, suggests that the plasma membrane SNARE acceptor complex between SNAP25 and syntaxin and the subsequent trans-SNARE complex with the v-SNARE synaptobrevin form within 100 ms before fusion.

Nonsense-mediated decay (NMD) is a translation-dependent RNA quality control mechanism that occurs in the cytoplasm. However, it is unknown how NMD regulates the stability of RNAs translated at the Endoplasmic Reticulum (ER). Here, we identify a localized NMD pathway dedicated to ER-translated mRNAs. We previously identified NBAS, a component of the Syntaxin 18 complex involved in Golgi-to-ER trafficking, as a novel NMD factor. Here, we show that NBAS fulfils an independent function in NMD. This ER-NMD pathway requires the interaction of NBAS with the core NMD factor UPF1, which is partially localized at the ER in the proximity of the translocon. NBAS and UPF1 co-regulate the stability of ER-associated transcripts, in particular those associated with the cellular stress response. We propose a model where NBAS recruits UPF1 to the membrane of the ER and activates an ER-dedicated NMD pathway, thus providing an ER protective function by ensuring quality control of ER-translated mRNAs.Graphical AbstractHIGHLIGHTSNBAS is an NMD factor that localizes to the membrane of the endoplasmic reticulum (ER)NBAS has dual, independent, roles in Golgi-to-ER retrograde transport and in ER-NMDNBAS recruits the core NMD factor UPF1 to the membrane of the ERThe ER-NMD pathway targets for degradation mRNAs that are translated at the ER

21 Pág., Filamentous fungal and oomycete plant pathogens that invade by direct penetration through the leaf epidermal cell wall cause devastating plant diseases. Plant preinvasive immunity toward nonadapted filamentous pathogens is highly effective and durable. Pre- and postinvasive immunity correlates with the formation of evolutionarily conserved and cell-autonomous cell wall structures, named papillae and encasements, respectively. Yet, it is still unresolved how papillae/encasements are formed and whether these defense structures prevent pathogen ingress. Here, we show that in Arabidopsis the two closely related members of the SYP12 clade of syntaxins (PEN1 and SYP122) are indispensable for the formation of papillae and encasements. Moreover, loss-of-function mutants were hampered in preinvasive immunity toward a range of phylogenetically distant nonadapted filamentous pathogens, underlining the versatility and efficacy of this defense. Complementation studies using SYP12s from the early diverging land plant, Marchantia polymorpha, showed that the SYP12 clade immunity function has survived 470 million years of independent evolution. These results suggest that ancestral land plants evolved the SYP12 clade to provide a broad and durable preinvasive immunity to facilitate their life on land and pave the way to a better understanding of how adapted pathogens overcome this ubiquitous plant defense strategy., This project was supported by grants from the Villum Foundation (MEN, VKR023502), Independent Research Fund Denmark, Technical and Production Sciences (MEN, 6111-00524B), Novo Nordisk Foundation (MEN, NNF19OC0056457), Agence Nationale de la Recherche (RJO, ANR-17-CAPS 0004-01), and China Scholarship Council (ML, No. 201906300075).

SummaryAssembly of SNARE complexes that mediate neurotransmitter release requires opening of a ‘closed’ conformation of UNC-64/syntaxin. Rescue of unc-13/Munc13 phenotypes by overexpressed open UNC-64/syntaxin suggested a specific function of UNC-13/Munc13 in opening UNC-64/ syntaxin. Here, we revisit the effects of open unc-64/syntaxin by generating knockin (KI) worms. The KI animals exhibited enhanced spontaneous and evoked exocytosis compared to wild-type animals. Unexpectedly, the open syntaxin KI partially suppressed exocytosis defects of various mutants, including snt-1/synaptotagmin, unc-2/P/Q/N-type Ca2+ channel alpha-subunit, and unc-31/CAPS in addition to unc-13/Munc13 and unc-10/RIM, and enhanced exocytosis in tom-1/Tomosyn mutants. However, open syntaxin aggravated the defects of unc-18/Munc18 mutants. Correspondingly, open syntaxin partially bypasses the requirement of Munc13 but not Munc18 for liposome fusion. Our results show that facilitating opening of syntaxin enhances exocytosis in a wide range of genetic backgrounds, and may provide a general means to enhance synaptic transmission in normal and disease states.

Due to limited free diffusion in the cytoplasm, viruses must use active transport mechanisms to move intracellularly. Nevertheless, how the plant ssDNA begomoviruses hijacks the host intracytoplasmic transport machinery to move from the nucleus to the plasmodesmata remains enigmatic. Here, we identified nuclear shuttle protein (NSP)-interacting proteins from Arabidopsis by probing a protein microarray and demonstrated that the Cabbage leaf curl virus (CabLCV) NSP, a facilitator of the nucleocytoplasmic trafficking of viral (v)DNA, interacts with an endosomal vesicle-localized plant-specific syntaxin-6 protein, designated NSP-interacting syntaxin-6 domain-containing protein (NISP) in planta. NISP displays a pro-viral function, but not the syntaxin-6 paralog AT2G18860 that failed to interact with NSP. Consistent with these findings, nisp-1 mutant plants were less susceptible to begomovirus infection, a phenotype reversed by NISP complementation. NISP-overexpressing lines accumulated higher levels of viral DNA than wild-type. Furthermore, NISP interacted with NIG, an NSP-interacting GTPase involved in NSP-vDNA nucleocytoplasmic translocation. The NISP-NIG interaction was enhanced by NSP. We also showed that NISP associates with vDNA and might assemble a NISP-NIG-NSP-vDNA-complex. NISP may function as a docking site for recruiting NIG and NSP into endosomes, providing a mechanism for the intracytoplasmic translocation of the NSP-vDNA complex towards to and from the cell periphery.Author SummaryAs viruses must use an active and directed intracellular movement, they hijack the intracellular host transport system for their own benefits. Therefore, the identification of interactions between host proteins and begomovirus movement proteins should target the intracellular transport machinery. This work focused on the identification of these protein-protein interactions; it addressed the molecular bases for the intracellular transport of begomoviruses. We used a protein microarray to identify cellular partners for the movement protein (MP) and the viral nuclear shuttle protein (NSP), which is a facilitator of the nucleocytoplasmic trafficking of viral (v)DNA. We identified relevant protein-protein interaction (PPI) hubs connecting host and viral proteins. We revealed a novel NSP-interacting protein, which functions in the intracytoplasmic transport of proteins and DNA from begomoviruses and was designated NSP-interacting syntaxin domain-containing protein (NISP). Our data suggest an intracellular route connecting the release of newly synthesized begomoviral DNA in the cytosol with the cell surface. Resolving viral DNA-host protein complexes led to the identification of a novel class of components of the cell machinery and a representative member, NISP, that functions as a susceptibility gene against begomoviruses. As geminiviruses pose a severe threat to agriculture and food security, this recessive gene can now be exploited as a target for engineering resistance by gene editing in crops.

How cells control the proper delivery of vesicles and their associated cargo to specific plasma membrane (PM) domains upon internal or external cues is a major question in plant cell biology. A widely held hypothesis is that expansion of plant exocytotic machinery components, such as SNARE proteins, has led to a diversification of exocytotic membrane trafficking pathways to function in specific biological processes. A key biological process that involves the creation of a specialized PM domain is the formation of a host–microbe interface (the peri-arbuscular membrane) in the symbiosis with arbuscular mycorrhizal fungi. We have previously shown that the ability to intracellularly host AM fungi correlates with the evolutionary expansion of both v- (VAMP721d/e) and t-SNARE (SYP132α) proteins, that are essential for arbuscule formation in Medicago truncatula. Here we studied to what extent the symbiotic SNAREs are different from their non-symbiotic family members and whether symbiotic SNAREs define a distinct symbiotic membrane trafficking pathway. We show that all tested SYP1 family proteins, and most of the non-symbiotic VAMP72 members, are able to complement the defect in arbuscule formation upon knock-down/-out of their symbiotic counterparts when expressed at sufficient levels. This functional redundancy is in line with the ability of all tested v- and t-SNARE combinations to form SNARE complexes. Interestingly, the symbiotic t-SNARE SYP132α appeared to occur less in complex with v-SNAREs compared to the non-symbiotic syntaxins in arbuscule-containing cells. This correlated with a preferential localization of SYP132α to functional branches of partially collapsing arbuscules, while non-symbiotic syntaxins accumulate at the degrading parts. Overexpression of VAMP721e caused a shift in SYP132α localization toward the degrading parts, suggesting an influence on its endocytic turn-over. These data indicate that the symbiotic SNAREs do not selectively interact to define a symbiotic vesicle trafficking pathway, but that symbiotic SNARE complexes are more rapidly disassembled resulting in a preferential localization of SYP132α at functional arbuscule branches.

Neurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigate the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.

Spatiotemporal coordination of protein trafficking among organelles is essential for eukaryotic cells. The post-Golgi interface, including the trans-Golgi network (TGN), is a pivotal hub for multiple trafficking pathways. The Golgi-released independent TGN (GI-TGN) is a compartment described only in plant cells, and its cellular and physiological roles remain elusive. In Arabidopsis (Arabidopsis thaliana), the SYNTAXIN OF PLANTS (SYP) 4 group Qa-SNARE (soluble N-ethylmaleimide) membrane fusion proteins are shared components of TGN and GI-TGN and regulate secretory and vacuolar transport. Here we reveal that GI-TGNs mediate the transport of the R-SNARE VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP) 721 to the plasma membrane. In interactions with a nonadapted powdery mildew pathogen, the SYP4 group of SNAREs is required for the dynamic relocation of VAMP721 to plant–fungus contact sites via GI-TGNs, thereby facilitating complex formation with its cognate SNARE partner PENETRATION1 to restrict pathogen entry. Furthermore, quantitative proteomic analysis of leaf apoplastic fluid revealed constitutive and pathogen-inducible secretion of cell wall-modification enzymes in a SYP4- and VAMP721-dependent manner. Hence, the GI-TGN acts as a transit compartment between the Golgi apparatus and the plasma membrane. We propose a model in which the GA-TGN matures into the GI-TGN and then into secretory vesicles by increasing the abundance of VAMP721-dependent secretory pathway components.

A major focus for our laboratory is identifying the molecules and mechanisms that regulate basolateral-to-apical transcytosis in polarized hepatocytes. Our most recent studies have focused on characterizing the biochemical and functional properties of the small rab17 GTPase. We determined that rab17 is a monosumoylated protein and that this modification likely mediates selective interactions with the apically located syntaxin 2. Using polarized hepatic WIF-B cells exogenously expressing wild-type, dominant active/guanosine triphosphate (GTP)-bound, dominant negative/guanosine diphosphate (GDP)-bound, or sumoylation-deficient/K68R rab17 proteins, we confirmed that rab17 regulates basolateral-to-apical transcytotic vesicle docking and fusion with the apical surface. We further confirmed that transcytosis is impaired from the subapical compartment to the apical surface and that GTP-bound and sumoylated rab17 are likely required for apical vesicle docking. Because expression of the GTP-bound rab17 led to impaired transcytosis, whereas wild type had no effect, we further propose that rab17 GTP hydrolysis is required for vesicle delivery. We also determined that transcytosis of three classes of newly synthesized apical residents showed similar responses to rab17 mutant expression, indicating that rab17 is a general component of the transcytotic machinery required for apically destined vesicle docking and fusion.

Background/Aims: Cystic fibrosis (CF) is a lethal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR). ΔF508, the most common mutation, is a misfolded protein that is retained in the endoplasmic reticulum and degraded, precluding delivery to the cell surface [1]. Methods: Here we use a combination of western blotting, immunoprecipitation, and short circuit current techniques combined with confocal microscopy to address whether the SNARE attachment protein, STX8 plays a role in ΔF508’s processing and movement out of the ER. Results: Although the SNARE protein STX8 is thought to be functionally related and primarily localized to early endosomes, we show that silencing of STX8, particularly in the presence of the Vertex corrector molecule C18, rescues ΔF508-CFTR, allowing it to reach the cell surface and increasing CFTR-dependent chloride currents by approximately 2.5-fold over control values. STX8 silencing reduced the binding of quality control protein, Hsp 27, a protein that targets ΔF508-CFTR for sumoylation and subsequent degradation, to ΔF508-CFTR. STX8 silencing increased the levels of Hsp 60 a protein involving in early events in protein folding. Conclusion: STX8 knockdown creates an environment favorable for mature ΔF508 to reach the cell surface. The data also suggest that when present at normal levels, STX8 functions as part of the cell’s quality control mechanism.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion and contribute to homoeostasis, pathogen defense, cell expansion, and growth in plants. In Arabidopsis (Arabidopsis thaliana), two homologous Qa-SNAREs, SYNTAXIN OF PLANTS121 (SYP121) and SYP122, facilitate the majority of secretory traffic to the plasma membrane, and the single mutants are indistinguishable from wild-type plants in the absence of stress, implying a redundancy in their functions. Nonetheless, several studies suggest differences among the secretory cargo of these SNAREs. To address this issue, we conducted an analysis of the proteins secreted by cultured wild-type, syp121, and syp122 mutant Arabidopsis seedlings. Here, we report that a number of cargo proteins were associated differentially with traffic mediated by SYP121 and SYP122. The data also indicated important overlaps between the SNAREs. Therefore, we conclude that the two Qa-SNAREs mediate distinct but complementary secretory pathways during vegetative plant growth.

The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins synaptobrevin (Syb), syntaxin, and SNAP-25 function in Ca2+-triggered exocytosis in both endocrine cells and neurons. The transmembrane domains (TMDs) of Syb and syntaxin span the vesicle and plasma membrane, respectively, and influence flux through fusion pores in endocrine cells as well as fusion pores formed during SNARE-mediated fusion of reconstituted membranes. These results support a model for exocytosis in which SNARE TMDs form the initial fusion pore. The present study sought to test this model in synaptic terminals. Patch-clamp recordings of miniature EPSCs (mEPSCs) were used to probe fusion pore properties in cultured hippocampal neurons from mice of both sexes. Mutants harboring tryptophan at four different sites in the Syb TMD reduced the rate-of-rise of mEPSCs. A computer model that simulates glutamate diffusion and receptor activation kinetics could account for this reduction in mEPSC rise rate by slowing the flux of glutamate through synaptic fusion pores. TMD mutations introducing positive charge also reduced the mEPSC rise rate, but negatively charged residues and glycine, which should have done the opposite, had no effect. The sensitivity of mEPSCs to pharmacological blockade of receptor desensitization was enhanced by a mutation that slowed the mEPSC rate-of-rise, suggesting that the mutation prolonged the residence of glutamate in the synaptic cleft. The same four Syb TMD residues found here to influence synaptic release were found previously to influence endocrine release, leading us to propose that a similar TMD-lined fusion pore functions widely in Ca2+-triggered exocytosis in mammalian cells.SIGNIFICANCE STATEMENT SNARE proteins function broadly in biological membrane fusion. Evidence from non-neuronal systems suggests that SNARE proteins initiate fusion by forming a fusion pore lined by transmembrane domains, but this model has not yet been tested in synapses. The present study addressed this question by testing mutations in the synaptic vesicle SNARE synaptobrevin for an influence on the rise rate of miniature synaptic currents. These results indicate that synaptobrevin's transmembrane domain interacts with glutamate as it passes through the fusion pore. The sites in synaptobrevin that influence this flux are identical to those shown previously to influence flux through endocrine fusion pores. Thus, SNARE transmembrane domains may function in the fusion pores of Ca2+-triggered exocytosis of both neurotransmitters and hormones.

Matsui et al. identify YKT6 as a novel autophagosomal SNARE protein. YKT6 is required for autophagosome–lysosome fusion independently of STX17, a known autophagosomal SNARE., Macroautophagy is an evolutionarily conserved catabolic mechanism that delivers intracellular constituents to lysosomes using autophagosomes. To achieve degradation, lysosomes must fuse with closed autophagosomes. We previously reported that the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein syntaxin (STX) 17 translocates to autophagosomes to mediate fusion with lysosomes. In this study, we report an additional mechanism. We found that autophagosome–lysosome fusion is retained to some extent even in STX17 knockout (KO) HeLa cells. By screening other human SNAREs, we identified YKT6 as a novel autophagosomal SNARE protein. Depletion of YKT6 inhibited autophagosome–lysosome fusion partially in wild-type and completely in STX17 KO cells, suggesting that YKT6 and STX17 are independently required for fusion. YKT6 formed a SNARE complex with SNAP29 and lysosomal STX7, both of which are required for autophagosomal fusion. Recruitment of YKT6 to autophagosomes depends on its N-terminal longin domain but not on the C-terminal palmitoylation and farnesylation that are essential for its Golgi localization. These findings suggest that two independent SNARE complexes mediate autophagosome–lysosome fusion.

The voltage-dependent K+ (Kv) channel Kv2.1 is a major delayed rectifier in many secretory cells, including pancreatic β cells. In addition, Kv2.1 has a direct role in exocytosis at an undefined step, involving SNARE proteins, that is independent of its ion-conducting pore function. Here, we elucidated the precise step in exocytosis. We previously reported that syntaxin-3 (Syn-3) is the key syntaxin that mediates exocytosis of newcomer secretory granules that spend minimal residence time on the plasma membrane before fusion. Using high-resolution total internal reflection fluorescence microscopy, we now show that Kv2.1 forms reservoir clusters on the β-cell plasma membrane and binds Syn-3 via its C-terminal C1b domain, which recruits newcomer insulin secretory granules into this large reservoir. Upon glucose stimulation, secretory granules were released from this reservoir to replenish the pool of newcomer secretory granules for subsequent fusion, occurring just adjacent to the plasma membrane Kv2.1 clusters. C1b deletion blocked the aforementioned Kv2.1-Syn-3–mediated events and reduced fusion of newcomer secretory granules. These insights have therapeutic implications, as Kv2.1 overexpression in type-2 diabetes rat islets restored biphasic insulin secretion.

Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in th ...

SNARE-mediated membrane fusion is a ubiquitous process responsible for intracellular vesicle trafficking, including membrane fusion in exocytosis that leads to hormone and neurotransmitter release. The proteins that facilitate this process are highly dynamic and adopt multiple conformations when they interact with other proteins and lipids as they form highly regulated molecular machines that operate on membranes. Solution NMR is an ideal method to capture high-resolution glimpses of the molecular transformations that take place when these proteins come together and work on membranes. Since solution NMR has limitations on the size of proteins and complexes that can be studied, lipid bilayer model membranes cannot be used in these approaches, so the relevant interactions are typically studied in various types of membrane-mimetics that are tractable by solution NMR methods. In this review we therefore first summarize different membrane-mimetic systems that are commonly used or that show promise for solution NMR studies of membrane-interacting proteins. We then summarize recent NMR studies on two SNARE proteins, syntaxin and synaptobrevin, and two related regulatory proteins, complexin and α-synuclein, and their interactions with membrane lipids. These studies provide a structural and dynamical framework for how these proteins might carry out their functions in the vicinity of lipid membranes. The common theme throughout these studies is that membrane interactions have major influences on the structural dynamics of these proteins that cannot be ignored when attempting to explain their functions in contemporary models of SNARE-mediated membrane fusion.

Although synaptic loss is thought to be core to the pathophysiology of schizophrenia, the nature, consistency and magnitude of synaptic protein and mRNA changes has not been systematically appraised. Our objective was thus to systematically review and meta-analyse findings. The entire PubMed database was searched for studies from inception date to the 1st of July 2017. We selected case-control postmortem studies in schizophrenia quantifying synaptic protein or mRNA levels in brain tissue. The difference in protein and mRNA levels between cases and controls was extracted and meta-analysis conducted. Among the results, we found a significant reduction in synaptophysin in schizophrenia in the hippocampus (effect size: −0.65, p p = 0.04), and cingulate cortices (effect size: −0.54, p = 0.02), but no significant changes for synaptophysin in occipital and temporal cortices, and no changes for SNAP-25, PSD-95, VAMP, and syntaxin in frontal cortex. There were insufficient studies for meta-analysis of complexins, synapsins, rab3A and synaptotagmin and mRNA measures. Findings are summarised for these, which generally show reductions in SNAP-25, PSD-95, synapsin and rab3A protein levels in the hippocampus but inconsistency in other regions. Our findings of moderate–large reductions in synaptophysin in hippocampus and frontal cortical regions, and a tendency for reductions in other pre- and postsynaptic proteins in the hippocampus are consistent with models that implicate synaptic loss in schizophrenia. However, they also identify potential differences between regions and proteins, suggesting synaptic loss is not uniform in nature or extent.

Platelets regulate vascular integrity by secreting a host of molecules that promote hemostasis and its sequelae. Given the importance of platelet exocytosis, it is critical to understand how it is controlled. The t-SNAREs, SNAP-23 and syntaxin-11, lack classical transmembrane domains (TMDs), yet both are associated with platelet membranes and redistributed into cholesterol-dependent lipid rafts when platelets are activated. Using metabolic labeling and hydroxylamine (HA)/HCl treatment, we showed that both contain thioester-linked acyl groups. Mass spectrometry mapping further showed that syntaxin-11 was modified on cysteine 275, 279, 280, 282, 283, and 285, and SNAP-23 was modified on cysteine 79, 80, 83, 85, and 87. Interestingly, metabolic labeling studies showed incorporation of [(3)H]palmitate into the t-SNAREs increased although the protein levels were unchanged, suggesting that acylation turns over on the two t-SNAREs in resting platelets. Exogenously added fatty acids did compete with [(3)H]palmitate for t-SNARE labeling. To determine the effects of acylation, we measured aggregation, ADP/ATP release, as well as P-selectin exposure in platelets treated with the acyltransferase inhibitor cerulenin or the thioesterase inhibitor palmostatin B. We found that cerulenin pretreatment inhibited t-SNARE acylation and platelet function in a dose- and time-dependent manner whereas palmostatin B had no detectable effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin, suggesting that maintaining the acylation state is important for platelet function. Thus, our work shows that t-SNARE acylation is actively cycling in platelets and suggests that the enzymes regulating protein acylation could be potential targets to control platelet exocytosis in vivo.

Supplemental Digital Content is available in the text., Objective— Endothelial cells store VWF (von Willebrand factor) in rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs). WPB exocytosis is coordinated by a complex network of Rab GTPases, Rab effectors, and SNARE (soluble NSF attachment protein receptor) proteins. We have previously identified STXBP1 as the link between the Rab27A-Slp4-a complex on WPBs and the SNARE proteins syntaxin-2 and -3. In this study, we investigate the function of syntaxin-3 in VWF secretion. Approach and Results— In human umbilical vein endothelial cells and in blood outgrowth endothelial cells (BOECs) from healthy controls, endogenous syntaxin-3 immunolocalized to WPBs. A detailed analysis of BOECs isolated from a patient with variant microvillus inclusion disease, carrying a homozygous mutation in STX3(STX3−/−), showed a loss of syntaxin-3 protein and absence of WPB-associated syntaxin-3 immunoreactivity. Ultrastructural analysis revealed no detectable differences in morphology or prevalence of immature or mature WPBs in control versus STX3−/− BOECs. VWF multimer analysis showed normal patterns in plasma of the microvillus inclusion disease patient, and media from STX3−/− BOECs, together indicating WPB formation and maturation are unaffected by absence of syntaxin-3. However, a defect in basal as well as Ca2+- and cAMP-mediated VWF secretion was found in the STX3−/− BOECs. We also show that syntaxin-3 interacts with the WPB-associated SNARE protein VAMP8 (vesicle-associated membrane protein-8). Conclusions— Our data reveal syntaxin-3 as a novel WPB-associated SNARE protein that controls WPB exocytosis.

The Drosophila extracellular matrix protein Dumpy (Dpy) is one of the largest proteins encoded by any animal. One class of dpy mutations produces a characteristic shortening of the wing blade known as oblique (dpyo), due to altered tension in the developing wing. We describe here the characterization of docked (doc), a gene originally named because of an allele producing a truncated wing. We show that doc corresponds to the gene model CG5484, which encodes a homolog of the yeast protein Yif1 and plays a key role in ER to Golgi vesicle transport. Genetic analysis is consistent with a similar role for Doc in vesicle trafficking: docked alleles interact not only with genes encoding the COPII core proteins sec23 and sec13, but also with the SNARE proteins synaptobrevin and syntaxin. Further, we demonstrate that the strong similarity between the doc1 and dpyo wing phenotypes reflects a functional connection between the two genes; we found that various dpy alleles are sensitive to changes in dosage of genes encoding other vesicle transport components such as sec13 and sar1. Doc’s effects on trafficking are not limited to Dpy; for example, reduced doc dosage disturbed Notch pathway signaling during wing blade and vein development. These results suggest a model in which the oblique wing phenotype in doc1 results from reduced transport of wild-type Dumpy protein; by extension, an additional implication is that the dpyo alleles can themselves be explained as hypomorphs.