The MCTP-SNARE Complex Regulates Florigen Transport in Arabidopsis.

Multiple flowering pathways in Arabidopsis (Arabidopsis thaliana) converge on the transcriptional regulation of FLOWERING LOCUS T (FT), encoding a mobile floral stimulus that moves from leaves to the shoot apex. Despite our progress in understanding FT movement, the mechanisms underlying its transport along the endoplasmic reticulum-plasmalemma pathway in phloem companion cells remain largely unclear. Here, we show that the plasma membrane-resident syntaxin-like glutamine-soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor (Q-SNARE), SYNTAXIN OF PLANTS121 (SYP121), interacts with QUIRKY (QKY), a member of the family of multiple C2 domain and transmembrane region proteins (MCTPs), to mediate FT transport in Arabidopsis. QKY and SYP121 coordinately regulate FT movement to the plasmalemma through the endosomal trafficking pathway and are required for FT export from companion cells to sieve elements, thus affecting FT transport through the phloem to the shoot apical meristem. These findings suggest that MCTP-SNARE complex-mediated endosomal trafficking is essential for the export of florigen from phloem companion cells to sieve elements to induce flowering.plantcell;31/10/2475/FX1F1fx1.

Q-SNARE protein FgSyn8 plays important role in growth, DON production and pathogenicity of Fusarium graminearum.

SNAREs (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) help intracellular vesicle trafficking and membrane fusion among eukaryotes. They are vital for growth and development of phyto-pathogenic fungi such as Fusarium graminearum which causes Fusarium Head Blight (FHB) of wheat and barley. The SNARE protein Syn8 and its homologues play many roles among different organisms. Here, we have characterized FgSyn8 in F. graminearum as a homologue of Syn8. We have integrated biochemical, microbiological and molecular genetic approaches to investigate the roles of this protein. Our results reveal that FgSyn8 is indispensable for normal vegetative growth, conidiation, conidial morphology and pathogenicity of F. graminearum. Deoxynivalenol (DON) biochemical assay reveals active participation of this protein in DON production of F. graminearum. This has further been confirmed by the production of bulbous structures among the intercalary hyphae. FgSyn8 mutant strain produced defects in perithecia formation which portrays its role in sexual reproduction. In summary, our results support that the SNARE protein FgSyn8 is required for vegetative growth, sexual reproduction, DON production and pathogenicity of F. graminearum.

The Vesicle Priming Factor CAPS Functions as a Homodimer via C2 Domain Interactions to Promote Regulated Vesicle Exocytosis.

Neurotransmitters and peptide hormones are secreted by regulated vesicle exocytosis. CAPS (also known as CADPS) is a 145-kDa cytosolic and peripheral membrane protein required for vesicle docking and priming steps that precede Ca2+-triggered vesicle exocytosis. CAPS binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and SNARE proteins and is proposed to promote SNARE protein complex assembly for vesicle docking and priming. We characterized purified soluble CAPS as mainly monomer in equilibrium with small amounts of dimer. However, the active form of CAPS bound to PC12 cell membranes or to liposomes containing PI(4,5)P2 and Q-SNARE proteins was mainly dimer. CAPS dimer formation required its C2 domain based on mutation or deletion studies. Moreover, C2 domain mutations or deletions resulted in a loss of CAPS function in regulated vesicle exocytosis, indicating that dimerization is essential for CAPS function. Comparison of the CAPS C2 domain to a structurally defined Munc13-1 C2A domain dimer revealed conserved residues involved in CAPS dimerization. We conclude that CAPS functions as a C2 domain-mediated dimer in regulated vesicle exocytosis. The unique tandem C2-PH domain of CAPS may serve as a PI(4,5)P2-triggered switch for dimerization. CAPS dimerization may be coupled to oligomeric SNARE complex assembly for vesicle docking and priming.

Deciphering dead-end docking of large dense core vesicles in bovine chromaffin cells.

Large dense core vesicle (LDCV) exocytosis in chromaffin cells follows a well characterized process consisting of docking, priming, and fusion. Total internal reflection fluorescence microscopy (TIRFM) studies suggest that some LDCVs, although being able to dock, are resistant to calcium-triggered release. This phenomenon termed dead-end docking has not been investigated until now. We characterized dead-end vesicles using a combination of membrane capacitance measurement and visualization of LDCVs with TIRFM. Stimulation of bovine chromaffin cells for 5 min with 6 µm free intracellular Ca2+ induced strong secretion and a large reduction of the LDCV density at the plasma membrane. Approximately 15% of the LDCVs were visible at the plasma membrane throughout experiments, indicating they were permanently docked dead-end vesicles. Overexpression of Munc18-2 or SNAP-25 reduced the fraction of dead-end vesicles. Conversely, expressing open-syntaxin increased the fraction of dead-end vesicles. These results indicate the existence of the unproductive target soluble N-ethylmaleimide-sensitive factor attachment protein receptor acceptor complex composed of 2:1 syntaxin-SNAP-25 in vivo. More importantly, they define a novel function for this acceptor complex in mediating dead-end docking.

Essential roles of snap-29 in C. elegans.

SNARE domain proteins are key molecules mediating intracellular fusion events. SNAP25 family proteins are unique target-SNAREs possessing two SNARE domains. Here we report the genetic, molecular, and cell biological characterization of C. elegans SNAP-29. We found that snap-29 is an essential gene required throughout the life-cycle. Depletion of snap-29 by RNAi in adults results in sterility associated with endomitotic oocytes and pre-meiotic maturation of the oocytes. Many of the embryos that are produced are multinucleated, indicating a defect in embryonic cytokinesis. A profound defect in secretion by oocytes and early embryos in animals lacking SNAP-29 appears to be the underlying defect connecting these phenotypes. Further analysis revealed defects in basolateral and apical secretion by intestinal epithelial cells in animals lacking SNAP-29, indicating a broad requirement for this protein in the secretory pathway. A SNAP-29-GFP fusion protein was enriched on recycling endosomes, and loss of SNAP-29 disrupted recycling endosome morphology. Taken together these results suggest a requirement for SNAP-29 in the fusion of post-Golgi vesicles with the recycling endosome for cargo to reach the cell surface.

O-GlcNAc-modification of SNAP-29 regulates autophagosome maturation.

The mechanism by which nutrient status regulates the fusion of autophagosomes with endosomes/lysosomes is poorly understood. Here, we report that O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates O-GlcNAcylation of the SNARE protein SNAP-29 and regulates autophagy in a nutrient-dependent manner. In mammalian cells, OGT knockdown, or mutating the O-GlcNAc sites in SNAP-29, promotes the formation of a SNAP-29-containing SNARE complex, increases fusion between autophagosomes and endosomes/lysosomes, and promotes autophagic flux. In Caenorhabditis elegans, depletion of ogt-1 has a similar effect on autophagy; moreover, expression of an O-GlcNAc-defective SNAP-29 mutant facilitates autophagic degradation of protein aggregates. O-GlcNAcylated SNAP-29 levels are reduced during starvation in mammalian cells and in C. elegans. Our study reveals a mechanism by which O-GlcNAc-modification integrates nutrient status with autophagosome maturation.

Caenorhabditis elegans SNAP-29 is required for organellar integrity of the endomembrane system and general exocytosis in intestinal epithelial cells.

It is generally accepted that soluble N-ethylmaleimide-sensitive factor attachment protein receptors mediate the docking and fusion of transport intermediates with target membranes. Our research identifies Caenorhabditis elegans homologue of synaptosomal-associated protein 29 (SNAP-29) as an essential regulator of membrane trafficking in polarized intestinal cells of living animals. We show that a depletion of SNAP-29 blocks yolk secretion and targeting of apical and basolateral plasma membrane proteins in the intestinal cells and results in a strong accumulation of small cargo-containing vesicles. The loss of SNAP-29 also blocks the transport of yolk receptor RME-2 to the plasma membrane in nonpolarized oocytes, indicating that its function is required in various cell types. SNAP-29 is essential for embryogenesis, animal growth, and viability. Functional fluorescent protein-tagged SNAP-29 mainly localizes to the plasma membrane and the late Golgi, although it also partially colocalizes with endosomal proteins. The loss of SNAP-29 leads to the vesiculation/fragmentation of the Golgi and endosomes, suggesting that SNAP-29 is involved in multiple transport pathways between the exocytic and endocytic organelles. These observations also suggest that organelles comprising the endomembrane system are highly dynamic structures based on the balance between membrane budding and fusion and that SNAP-29-mediated fusion is required to maintain proper organellar morphology and functions.

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

Membrane fusion within the endomembrane system follows a defined order of events: membrane tethering, mediated by Rabs and tethers, assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes, and lipid bilayer mixing. Here we present evidence that the vacuolar HOPS tethering complex controls fusion through specific interactions with the vacuolar SNARE complex (consisting of Vam3, Vam7, Vti1, and Nyv1) and the N-terminal domains of Vam7 and Vam3. We show that homotypic fusion and protein sorting (HOPS) binds Vam7 via its subunits Vps16 and Vps18. In addition, we observed that Vps16, Vps18, and the Sec1/Munc18 protein Vps33, which is also part of the HOPS complex, bind to the Q-SNARE complex. In agreement with this observation, HOPS-stimulated fusion was inhibited if HOPS was preincubated with the minimal Q-SNARE complex. Importantly, artificial targeting of Vam7 without its PX domain to membranes rescued vacuole morphology in vivo, but resulted in a cytokinesis defect if the N-terminal domain of Vam3 was also removed. Our data thus support a model of HOPS-controlled membrane fusion by recognizing different elements of the SNARE complex.

Identification of the neuroblastoma-amplified gene product as a component of the syntaxin 18 complex implicated in Golgi-to-endoplasmic reticulum retrograde transport.

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

AQP2 exocytosis in the renal collecting duct -- involvement of SNARE isoforms and the regulatory role of Munc18b.

Vasopressin regulates the fusion of the water channel aquaporin 2 (AQP2) to the apical membrane of the renal collecting-duct principal cells and several lines of evidence indicate that SNARE proteins mediate this process. In this work MCD4 renal cells were used to investigate the functional role of a set of Q- and R-SNAREs, together with that of Munc18b as a negative regulator of the formation of the SNARE complex. Both VAMP2 and VAMP3 were associated with immunoisolated AQP2 vesicles, whereas syntaxin 3 (Stx3), SNAP23 and Munc18 were associated with the apical plasma membrane. Co-immunoprecipitation experiments indicated that Stx3 forms complexes with VAMP2, VAMP3, SNAP23 and Munc18b. Protein knockdown coupled to apical surface biotinylation demonstrated that reduced levels of the R-SNAREs VAMP2 and VAMP3, and the Q-SNAREs Stx3 and SNAP23 strongly inhibited AQP2 fusion at the apical membrane. In addition, knockdown of Munc18b promoted a sevenfold increase of AQP2 fused at the plasma membrane without forskolin stimulation. Taken together these findings propose VAMP2, VAMP3, Stx3 and SNAP23 as the complementary set of SNAREs responsible for AQP2-vesicle fusion into the apical membrane, and Munc18b as a negative regulator of SNARE-complex formation in renal collecting-duct principal cells.

Munc18c interaction with syntaxin 4 monomers and SNARE complex intermediates in GLUT4 vesicle trafficking.

In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins. However, both N and C termini of syntaxin 4 are required for Munc18c binding, since a mutation in the syntaxin 4 SNARE domain (I241A) reduces the interaction, irrespective of syntaxin 4 conformation. Using an optical reporter for syntaxin 4-SNARE pairings in vivo, we demonstrate that Munc18c blocks recruitment of SNAP23 to wild type syntaxin 4 yet associates with syntaxin 4LE-SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4LE interaction with the I241A mutation, indicating that Munc18c facilitates vesicle docking. Therefore, in contradiction to previous models, our data indicates that the conformational "opening" of syntaxin 4 rather than the dissociation of Munc18c is the critical event required for GLUT4 vesicle docking.

Reversible, cooperative reactions of yeast vacuole docking.

Homotypic yeast vacuole fusion occurs in three stages: (i) priming reactions, which are independent of vacuole clustering, (ii) docking, in which vacuoles cluster and accumulate fusion proteins and fusion regulatory lipids at a ring-shaped microdomain surrounding the apposed membranes of each docked vacuole, where fusion will occur, and (iii) bilayer fusion/compartment mixing. These stages require vacuolar SNAREs, SNARE-chaperones, GTPases, effector complexes, and chemically minor but functionally important lipids. For each, we have developed specific ligands that block fusion and conditions that reverse each block. Using them, we test whether docking entails a linearly ordered series of catalytic events, marked by sequential acquisition of resistance to inhibitors, or whether docking subreactions are cooperative and/or reversible. We find that each fusion protein and regulatory lipid is needed throughout docking, indicative of a reversible or highly cooperative assembly of the fusion-competent vertex ring. In accord with this cooperativity, vertices enriched in one fusion catalyst are enriched in others. Docked vacuoles finally assemble SNARE complexes, yet still require physiological temperature and lipid rearrangements to complete fusion.

Identification, localization and interaction of SNARE proteins in atrial cardiac myocytes.

Atrial cardiac myocytes secrete the vasoactive hormone atrial natriuretic peptide (ANP) by both constitutive and regulated exocytotic fusion of ANP-containing large dense core vesicles (LDCV) with the sarcolemma. Detailed information, however, regarding the identity and function of specific membrane fusion proteins (SNARE proteins) involved in exocytosis in the endocrine heart is lacking. In the current study, we identified SNARE proteins and determined their association with ANP-containing secretory granules using primary cultures of neonatal and adult rat atrial cardiac myocytes. Using RT-PCR, cardiac myocytes were screened for SNARE and SNARE-associated transcripts. Identified SNARE proteins that have been implicated in exocytosis in neuroendocrine cells were further characterized by Western blot analysis. Functional interaction between SNARE proteins was demonstrated using immunoprecipitation. Using cell fractionation and immunocytochemical methods, it was revealed that VAMP-1, VAMP-2 and synaptotagmin-1 (the putative Ca(2+) sensor) localized to subpopulations of ANP-containing secretory granules in atrial myocytes. Currently, there is conflicting data regarding the role of Ca(2+) in ANP exocytosis. To judge whether secretory activity could be evoked by intracellular Ca(2+) elevation, time-resolved membrane capacitance measurements were used in combination with the flash photolysis of caged compounds to follow the exocytotic activity of single neonatal atrial myocytes. These studies demonstrated that multiple SNARE proteins are present in neonatal and adult cardiac myocytes and suggest the importance of Ca(2+) in exocytosis of ANP from neonatal atrial cardiac myocytes.