The SNAREs vti1a and vti1b have distinct localization and SNARE complex partners

Two mammalian proteins, vtila and vtilb, are homologous to the yeast Q-SNARE Vtilp which is part of several SNARE complexes in different transport steps. In vitro experiments suggest distinct functions for vtila and vtilb. Here we compared the subcellular localization of endogenous vtila and vtilb by immunofluorescence and immuno-electron microscopy. Both proteins had a distinct but overlapping localization. vtila was found predominantly on the Golgi and the TGN, vtilb mostly on tubules and vesicles in the TGN area and on endosomes. vti1a coimmunoprecipitated with VAMP-4, syntaxin 6, and syntaxin 16. These four SNAREs could assemble into a SNARE complex of conserved structure because one SNARE motif of each subgroup is present. vtila-beta, VAMP-4, syntaxin 6, and syntaxin 16 are coenriched with small synaptic vesicles and with clathrin-coated vesicles isolated from rat brain synaptosomes. Therefore, this SNARE complex may have a role in synaptic vesicle biogenesis or recycling.     

The N-terminal domains of syntaxin 7 and vti1b form three-helix bundles that differ in their ability to regulate SNARE complex assembly

The SNAREs syntaxin 7, syntaxin 8, vti1b, and endobrevin/VAMP8 function in the fusion of late endosomes. Although the core complex formed by these SNAREs is very similar to the neuronal SNARE complex, it differs from the neuronal complex in that three of the four SNAREs contain extended N-terminal regions of unknown structure and function. Here we show that the N-terminal regions of syntaxin 7, syntaxin 8, and vti1b contain well folded alpha-helical domains. Multidimensional NMR spectroscopy revealed that in syntaxin 7 and vti1b, the domains form three-helix bundles resembling those of syntaxin 1, Sso1p, and Vam3p. The three-helix bundle domain of vti1b is the first of its kind identified in a SNARE outside the syntaxin family. Only syntaxin 7 adopts a closed conformation, whereas in vti1b and syntaxin 8, the N-terminal domains do not interact with the adjacent SNARE motifs. Accordingly, the rate of SNARE complex assembly is retarded about 7-fold when syntaxin 7 contains its N-terminal domain, whereas the N-terminal domains of vti1b and syntaxin 8 have no influence on assembly kinetics. We conclude that three-helix bundles represent a common fold for SNARE N-terminal domains, not restricted to the syntaxin family. However, they differ in their ability to adopt closed conformations and thus to regulate the assembly of SNARE complexes.     

The SNARE protein vti1a functions in dense‐core vesicle biogenesis

The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans‐Golgi network, partially overlapping with syntaxin‐6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca²⁺‐channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin‐2 content. In contrast, release kinetics and Ca²⁺‐sensitivity remain unchanged, indicating that the final fusion reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long‐term re‐expression of vti1a (days) was necessary for restoration of secretory capacity, whereas strong short‐term expression (hours) was ineffective, consistent with vti1a involvement in an upstream step related to vesicle generation, rather than in fusion. We conclude that vti1a functions in vesicle generation and Ca²⁺‐channel trafficking, but is dispensable for transmitter release.     

Regulation of the endosomal SNARE protein syntaxin 7 by colony-stimulating factor 1 in macrophages

Colony-stimulating factor 1 (CSF-1) is the main growth factor controlling the development of macrophages from myeloid progenitor cells. However, CSF-1 also regulates some of the key effector functions of macrophages (e.g., phagocytosis and cytokine secretion). The endosomal SNARE protein syntaxin 7 (Stx7) regulates vesicle trafficking events involved in phagocytosis and cytokine secretion. Therefore, we investigated the ability of CSF-1 to regulate Stx7. CSF-1 upregulated Stx7 expression in primary mouse macrophages; it also upregulated expression of its SNARE partners Vti1b and VAMP8 but not Stx8. Additionally, CSF-1 induced the rapid serine phosphorylation of Stx7 and enhanced its binding to Vti1b, Stx8, and VAMP8. Bioinformatics analysis and results from experiments with kinase inhibitors suggested the CSF-1-induced phosphorylation of Stx7 was mediated by protein kinase C and Akt in response to phosphatidylinositol 3-kinase activation. Based on mutagenesis studies, CSF-1 appeared to increase the binding of Stx7 to its SNARE partners by inducing the phosphorylation of serine residues in the Habc domain and/or “linker” region of Stx7. Thus, CSF-1 is a key regulator of Stx7 expression and function in macrophages. Furthermore, the effects of CSF-1 on Stx7 may provide a mechanism for the regulation of macrophage effector functions by CSF-1.     

Mechanical coupling via the membrane fusion SNARE protein syntaxin 1A: a molecular dynamics study

SNARE trans complexes between membranes likely promote membrane fusion. For the t-SNARE syntaxin 1A involved in synaptic transmission, the secondary structure and bending stiffness of the five-residue juxtamembrane linker is assumed to determine the required mechanical energy transfer from the cytosolic core complex to the membrane. These properties have here been studied by molecular dynamics and annealing simulations for the wild-type and a C-terminal-prolongated mutant within a neutral and an acidic bilayer, suggesting linker stiffnesses above 1.7 but below 50 x 10(-3) kcal mol(-1) deg(-2). The transmembrane helix was found to be tilted by 15 degrees and tightly anchored within the membrane with a stiffness of 4-5 kcal mol(-1) A(-2). The linker turned out to be marginally helical and strongly influenced by its lipid environment. Charged lipids increased the helicity and H3 helix tilt stiffness. For the wild type, the linker was seen embedded deeply within the polar region of the bilayer, whereas the prolongation shifted the linker outward. This reduced its helicity and increased its average tilt, thereby presumably reducing fusion efficiency. Our results suggest that partially unstructured linkers provide considerable mechanical coupling; the energy transduced cooperatively by the linkers in a native fusion event is thus estimated to be 3-8 kcal/mol, implying a two-to-five orders of magnitude fusion rate increase.     

Crystal structure and biophysical properties of a complex between the N-terminal SNARE region of SNAP25 and syntaxin 1a

SNARE proteins are required for intracellular membrane fusion. In the neuron, the plasma membrane SNAREs syntaxin 1a and SNAP25 bind to VAMP2 found on neurotransmitter-containing vesicles. These three proteins contain "SNARE regions" that mediate their association into stable tetrameric coiled-coil structures. Syntaxin 1a contributes one such region, designated H3, and SNAP25 contributes two SNARE regions to the fusogenic complex with VAMP2. Syntaxin 1a H3 (syn1aH3) and SNAP25 can form a stable assembly, which can then be bound by VAMP2 to form the full SNARE complex. Here we show that syn1aH3 can also form a stable but kinetically trapped complex with the N-terminal SNARE region of SNAP25 (S25N). The crystal structure of this complex reveals an extended parallel four-helix bundle similar to that of the core SNARE and the syn1aH3-SNAP25 complexes. The inherent ability of syn1aH3 and S25N to associate stably in vitro implies that the intracellular fusion machinery must prevent formation of, or remove, any non-productive complexes. Comparison with the syn1aH3-SNAP25 complex suggests that the linkage of the N- and C-terminal SNAP25 SNARE regions is kinetically advantageous in preventing formation of the non-productive syn1aH3-S25N complex. We also demonstrate that the syn1aH3-S25N complex can be disassembled by alpha-SNAP and N-ethylmaleimide-sensitive factor.     

Characterization of the interactions between the glycine transporters GLYT1 and GLYT2 and the SNARE protein syntaxin 1A

In this study we have examined the effect of the SNARE protein syntaxin 1A on the glycine transporters GLYT1 and GLYT2. Our results demonstrate a functional and physical interaction between both glycine transporters and syntaxin 1A. Co-transfection of syntaxin 1A with GLYT1 or GLYT2 in COS cells resulted in approximately 40% inhibition in glycine transport. This inhibition was reversed by the syntaxin 1A-binding protein, Munc18. Furthermore, immunoprecipitation studies showed a physical interaction between syntaxin 1A and both transporters in COS cells and in rat brain tissue. Finally, we conclude that this physical interaction resulted in a partial removal of the glycine transporters from the plasma membrane as demonstrated by biotinylation studies.     

Self-association of the H3 region of syntaxin 1A. Implications for intermediates in SNARE complex assembly

Intracellular membrane fusion requires SNARE proteins found on the vesicle and target membranes. SNAREs associate by formation of a parallel four-helix bundle, and it has been suggested that formation of this complex promotes membrane fusion. The membrane proximal region of the cytoplasmic domain of the SNARE syntaxin 1A, designated H3, contributes one of the four helices to the SNARE complex. In the crystal structure of syntaxin 1A H3, four molecules associate as a homotetramer composed of two pairs of parallel helices that are anti-parallel to each other. The H3 oligomer observed in the crystals is also found in solution, as assessed by gel filtration and chemical cross-linking studies. The crystal structure reveals that the highly conserved Phe-216 packs against conserved Gln-226 residues present on the anti-parallel pair of helices. Modeling indicates that Phe-216 prevents parallel tetramer formation. Mutation of Phe-216 to Leu appears to allow formation of parallel tetramers, whereas mutation to Ala destabilizes the protein. These results indicate that Phe-216 has a role in preventing formation of stable parallel helical bundles, thus favoring the interaction of the H3 region of syntaxin 1a with other proteins involved in membrane fusion.     

SNARE bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of membrane fusion

Sec1/Munc18 (SM) proteins activate intracellular membrane fusion through binding to cognate SNAP receptor (SNARE) complexes. The synaptic target membrane SNARE syntaxin 1 contains a highly conserved H_abc domain, which connects an N-peptide motif to the SNARE core domain and is thought to participate in the binding of Munc18-1 (the neuronal SM protein) to the SNARE complex. Unexpectedly, we found that mutation or complete removal of the H_abc domain had no effect on Munc18-1 stimulation of fusion. The central cavity region of Munc18-1 is required to stimulate fusion but not through its binding to the syntaxin H_abc domain. SNAP-25, another synaptic SNARE subunit, contains a flexible linker and exhibits an atypical conjoined Q_bc configuration. We found that neither the linker nor the Q_bc configuration is necessary for Munc18-1 promotion of fusion. As a result, Munc18-1 activates a SNARE complex with the typical configuration, in which each of the SNARE core domains is individually rooted in the membrane bilayer. Thus, the SNARE four-helix bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of fusion.     

Deletion of Cdkn1b ameliorates hyperglycemia by maintaining compensatory hyperinsulinemia in diabetic mice

The protein p27(Kip1) regulates cell cycle progression in mammals by inhibiting the activity of cyclin-dependent kinases (CDKs). Here we show that p27(Kip1) progressively accumulates in the nucleus of pancreatic beta cells in mice that lack either insulin receptor substrate 2 (Irs2(-/-)) or the long form of the leptin receptor (Lepr(-/-) or db/db). Deletion of the gene encoding p27(Kip1) (Cdkn1b) ameliorated hyperglycemia in these animal models of type 2 diabetes mellitus by increasing islet mass and maintaining compensatory hyperinsulinemia, effects that were attributable predominantly to stimulation of pancreatic beta-cell proliferation. Thus, p27(Kip1) contributes to beta-cell failure during the development of type 2 diabetes in Irs2(-/-) and Lepr(-/-) mice and represents a potential new target for the treatment of this condition.     

Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport

The yeast SNARE Ykt6p has been implicated in several trafficking steps, including vesicular transport from the endoplasmic reticulum (ER) to the Golgi, intra-Golgi transport, and homotypic vacuole fusion. The functional role of its mammalian homologue (Ykt6) has not been established. Using antibodies specific for mammalian Ykt6, it is revealed that it is found mainly in Golgi-enriched membranes. Three SNAREs, syntaxin 5, GS28, and Bet1, are specifically associated with Ykt6 as revealed by co-immunoprecipitation, suggesting that these four SNAREs form a SNARE complex. Double labeling of Ykt6 and the Golgi marker mannosidase II or the ER-Golgi recycling marker KDEL receptor suggests that Ykt6 is primarily associated with the Golgi apparatus. Unlike the KDEL receptor, Ykt6 does not cycle back to the peripheral ER exit sites. Antibodies against Ykt6 inhibit in vitro ER-Golgi transport of vesicular stomatitis virus envelope glycoprotein (VSVG) only when they are added before the EGTA-sensitive stage. ER-Golgi transport of VSVG in vitro is also inhibited by recombinant Ykt6. In the presence of antibodies against Ykt6, VSVG accumulates in peri-Golgi vesicular structures and is prevented from entering the mannosidase II compartment, suggesting that Ykt6 functions at a late stage in ER-Golgi transport. Golgi apparatus marked by mannosidase II is fragmented into vesicular structures in cells microinjected with Ykt6 antibodies. It is concluded that Ykt6 functions in a late step of ER-Golgi transport, and this role may be important for the integrity of the Golgi complex.     

The SNARE motif is essential for the formation of syntaxin clusters in the plasma membrane

In the plasma membrane, syntaxin 1 and syntaxin 4 clusters define sites at which secretory granules and caveolae fuse, respectively. It is widely believed that lipid phases are mandatory for cluster formation, as cluster integrity depends on cholesterol. Here we report that the native lipid environment is not sufficient for correct syntaxin 1 clustering and that additional cytoplasmic protein-protein interactions, primarily involving the SNARE motif, are required. Apparently no specific cofactors are needed because i), clusters form equally well in nonneuronal cells, and ii), as revealed by nanoscale subdiffraction resolution provided by STED microscopy, the number of clusters directly depends on the syntaxin 1 concentration. For syntaxin 4 clustering the N-terminal domain and the linker region are also dispensable. Moreover, clustering is specific because in both cluster types syntaxins mutually exclude one another at endogenous levels. We suggest that the SNARE motifs of syntaxin 1 and 4 mediate specific syntaxin clustering by homooligomerization, thereby spatially separating sites for different biological activities. Thus, syntaxin clustering represents a mechanism of membrane patterning that is based on protein-protein interactions.     

Thymic alterations in mice deficient for the SNARE protein VAMP8/endobrevin

SNARE (soluble-N-ethylmaleimide-sensitive factor attachment receptor) proteins mediate the recognition and fusion of transport vesicles in eukaryotic cells. The SNARE protein VAMP8 (also called endobrevin) is involved in the fusion of late endosomes and in some pathways of regulated exocytosis. In a subset of mice deficient for the SNARE protein VAMP8, a severe alteration of the thymus and in T lymphocyte development was observed and characterized. The size of the thymus and the number of thymocytes were dramatically reduced compared with those in heterozygous littermates. Further, the compartmentalization into cortex and medulla and the organization of the thymus epithelium were disturbed. The numbers of all thymocyte subpopulations were reduced, with the CD4 and CD8 double-positive thymocytes being most severely affected. The proportion of proliferating thymocytes was reduced, and the staining of apoptotic cells in situ and ex vivo indicated an increased number of apoptotic cells. Isolated thymocytes of Vamp8 ^−/− mice were more susceptible to various apoptotic stimuli including glucocorticoids, FAS receptor, and CD3/CD28-mediated signaling in vitro, even before an increased number of apoptotic cells was detectable in situ. However, bone marrow of phenotypically affected Vamp8 ^−/− mice was readily able to repopulate immunodeficient hosts suggesting that the SNARE protein VAMP8 has a specific function in the thymic stroma affecting the proliferation and apoptosis of T lymphocytes during maturation in the thymus. This work was supported by a Georg Christoph Lichtenberg Stipend from the Ministry for Science and Culture of Lower Saxony (to N.K.) and in part by grant MRTN-CT-2004-512253 (TRANS-NET) from the European Union (to R.D.).     

Deletion of multimerin-1 in alpha-synuclein-deficient mice

A deletion of the murine Snca gene has been discovered in C57BL/6JOlaHsd, a population of the inbred strain C57BL/6J. We now characterize the exact nature of this deletion, Del(6)Snca1Slab. Detailed mapping and sequencing of the breakpoint revealed the absence of 365 kb, encompassing the Mmrn1 gene in addition to Snca. Despite the lack of alpha-synuclein and multimerin-1 C57BL/6JOlaHsd animals do not display obvious phenotypes. Sequence comparisons revealed that the chromosomal organization of Sncg and Mmrn2 is highly reminiscent of the region containing Snca and Mmrn1, suggesting a duplication event of a cluster of apparently unrelated genes during evolution.     

Involvement of syntaxin 18, an endoplasmic reticulum (ER)-localized SNARE protein, in ER-mediated phagocytosis

The endoplasmic reticulum (ER) is thought to play an important structural and functional role in phagocytosis. According to this model, direct membrane fusion between the ER and the plasma or phagosomal membrane must precede further invagination, but the exact mechanisms remain elusive. Here, we investigated whether various ER-localized SNARE proteins are involved in this fusion process. When phagosomes were isolated from murine J774 macrophages, we found that ER-localized SNARE proteins (syntaxin 18, D12, and Sec22b) were significantly enriched in the phagosomes. Fluorescence and immuno-EM analyses confirmed the localization of syntaxin 18 in the phagosomal membranes of J774 cells stably expressing this protein tagged to a GFP variant. To examine whether these SNARE proteins are required for phagocytosis, we generated 293T cells stably expressing the Fc gamma receptor, in which phagocytosis occurs in an IgG-mediated manner. Expression in these cells of dominant-negative mutants of syntaxin 18 or D12 lacking the transmembrane domain, but not a Sec22b mutant, impaired phagocytosis. Syntaxin 18 small interfering RNA (siRNA) selectively decreased the efficiency of phagocytosis, and the rate of phagocytosis was markedly enhanced by stable overexpression of syntaxin 18 in J774 cells. Therefore, we conclude that syntaxin 18 is involved in ER-mediated phagocytosis, presumably by regulating the specific and direct fusion of the ER and plasma or phagosomal membranes.     

Munc18-2, a functional partner of syntaxin 3, controls apical membrane trafficking in epithelial cells

The Sec1-related proteins bind to syntaxin family t-SNAREs with high affinity, thus controlling the interaction of syntaxins with their cognate SNARE partners. Munc18-2 is a Sec1 homologue enriched in epithelial cells and forms a complex with syntaxin 3, a t-SNARE localized to the apical plasma membrane. We generated here a set of Munc18-2 point mutants with substitutions in conserved amino acid residues. The mutants displayed a spectrum of different syntaxin binding efficiencies. The in vitro and in vivo binding patterns were highly similar, and the association of the Munc18-2 variants with syntaxin 3 correlated well with their ability to displace SNAP-23 from syntaxin 3 complexes when overexpressed in Caco-2 cells. Even the Munc18-2 mutants that do not detectably bind syntaxin 3 were membrane associated in Caco-2 cells, suggesting that the syntaxin interaction is not the sole determinant of Sec1 protein membrane attachment. Overexpression of the wild-type Munc18-2 was shown to inhibit the apical delivery of influenza virus hemagglutinin (HA). Interestingly, mutants unable to bind syntaxin 3 behaved differently in the HA transport assay. While one of the mutants tested had no effect, one inhibited and one enhanced the apical transport of HA. This implies that Munc18-2 function in apical membrane trafficking involves aspects independent of the syntaxin 3 interaction.     

Deletion of SNAP-23 results in pre-implantation embryonic lethality in mice

SNARE-mediated membrane fusion is a pivotal event for a wide-variety of biological processes. SNAP-25, a neuron-specific SNARE protein, has been well-characterized and mouse embryos lacking Snap25 are viable. However, the phenotype of mice lacking SNAP-23, the ubiquitously expressed SNAP-25 homolog, remains unknown. To reveal the importance of SNAP-23 function in mouse development, we generated Snap23-null mice by homologous recombination. We were unable to obtain newborn SNAP-23-deficient mice, and analysis of pre-implantation embryos from Snap23(Δ/wt) matings revealed that Snap23-null blastocysts were dying prior to implantation at embryonic day E3.5. Thus these data reveal a critical role for SNAP-23 during embryogenesis.     

Rab8b and its interacting partner TRIP8b are involved in regulated secretion in AtT20 cells

Rab proteins are a family of small GTPases that regulate intracellular vesicle traffic. Rab8b, because of its homology with Rab8, has been suggested to function in vesicle transport to the plasma membrane. Using the yeast two-hybrid system, we identified a Rab8b interacting clone, termed TRIP8b, from a rat brain cDNA library. The gene encodes a 66-kDa protein with homology to the peroxisomal targeting signal 1 receptor. The interaction between Rab8b and TRIP8b was further verified by in vitro binding assays and co-immunoprecipitation studies. Additional experiments with Rab8b mutants demonstrated that Rab8b requires a guanine nucleotide but not prenylation for its interaction with TRIP8b. Western immunoblot analysis showed that TRIP8b was primarily expressed in brain. Subcellular fractionation of AtT20 cells revealed that TRIP8b was present in both cytosolic and membrane fractions. To investigate the function of Rab8b and TRIP8b in secretion, we examined the release of ACTH from AtT20 cells. Results from stable cell lines expressing Rab8b or TRIP8b indicated that both proteins had a stimulatory effect on cAMP-induced secretion of ACTH. In summary, these data suggest that Rab8b and TRIP8b interact with each other and are involved in the regulated secretory pathway in AtT20 cells.     

Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in b16 melanoma cells

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.