Ca2+-dependent and -independent interactions of the isoforms of the α1A subunit of brain Ca2+ channels with presynaptic SNARE proteins

Fast neurotransmission requires that docked synaptic vesicles be located near the presynaptic N-type or P/Q-type calcium channels. Specific protein–protein interactions between a synaptic protein interaction (synprint) site on N-type and P/Q-type channels and the presynaptic SNARE proteins syntaxin, SNAP-25, and synaptotagmin are required for efficient, synchronous neurotransmitter release. Interaction of the synprint site of N-type calcium channels with syntaxin and SNAP-25 has a biphasic calcium dependence with maximal binding at 10–20 μM. We report here that the synprint sites of the BI and rbA isoforms of the α1A subunit of P/Q-type Ca2+ channels have different patterns of interactions with synaptic proteins. The BI isoform of α1A specifically interacts with syntaxin, SNAP-25, and synaptotagmin independent of Ca2+ concentration and binds with high affinity to the C2B domain of synaptotagmin but not the C2A domain. The rbA isoform of α1A interacts specifically with synaptotagmin and SNAP-25 but not with syntaxin. Binding of synaptotagmin to the rbA isoform of α1A is Ca2+-dependent, with maximum affinity at 10–20 μM Ca2+. Although the rbA isoform of α1A binds well to both the C2A and C2B domains of synaptotagmin, only the interaction with the C2A domain is Ca2+-dependent. These differential, Ca2+-dependent interactions of Ca2+ channel synprint sites with SNARE proteins may modulate the efficiency of transmitter release triggered by Ca2+ influx through these channels.

Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs

SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four α-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.

SNARE proteins are highly enriched in lipid rafts in PC12 cells: Implications for the spatial control of exocytosis

Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells. The target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to be highly enriched in lipid rafts (≈25-fold). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A/SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected only in nonraft fractions. SDS-resistance assays revealed that raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact with VAMP2. Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.

Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins

A major physiological role of insulin is the regulation of glucose uptake into skeletal and cardiac muscle and adipose tissue, mediated by an insulin-stimulated translocation of GLUT4 glucose transporters from an intracellular vesicular pool to the plasma membrane. This process is similar to the regulated docking and fusion of vesicles in neuroendocrine cells, a process that involves SNARE-complex proteins. Recently, several SNARE proteins were found in adipocytes: vesicle-associated membrane protein (VAMP-2), its related homologue cellubrevin, and syntaxin-4. In this report we show that treatment of permeabilized 3T3-L1 adipocytes with botulinum neurotoxin D, which selectively cleaves VAMP-2 and cellubrevin, inhibited the ability of insulin to stimulate translocation of GLUT4 vesicles to the plasma membrane. Furthermore, treatment of the permeabilized adipocytes with glutathione S-transferase fusion proteins encoding soluble forms of VAMP-2 or syntaxin-4 also effectively blocked insulin-regulated GLUT4 translocation. These results provide evidence of a functional role for SNARE-complex proteins in insulin-stimulated glucose uptake and suggest that adipocytes utilize a mechanism of regulating vesicle docking and fusion analogous to that found in neuroendocrine tissues.

Syntaxin 6 and Vti1b form a novel SNARE complex, which is upregulated in activated macrophages to facilitate exocytosis of TNF.

A key function of activated macrophages is to secrete proinflammatory cytokines such as TNF; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNF� vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNF� trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.

Structural analysis of SNARE motifs from sea perch, Lateolabrax japonicus by computerized approaches

Three cDNA sequences encoding four SNARE (N-ethylmaleimide-sensitive fusion protein attachment protein receptors) motifs were cloned from sea perch, and the deduced peptide sequences were analyzed for structural prediction by using 14 different web servers and softwares. The "ionic layer" structure, the three dimensional extension and conformational characters of the SNARE 7S core complex by using bioinformatics approaches were compared respectively with those from mammalian X-ray crystallographic investigations. The result suggested that the formation and stabilization of fish SNARE core complex might be driven by hydrophobic association, hydrogen bond among R group of core amino acids and electrostatic attraction at molecular level. This revealed that the SNARE proteins interaction of the fish may share the same molecular mechanism with that of mammal, indicating the universality and solidity of SNARE core complex theory. This work is also an attempt to get the protein 3D structural information which appears to be similar to that obtained through X-ray crystallography, only by using computerized approaches. (C) 2007 Elsevier Ltd. All rights reserved.

Syntaxin 3b is a t-SNARE specific for ribbon synapses of the retina.

Previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the retina contain the related isoform syntaxin 3. In addition to its localization in ribbon synapses, syntaxin 3 is also found in nonneuronal cells, where it has been implicated in the trafficking of transport vesicles to the apical plasma membrane of polarized cells. The syntaxin 3 gene codes for four different splice forms, syntaxins 3A, 3B, 3C, and 3D. We demonstrate here by using analysis of EST databases, RT-PCR, in situ hybridization, and Northern blot analysis that cells in the mouse retina express only syntaxin 3B. In contrast, nonneuronal tissues, such as kidney, express only syntaxin 3A. The two major syntaxin isoforms (3A and 3B) have an identical N-terminal domain but differ in the C-terminal half of the SNARE domain and the C-terminal transmembrane domain. These two domains are thought to be directly involved in synaptic vesicle fusion. The interaction of syntaxin 1A and syntaxin 3B with other synaptic proteins was examined. We found that both proteins bind Munc18/N-sec1 with similar affinity. In contrast, syntaxin 3B had a much lower binding affinity for the t-SNARE SNAP25 compared with syntaxin 1A. By using an in vitro fusion assay, we could demonstrate that vesicles containing syntaxin 3B and SNAP25 could fuse with vesicles containing synaptobrevin2/VAMP2, demonstrating that syntaxin 3B can function as a t-SNARE.

Phosphorylation of SNAP-23 by the Novel Kinase SNAK Regulates t-SNARE Complex Assembly

The docking and fusion of cargo-containing vesicles with target membranes of eukaryotic cells is mediated by the interaction of SNARE proteins present on both vesicle and target membranes. In many cases, the target membrane SNARE, or t-SNARE, exists as a complex of syntaxin with a member of the SNAP-25 family of palmitoylated proteins. We have identified a novel human kinase SNAK (SNARE kinase) that specifically phosphorylates the nonneuronal t-SNARE SNAP-23 in vivo. Interestingly, only SNAP-23 that is not assembled into t-SNARE complexes is phosphorylated by SNAK, and phosphorylated SNAP-23 resides exclusively in the cytosol. Coexpression with SNAK significantly enhances the stability of unassembled SNAP-23, and as a consequence, the assembly of newly synthesized SNAP-23 with syntaxin is augmented. These data demonstrate that phosphorylation of SNAP-23 by SNAK enhances the kinetics of t-SNARE assembly in vivo.

Selective Formation of Sed5p-containing SNARE Complexes Is Mediated by Combinatorial Binding Interactions

Sed5p is the only syntaxin family member required for protein transport through the yeast Golgi and it is known to bind up to nine other soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in vivo. We describe in vitro binding experiments in which we identify ternary and quaternary Sed5p-containing SNARE complexes. The formation of SNARE complexes among these endoplasmic reticulum- and Golgi-localized proteins requires Sed5p and is syntaxin-selective. In addition, Sed5p-containing SNARE complexes form selectively and this selectivity is mediated by Sed5p-containing intermediates that discriminate among subsequent binding partners. Although many of these SNAREs have overlapping distributions in vivo, the SNAREs that form complexes with Sed5p in vitro reflect their functionally distinct locales. Although SNARE–SNARE interactions are promiscuous and a single SNARE protein is often found in more than one complex, both the biochemical as well as genetic analyses reported here suggest that this is not a result of nonselective direct substitution of one SNARE for another. Rather our data are consistent with the existence of multiple (perhaps parallel) trafficking pathways where Sed5p-containing SNARE complexes play overlapping and/or distinct functional roles.

Three SNARE complexes cooperate to mediate membrane fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the syntaxin, SNAP-25, and VAMP families mediate intracellular membrane fusion through the formation of helical bundles that span opposing membranes. Soluble SNARE domains that lack their integral membrane anchors inhibit membrane fusion by forming nonfunctional complexes with endogenous SNARE proteins. In this study we investigate the dependence of membrane fusion on the concentration of a soluble SNARE coil domain derived from VAMP2. The increase in the inhibition of fusion observed with increasing concentration of inhibitor is best fit to a function that suggests three SNARE complexes cooperate to mediate fusion of a single vesicle. These three complexes likely contribute part of a protein and lipidic fusion pore.

Syntaxin 6 and Vti1b form a novel SNARE complex, which is up-regulated in activated macrophages to facilitate exocytosis of tumor necrosis factor-alpha

A key function of activated macrophages is to secrete proinflammatory cytokines such as TNF alpha; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNF alpha vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNF alpha trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor α (TNFα) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNFα is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasmamembrane, and we investigated a possible role for lipid rafts in TNFα trafficking and secretion. TNFα surface delivery and secretion were found to be cholesterol- dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasmamembrane, particularly on filopodia. Imaging the early stages of TNFα surface distribution revealed these puncta to be the initial points of TNFα delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.

Cholesterol regulates Syntaxin 6 trafficking at trans-Golgi network endosomal boundaries.

Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN). Here, using Chinese hamster ovary (CHO) Niemann-Pick type C1 (NPC1) mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6) accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs). This increases Stx6/VAMP3 interaction and interferes with the recycling of αVβ3 and α5β1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.

The cross-talk of LDL-cholesterol with cell migration: Insights from the Niemann Pick Type C1 mutation

Cholesterol is considered indispensible for the recruitment and functioning of integrins in focal adhesions for cell migration. However, the physiological cholesterol pools that control integrin trafficking and focal adhesion assembly remain unclear. Using Niemann Pick Type C1 (NPC) mutant cells, which accumulate Low Density lipoprotein (LDL)-derived cholesterol in late endosomes (LE), several recent studies indicate that LDL-cholesterol has multiple roles in regulating focal adhesion dynamics. Firstly, targeting of endocytosed LDL-cholesterol from LE to focal adhesions controls their formation at the leading edge of migrating cells. Other newly emerging literature suggests that this may be coupled to vesicular transport of integrins, Src kinase and metalloproteases from the LE compartment to focal adhesions. Secondly, our recent work identified LDL-cholesterol as a key factor that determines the distribution and ability of several Soluble NSF Attachment Protein (SNAP) Receptor (SNARE) proteins, key players in vesicle transport, to control integrin trafficking to the cell surface and extracellular matrix (ECM) secretion. Collectively, dietary, genetic and pathological changes in cholesterol metabolism may link with efficiency and speed of integrin and ECM cell surface delivery in metastatic cancer cells. This commentary will summarize how direct and indirect pathways enable LDL-cholesterol to modulate cell motility.