Soluble NSF Attachment Protein Research Articles

Activation of Syntaxin 1C, an Alternative Splice Variant of HPC-1/Syntaxin 1A, by Phorbol 12-Myristate 13-Acetate (PMA) Suppresses Glucose Transport into Astroglioma Cells via the Glucose Transporter-1 (GLUT-1)

Syntaxin 1C is an alternative splice variant lacking the transmembrane domain of HPC-1/syntaxin 1A. We found previously that syntaxin 1C is expressed as a soluble protein in human astroglioma (T98G) cells, and syntaxin 1C expression is enhanced by stimulation with phorbol 12-myristate 13-acetate (PMA). However, the physiological function of syntaxin 1C is not known. In this study, we examined the relationship between syntaxin 1C and glucose transport. First, we discovered that glucose transporter-1 (GLUT-1) was the primary isoform in T98G cells. Second, we demonstrated that glucose uptake in T98G cells was suppressed following an increase in endogenous syntaxin 1C after stimulation with PMA, which did not alter the expression levels of other plasma membrane syntaxins. We further examined glucose uptake and intracellular localization of GLUT-1 in cells that overexpressed exogenous syntaxin 1C; glucose uptake via GLUT-1 was inhibited without affecting sodium-dependent glucose transport. The value of Vmax for the dose-dependent uptake of glucose was reduced in syntaxin 1C-expressing cells, whereas there was no change in Km. Immunofluorescence studies revealed a reduction in the amount of GLUT-1 in the plasma membrane in cells that expressed syntaxin 1C. Based on these results, we postulate that syntaxin 1C regulates glucose transport in astroglioma cells by changing the intracellular trafficking of GLUT-1. This is the first report to indicate that a syntaxin isoform that lacks a transmembrane domain can regulate the intracellular transport of a plasma membrane protein.

Genetic analysis of soluble N-ethylmaleimide-sensitive factor attachment protein function in Drosophila reveals positive and negative secretory roles.

The N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (SNAP) are cytosolic factors that promote vesicle fusion with a target membrane in both the constitutive and regulated secretory pathways. NSF and SNAP are thought to function by catalyzing the disassembly of a SNAP receptor (SNARE) complex consisting of membrane proteins of the secretory vesicle and target membrane. Although studies of NSF function have provided strong support for this model, the precise biochemical role of SNAP remains controversial. To further explore the function of SNAP, we have used mutational and transgenic approaches in Drosophila to investigate the effect of altered SNAP dosage on neurotransmitter release and SNARE complex metabolism. Our results indicate that reduced SNAP activity results in diminished neurotransmitter release and accumulation of a neural SNARE complex. Increased SNAP dosage results in defective synapse formation and a variety of tissue morphological defects without detectably altering the abundance of neural SNARE complexes. The SNAP overexpression phenotypes are enhanced by mutations in other secretory components and are at least partially overcome by co-overexpression of NSF, suggesting that these phenotypes derive from a specific perturbation of the secretory pathway. Our results indicate that SNAP promotes neurotransmitter release and SNARE complex disassembly but inhibits secretion when present at high abundance relative to NSF.

The maintenance and generation of membrane polarity in hepatocytes.

Hepatocytes, the major epithelial cells in the liver, are highly polarized. Their plasma membranes are separated by tight junctions into sinusoidal– basolateral and canalicular–apical domains, which contain distinct sets of proteins and lipids. A normal membrane polarity is vital for hepatocytes to perform many diverse functions, such as canalicular bile secretion and simultaneous sinusoidal secretion of large quantities of serum proteins into blood. Hepatocyte polarity is lost in many diseases like cholestasis. A complete understanding of the molecular mechanisms involved in hepatocyte polarity therefore is of considerable significance to both liver cell biology and the pathogenesis of liver diseases. To maintain the distinct distribution of proteins and lipids in the canalicular and sinusoidal membranes, hepatocytes have developed complex polarized trafficking and retention mechanisms. The establishment of membrane polarity in hepatocytes begins during liver embryogenesis. During hepatocyte differentiation, specific routes and mechanisms are defined for the delivery of plasma membrane proteins. How this polarity is maintained in adult hepatocytes and is generated during development are two major unanswered questions in this field. In this review, we first give an overview of the current knowledge about how membrane polarity is maintained in hepatocytes with the focus on recent developments in polarized protein trafficking.We then present a hypothetical model about how membrane polarity may be generated during liver development, and finally, we present an example of how membrane polarity is altered in cholestasis. Readers are directed to recent reviews for discussion of lipid trafficking in hepatocytes.1,2

Synaptotagmin Interaction with the Syntaxin/SNAP-25 Dimer Is Mediated by an Evolutionarily Conserved Motif and Is Sensitive to Inositol Hexakisphosphate

Synaptotagmins are membrane proteins that possess tandem C2 domains and play an important role in regulated membrane fusion in metazoan organisms. Here we show that both synaptotagmins I and II, the two major neuronal isoforms, can interact with the syntaxin/synaptosomal-associated protein of 25 kDa (SNAP-25) dimer, the immediate precursor of the soluble NSF attachment protein receptor (SNARE) fusion complex. A stretch of basic amino acids highly conserved throughout the animal kingdom is responsible for this calcium-independent interaction. Inositol hexakisphosphate modulates synaptotagmin coupling to the syntaxin/SNAP-25 dimer, which is mirrored by changes in chromaffin cell exocytosis. Our results shed new light on the functional importance of the conserved polybasic synaptotagmin motif, suggesting that synaptotagmin interacts with the t-SNARE dimer to up-regulate the probability of SNARE-mediated membrane fusion.

Regional and progressive changes in brain expression of complexin II in a mouse transgenic for the Huntington’s Disease mutation

Changes in mRNA expression of soluble NSF-attachment protein receptors (SNAREs) and SNARE-associated proteins have been shown to occur in a number of disorders such as schizophrenia, Alzheimer’s disease and Parkinson’s disease. We have shown previously that there is a decrease in protein levels of the SNARE-associated protein, complexin II (CPLXII) in Huntington’s disease brain and in the R6/2 mouse model of Huntington’s disease. In the current study, we used quantitative in situ hybridisation to examine mRNA expression of SNAREs (25 kDa synaptosome-associated protein (SNAP-25), syntaxin-1A and synaptobrevin-2) and SNARE-associated proteins (α-SNAP, CPLXI and CPLXII) in brain of R6/2 mice and their wild type littermates between 3 and 15 weeks of age. We found an early and progressive decrease of CPLXII expression in R6/2 mice brains. In contrast, no changes in SNARE expression were seen in R6/2 brains compared with wild type brain. Further, while decreased expression of α-SNAP and CPLXI was seen, this was not until 15 weeks of age and even then the changes were small. We suggest that downregulation of expression of mRNA encoding SNARE-associated proteins, first CPLXII and later CPLXI and α-SNAP, contributes to the progressive neuropathology of the R6/2 mouse model of Huntington’s disease.

Minimum essential factors required for vesicle mobilization at hippocampal synapses.

Studies on the mechanisms that underlie the function of small central presynaptic terminals have been hampered by the inaccessibility of these synapses to soluble reagents. Here, we permeabilized hippocampal synapses in culture, manipulated their interior, and monitored the resulting changes in vesicle mobilization with the styryl dye FM2-10. Using this method, we found that 1 microm Ca2+ after incubation with GTP or GTP-gamma-S could mobilize approximately 90% of the total recycling pool, whereas 1 microm Ca2+ application after dialysis of permeabilized synapses with GDP-beta-S mobilized approximately 30% of the recycling vesicles, presumably corresponding to the readily releasable pool. In electron micrographs of permeabilized hippocampal synapses stimulated with 1 microm Ca2+, we could detect significant vesicle depletion after preincubation with GTP-gamma-S, whereas preincubation with GDP-beta-S left the total vesicle pool relatively intact. Taken together, in this system replenishment of the readily releasable pool by the reserve vesicles was strictly GTP dependent. In contrast, vesicle replenishment and release did not require ATP or N-ethylmaleimide-sensitive factor (NSF); however, this process involved formation of new soluble NSF-attachment protein receptor (SNARE) complexes as judged by its sensitivity to tetanus toxin. These results suggest that in hippocampal synapses, vesicle mobilization and replenishment of the readily releasable pool require GTP and Ca2+ but do not necessitate ATP-dependent priming and SNARE recycling.

Differential messenger RNA expression of complexins in mouse brain

Complexins (CPLXs) are small isomeric proteins that bind to the soluble NSF-attachment protein receptor (SNARE) complex and modulate neurotransmitter release. Two isoforms of CPLX exist in the brain, CPLXI and CPLXII. These are differentially distributed in the cortex and cerebellum, with CPLXI found in axosomatic terminals and CPLXII in axodendritic terminals. Since in cortex and cerebellum axosomatic terminals are inhibitory and axodendritic terminals are excitatory, it has been assumed that CPLXI modulates inhibitory and CPLXII modulates excitatory transmitter release. Here we used in situ hybridisation to study the mRNA distribution of CPLXI and CPLXII in mouse brain. We show that while CPLXs are expressed in distinct cell populations, they do not segregate with either particular neurotransmitters, or different classes of transmitter action. For example, while CPLXII is the dominant isoform in the output (glutamatergic excitatory) neurons of the cortex, it is also the dominant isoform in medium spiny (GABAergic inhibitory) neurons of the striatum. We suggest that the functional role of CPLXs depends not only on the identity of the neurotransmitter, but also upon the circuitry connecting the neurons in which they are expressed. Thus, the predominant expression of CPLXII in neurons of the basal ganglia and cortex suggests a role in cognition, emotional behaviour and control of voluntary movement, while the pattern of CPLXI expression suggests a primary role in motor learning programs and sensory processing.

Expression of SNARE proteins as potential regulators of exocytosis in the basophil-like cell line KU-812

Rationale SNARE (soluble nSF attachment protein receptor) proteins have been implicated in regulating granular and vesicular docking in many inflammatory cells undergoing regulated and constitutive exocytosis. SNARE complex expression has been demonstrated in all inflammatory cells, except basophils. Basophils contribute to allergic inflammation following degranulation and release of histamine, proteases and regulatory cytokines including IL-4 and IL-13.SNARE molecules are divided into two groups: vesicular SNAREs (v-SNARE), located on the granule or vesicle membrane and target SNARE (t-SNARE) associated with the target plasma membrane. Methods We used KU-812, a human prebasophilic leukemic cell line, as a model system to study the process of exocytosis in basophil-like cells. RT-PCR and Western blot analysis, coupled with confocal microscopy were used to identify mRNA, protein expression and distribution, respectively of distinct isoforms of SNARE complex. Results KU-812 cells expressed mRNA for the v-SNARE isoforms of VAMP (vesicle-associated membrane protein)-1, 2, 3, 7 and 8. As well, KU-812 cells encoded for mRNA for t-SNAREs SNAP-23 and syntaxin-3, 4, 6. Syntaxin-4 protein and SNAP-23 were also detected by Western blot analysis of KU-812 lysates. The presence of SNARE proteins was confirmed by confocal microscopy and was localized to membrane bound vesicular structures. Conclusions Exocytosis in basophil-like cells and, by inference, human basophils, may be potentially regulated by SNARE proteins. The identification of a range of SNARE isoforms in these cells may contribute to a more cell-specific targeting of SNARE molecules during exocytosis.

Rab and SNARE isoform expression in cytotoxic T-cells (CTL): Potential players in regulated release of granzyme-B (GrB)

Rationale Cytotoxic T-cells (CTLs, CD8 + lymphocytes) have been implicated in asthma and chronic obstructive pulmonary disease (COPD), however the mechanism of CTL cytotoxic granule secretion of granzyme-B (GrB) is uncertain. Secretory cell exocytosis is dependent on interactions between SNARE (soluble nSF attachment protein receptor) proteins on vesicular (v-SNAREs) and target plasma membrane (t-SNARE). Rab proteins regulate intracellular organelle transport, matching appropriate v- with t-SNAREs. We hypothesized that SNARE and Rab proteins are involved in the regulated release of GrB stored in CTLs and natural killer (NK) cells. Methods Specific primer sets determined SNARE and Rab mRNA expression in human CD8 + cells and YT-Indy, a human NK cell line, by RT-PCR. Western blot analysis and immunocytochemistry, using specific antibodies, confirmed SNARE and Rab protein expression. Results CTLs and YT-Indy were shown to express mRNA for the Rab proteins, Rab 4a, 5a, and 27a. CTLs encoded for the v-SNAREs, VAMP 1, 2, 8, and the t-SNAREs, syntaxin 3, 4, 6, and SNAP-23. YT-Indy cells expressed mRNA for the SNAREs, VAMP 2, 7, 8, syntaxin-3, and SNAP-23. Expression of syntaxin-4, and 6 in the YT-Indy cell line was, however, variable. Interestingly, mRNA for VAMP-1 was expressed in CTLs, but not in YT-Indy, which may suggest cell-specific differences in regulation of secretion. Protein expression for SNAP-23, syntaxin-4, syntaxin-6, and VAMP-8 on GrB + granule membranes in CTLs was detected by Western blot analysis. Conclusions Our data suggest that SNARE proteins and Rabs may be important in the regulation of CTL mediated release of GrB.

The gene for soluble N-ethylmaleimide sensitive factor attachment protein alpha is mutated in hydrocephaly with hop gait (hyh) mice.

The spontaneous autosomal recessive mouse mutant for hydrocephaly with hop gait (hyh) exhibits dramatic cystic dilation of the ventricles at birth and invariably develops hopping gait. We show that the gene for soluble N-ethylmaleimide sensitive factor attachment protein alpha, also known as alpha-SNAP, is mutated in hyh mice. alpha-SNAP plays a key role in a wide variety of membrane fusion events in eukaryotic cells, including the regulated exocytosis of neurotransmitters. Homozygous mutant mice harbor a missense mutation M105I in a conserved residue in one of the alpha-helical domains. We demonstrate that the hyh mutant is not a null allele and is expressed; however, the mutant protein is 40% less abundant in hyh mice. The hyh mutant provides a valuable in vivo model to study vesicle/membrane trafficking and provides insight into the potential roles of alpha-SNAP in embryogenesis and brain development.

Multiple binding proteins suggest diverse functions for the N-ethylmaleimide sensitive factor

The hexameric ATPase, N-ethylmaleimide sensitive factor (NSF), is essential to vesicular transport and membrane fusion because it affects the conformations and associations of the soluble NSF attachment protein receptor (SNARE) proteins. NSF binds SNAREs through adaptors called soluble NSF attachment proteins (α- or β-SNAP) and disassembles SNARE complexes to recycle the monomers. NSF contains three domains, two nucleotide-binding domains (NSF-D1 and -D2) and an amino terminal domain (NSF-N) that is required for SNAP–SNARE complex binding. Mutagenesis studies indicate that a cleft between the two sub-domains of NSF-N is critical for binding. The structural conservation of N domains in NSF, p97/VCP, and VAT suggests that a similar type of binding site could mediate substrate recognition by other AAA proteins. In addition to SNAP–SNARE complexes, NSF also binds other proteins and protein complexes such as AMPA receptor subunits (GluR2), β2-adrenergic receptor, β-Arrestin1, GATE-16, LMA1, rabs, and rab-containing complexes. The potential for these interactions indicates a broader role for NSF in the assembly/disassembly cycles of several cellular complexes and suggests that NSF may have specific regulatory effects on the functions of the proteins involved in these complexes. The structural requirements for these interactions and their physiological significance will be discussed.

Nitric Oxide Regulates Exocytosis by S-Nitrosylation of N-ethylmaleimide-Sensitive Factor

Nitric oxide (NO) inhibits vascular inflammation, but the molecular basis for its anti-inflammatory properties is unknown. We show that NO inhibits exocytosis of Weibel-Palade bodies, endothelial granules that mediate vascular inflammation and thrombosis, by regulating the activity of N-ethylmaleimide-sensitive factor (NSF). NO inhibits NSF disassembly of soluble NSF attachment protein receptor (SNARE) complexes by nitrosylating critical cysteine residues of NSF. NO may regulate exocytosis in a variety of physiological processes, including vascular inflammation, neurotransmission, thrombosis, and cytotoxic T lymphocyte cell killing.

Evidence for SNARE zippering during Ca 2+-triggered exocytosis in PC12 cells

SNAREs ( soluble NSF attachment protein receptors) are membrane proteins that catalyze membrane fusion. SNAREs are defined by a characteristic 70 residue sequence called the SNARE motif. During synaptic vesicle fusion, the single SNARE motif of the synaptic vesicle SNARE protein synaptobrevin/VAMP associates into a four-helical bundle with SNARE motifs from the plasma membrane SNARE proteins syntaxin 1 and SNAP-25. The four SNARE motifs (one each from synaptobrevin and syntaxin, and two from SNAP-25) assume a parallel orientation in the complex, suggesting that formation of the complex initiates fusion by forcing the membranes containing the SNAREs into close proximity. It has been proposed that SNARE complexes assemble in an N- to C-terminal progression, a process referred to as zippering, but little direct evidence for zippering exists. Furthermore, the SM protein Munc18-1, which binds to syntaxin 1 and is essential for synaptic fusion, is thought to prepare SNAREs for complex formation by an unknown mechanism, possibly by nucleating zippering. We now show that fragments containing the N- and C-terminal regions of the SNARE motif from syntaxin 1A bind SNAP-25 similarly. However, in permeabilized PC12 cells which are used as a biochemical model system to study synaptic fusion, only fragments containing the N-terminal region are powerful inhibitors of fusion. Furthermore, mutations in the N-terminal part of the Syntaxin SNARE motif have only a moderate effect on SNAP-25 binding but abolish the inhibitory activity of the SNARE motif. Finally, larger fragments of syntaxin 1A that strongly bind to Munc18-1 but do not readily assemble into SNARE complexes had no effect on exocytosis in permeabilized PC12 cells. Together these results suggest that Munc18-1 acts before SNARE complex assembly, and is no longer required at the stage of fusion assayed in permeabilized PC12 cells. The selective effect of the N-terminal half of the syntaxin 1A SNARE motif on PC12 cell exocytosis shows that the SNARE motif is functionally polarized, and supports the notion that SNARE complexes assemble in an N- to C-terminal zippering reaction during fusion without a stable, partially assembled intermediate.

Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 A resolution.

N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three-dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP-ADP mixture at 11 A resolution by electron cryomicroscopy and single-particle averaging of NSF.alpha-SNAP.SNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF-N) emerge from the sides of the D1 domain ring. The density corresponding to alpha-SNAP and SNAREs is located on the 6-fold axis of the structure, near the NSF-N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF.

A role for Sec1/Munc18 proteins in platelet exocytosis.

A critical aspect of haemostasis is the release of clot-forming components from the three intra-platelet stores: dense-core granules, alpha granules and lysosomes. Exocytosis from these granules is mediated by soluble proteins [N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs)] and integral membrane proteins [vesicle and target SNAP receptors (v- and t-SNAREs)]. Three Sec1/Munc18 proteins (SM proteins) are present in platelets (Munc18a, Munc18b and Munc18c) and they bind to and potentially regulate specific syntaxin t-SNAREs. In resting platelets, these SM proteins associate with granules and open canalicular system membranes predominantly but not with the plasma membrane. Munc18a binds to syntaxin 2 alone and does not associate with other members of the core SNARE complex. Munc18b associates with a larger complex that contains synaptosome-associated protein of 23 kDa (SNAP-23) and cellubrevin/vesicle-associated membrane protein 3. Munc18c associates with both syntaxins 2 and 4, with synaptosome-associated protein of 23 kDa (SNAP-23) and with a v-SNARE. On stimulation, most of the platelet SM proteins are still found in membrane fractions. Phosphorylation of each Munc18 increases in thrombin-treated cells and phosphorylated Munc18c remains associated with syntaxins 2 and 4, but its affinity for the SNAREs appears to be reduced. To determine the functional role of the platelet SM proteins, we examined the effects of Munc18-based peptides (Munc18a peptide 3 and Munc18c peptide 3). Addition of the peptides to permeabilized platelets inhibits secretion from all three platelet granules. These peptides also inhibit agonist-induced aggregation in saponin-permeabilized platelets. These studies demonstrate a clear role for SM proteins in platelet exocytosis and aggregation and suggest a dominant role for Munc18c in all three granule-release events.

Calcium-triggered Membrane Fusion Proceeds Independently of Specific Presynaptic Proteins

Complexes of specific presynaptic proteins have been hypothesized to drive or catalyze the membrane fusion steps of exocytosis. Here we use a stage-specific preparation to test the roles of SNAREs, synaptotagmin, and SNARE-binding proteins in the mechanism of Ca2+-triggered membrane fusion. Excess exogenous proteins, sufficient to block SNARE interactions, did not inhibit either the Ca2+ sensitivity, extent, or kinetics of fusion. In contrast, despite a limited effect on SNARE and synaptotagmin densities, treatments with high doses of chymotrypsin markedly inhibited fusion. Conversely, low doses of chymotrypsin had no effect on the Ca2+ sensitivity or extent of fusion but did alter the kinetic profile, indicating a more direct involvement of other proteins in the triggered fusion pathway. SNAREs, synaptotagmin, and their immediate binding partners are critical to exocytosis at a stage other than membrane fusion, although they may still influence the triggered steps.

Defining the SNARE Complex Binding Surface of α-SNAP: IMPLICATIONS FOR SNARE COMPLEX DISASSEMBLY

N-Ethylmaleimide-sensitive factor (NSF) and its adaptor protein alpha-soluble NSF attachment protein (alpha-SNAP) sustain membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE) complexes that form during membrane fusion. To better understand the role of alpha-SNAP in this process, we used site-directed mutagenesis to identify residues in alpha-SNAP that interact with SNARE complexes. We find that mutations in charged residues distributed over a concave surface formed by the N-terminal nine alpha-helices of alpha-SNAP affect its ability to bind synaptic SNARE complex and promote its disassembly by NSF. Replacing basic residues on this surface with alanines reduced SNARE complex binding and disassembly, whereas replacing acidic residues with alanines enhanced alpha-SNAP efficacy in both assays. These findings show that the ability of NSF to take apart SNARE complexes depends upon electrostatic interactions between alpha-SNAP and the acidic surface of the SNARE complex and provide insight into how NSF and alpha-SNAP work together to drive disassembly.

Syntaxin specificity of cytokinesis in Arabidopsis.

Syntaxins interact with other SNAREs (soluble NSF-attachment protein receptors) to form structurally related complexes that mediate membrane fusion in diverse intracellular trafficking pathways. The original SNARE hypothesis postulated that each type of transport vesicle has its own distinct vesicle-SNARE that pairs up with a unique target-SNARE, or syntaxin, on the target membrane. However, recent evidence suggests that small G-proteins of the Rab family and their effectors mediate the initial contact between donor and acceptor membranes, providing complementary specificity to SNARE pairing at a later step towards membrane fusion. To assess the role of syntaxin specificity in membrane recognition requires a biological assay in which one syntaxin is replaced by other family members that do not normally function in that trafficking pathway. Here, we examine whether membrane fusion in Arabidopsis thaliana cytokinesis, which involves a plant-specific syntaxin, the cell-cycle-regulated KNOLLE (KN) protein, can be mediated by other syntaxins if expressed under the control of KN cis-regulatory sequences. Only a non-essential syntaxin was targeted to the plane of cell division and sufficiently related to KN to perform its function, thus revealing syntaxin specificity of cytokinesis.

The Yip1p·Yif1p Complex Is Required for the Fusion Competence of Endoplasmic Reticulum-derived Vesicles

Here we report that Yip1p and Yif1p, two members of an integral membrane protein complex that bind to the Rab Ypt1p, are required for membrane fusion with the Golgi in vitro. To block fusion, anti-Yip1p or anti-Yif1p antibodies must be added before vesicles bud from the endoplasmic reticulum (ER). These antibodies do not block the packaging of Yip1p, Yif1p, or the soluble NSF attachment protein receptor (SNAREs) into vesicles. We propose that Yip1p and Yif1p perform a critical role in establishing the fusion competence of ER to Golgi vesicles at the time of budding. Consistent with this proposal, we observe that the Yip1p.Yif1p complex binds to the ER to Golgi SNAREs Bos1p and Sec22p, two components of the membrane fusion machinery.

Characterization of alpha-soluble N-ethylmaleimide-sensitive fusion attachment protein in alveolar type II cells: implications in lung surfactant secretion.

N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (alpha-SNAP) are thought to be soluble factors that transiently bind and disassemble SNAP receptor complex during exocytosis in neuronal and endocrine cells. Lung surfactant is secreted via exocytosis of lamellar bodies from alveolar epithelial type II cells. However, the secretion of lung surfactant is a relatively slow process, and involvement of SNAP receptor and its cofactors (NSF and alpha-SNAP) in this process has not been demonstrated. In this study, we investigated a possible role of alpha-SNAP in surfactant secretion. alpha-SNAP was predominantly associated with the membranes in alveolar type II cells as determined by Western blot and immunocytochemical analysis using confocal microscope. Membrane-associated alpha-SNAP was not released from the membrane fraction when the cells were lyzed in the presence of Ca2+ or Mg2+ATP. The alkaline condition (0.1 M Na2CO3, pH 12), known to extract peripheral membrane proteins also failed to release it from the membrane. Phase separation using Triton X-114 showed that alpha-SNAP partitioned into both aqueous and detergent phases. NSF had membrane-bound characteristics similar to alpha-SNAP in type II cells. Permeabilization of type II cells with beta-escin resulted in a partial loss of alpha-SNAP from the cells, but cellular NSF was relatively unchanged. Addition of exogenous alpha-SNAP to the permeabilized cells increased surfactant secretion in a dose-dependent manner, whereas exogenous NSF has much less effects. An alpha-SNAP antisense oligonucleotide decreased its protein level and inhibited surfactant secretion. Our results suggest a role of alpha-SNAP in lung surfactant secretion.