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At least 163 records · Page 9Linked to original sources
PubMed2006

N-ethylmaleimide sensitive factor is required for fusion of the C. elegans uterine anchor cell.

The fusion of the Caenorhabditis elegans uterine anchor cell (AC) with the uterine-seam cell (utse) is an excellent model system for studying cell-cell fusion, which is essential to animal development. We obtained an egg-laying defective (Egl) mutant in which the AC fails to fuse with the utse. This defect is highly specific: other aspects of utse development and other cell fusions appear to occur normally. We find that defect is due to a missense mutation in the nsf-1 gene, which encodes N-ethylmaleimide-sensitive factor (NSF), an intracellular membrane fusion factor. There are two NSF-1 isoforms, which are expressed in distinct tissues through two separate promoters. NSF-1L is expressed in the uterus, including the AC. We find that nsf-1 is required cell-autonomously in the AC for its fusion with the utse. Our results establish AC fusion as a paradigm for studying cell fusion at single cell resolution and demonstrate that the NSF ATPase is a key player in this process.

Animals↗
PubMed1998

Selective stimulation of the D1 ATPase domain of N-ethylmaleimide-sensitive fusion protein (NSF) by soluble NSF attachment proteins.

N-Ethylmaleimide-sensitive fusion protein (NSF) is required for most intracellular membrane fusion events. NSF is recruited to membranes by soluble NSF attachment proteins (SNAPs) and membrane-resident SNAP receptor (SNARE) proteins. The 20 S complex of NSF/SNAPs/SNAREs disassembles when NSF hydrolyses ATP, and this disassembly event is believed to be essential for membrane fusion. SNAPs stimulate NSF ATPase activity, but it is not known which of NSF's two ATPase domains (D1 or D2) is affected. Using recombinant mutant NSFs defective in ATP hydrolysis in one domain only, we found that SNAPs stimulate NSF ATPase activity by a selective action on the D1 domain, yet had no effect on the D2 domain. Since the D1 domain of NSF is implicated in 20 S complex disassembly, this supports the idea that SNAP stimulation of NSF ATPase activity is required for membrane fusion.

Adenosine Triphosphatases↗
PubMed2001

N-ethylmaleimide sensitive factor (NSF) structure and function.

Our understanding of the molecular mechanisms of membrane trafficking advanced at a rapid rate during the 1990s. As one of the initial protein components of the trafficking machinery to be identified, N-ethylmaleimide sensitive factor (NSF) has served as a reference point in many of these recent studies. This hexameric ATPase is essential for most of the membrane-trafficking events in a cell. Initially, due to its ATPase activity, NSF was thought to be the motor that drove membrane fusion. Subsequent studies have shown that NSF actually plays the role of a chaperone by activating SNAP receptor proteins (SNAREs) so that they can participate in membrane fusion. In this review we will examine the initial characterization of NSF, its role in membrane fusion events, and what new structural information can tell us about NSF's mechanism of action.

Adenosine Triphosphatases↗
PubMed1999

Crystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein.

The cytosolic ATPase N-ethylmaleimide-sensitive fusion protein (NSF) disassembles complexes of membrane-bound proteins known as SNAREs, an activity essential for vesicular trafficking. The amino-terminal domain of NSF (NSF-N) is required for the interaction of NSF with the SNARE complex through the adaptor protein alpha-SNAP. The crystal structure of NSF-N reveals two subdomains linked by a single stretch of polypeptide. A polar interface between the two subdomains indicates that they can move with respect to one another during the catalytic cycle of NSF. Structure-based sequence alignments indicate that in addition to NSF orthologues, the p97 family of ATPases contain an amino-terminal domain of similar structure.

Adenosine Triphosphatases↗
PubMed1995

The N-ethylmaleimide-sensitive fusion protein and alpha-SNAP induce a conformational change in syntaxin.

The N-ethylmaleimide-sensitive fusion protein (NSF) plays an essential role in intracellular membrane fusion events and has been implicated in the exocytosis of synaptic vesicles. NSF binds through soluble NSF attachment proteins (SNAPs) to a complex of neuronal membrane proteins comprised of synaptobrevin, syntaxin, and SNAP-25. Disassembly of this complex by NSF is thought to be a critical step in the molecular events which lead to vesicle fusion with the plasma membrane. Here we have studied the interaction of alpha-SNAP and NSF with individual components of this complex and have identified syntaxin as a primary substrate for NSF/alpha-SNAP. We find that alpha-SNAP binds directly to syntaxin 1A as well as weakly to SNAP-25, while it does not bind to synaptobrevin II. NSF binds to syntaxin through alpha-SNAP and in the presence of ATP catalyzes a conformational rearrangement which abolishes binding of itself and alpha-SNAP. This reaction leads to the previously described disassembly of the fusion complex, since synaptobrevin binding to syntaxin is also reduced. alpha-SNAP binds to a carboxyl-terminal syntaxin fragment (residues 194-288) that also binds synaptobrevin and SNAP-25. However, NSF action on this syntaxin fragment has no effect on the binding of alpha-SNAP or synaptobrevin. This suggests that the conformational change normally induced by NSF in syntaxin depends on an interaction between carboxyl- and amino-terminal domains of syntaxin.

Base Sequence↗
PubMed1996

SNAP prevents Mg(2+)-ATP-induced release of N-ethylmaleimide-sensitive factor from the Golgi apparatus in digitonin-permeabilized PC12 cells.

The N-ethylmaleimide-sensitive factor (NSF), which is involved in the multisteps of protein transport, is released from Golgi membranes on in vitro incubation with Mg(2+)-ATP. However, several lines of evidence suggest that NSF is associated with membranes in spite of the presence of Mg2+ and ATP in vivo. We have used digitonin-permeabilized PC12 cells to investigate the mechanism underlying the association of NSF with membranes. In PC12 cells, immunoreactivity for NSF was observed in the nuclear membranes, the Golgi apparatus, and neuronal growth cones, where synaptic vesicles are concentrated. NSF associated with the Golgi apparatus was released on incubation with Mg(2+)-ATP, whereas NSF in the nuclear membranes and neuronal growth cones was not released on the same treatment. The addition of cytosol blocked the Mg(2+)-ATP-induced release of NSF from the Golgi apparatus. Chromatographic analyses revealed that the factor(s) that prevents NSF release from the Golgi apparatus was eluted at the same position as the soluble NSF attachment proteins (SNAPs). Purified His6-tagged alpha-SNAP exhibited such activity. His6-tagged alpha-SNAP also prevented the Mg(2+)-ATP-induced release of NSF from isolated Golgi membranes.

Adenosine Triphosphate↗
PubMed1996

A possible predocking attachment site for N-ethylmaleimide-sensitive fusion protein. Insights from in vitro endosome fusion.

N-Ethylmaleimide-sensitive fusion protein (NSF) is an ubiquitous protein required for multiple vesicular transport events. We have investigated the role of the two nucleotide-binding regions of NSF in endosomal fusion by analyzing NSF mutants in a cell-free system. Our results indicate that mutations on the first ATP-binding domain, that render a protein defective in either ATP binding or ATP hydrolysis, results in almost complete inhibition of endosomal fusion. A mutation in the second ATP-binding site of NSF was only slightly inhibitory. The inhibitory effect was observed only when the mutant proteins were added at early times during the fusion reaction indicating that NSF may be required for an early step during the docking/fusion process. Binding studies using Western blotting reveal that the binding of NSF mutants to endosomal membranes is differentially affected by Ca2+. Our results indicate that NSF, depending on its nucleotide state, may interact with membranes via an alternate mechanism. Our findings suggest the existence of a predocking binding site either independent of the docking complex or a site that leads to the formation of the SNAP-SNARE complex (e.g. 20 S particle).

Adenosine Triphosphate↗
PubMed1997

GS15, a 15-kilodalton Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) homologous to rbet1.

Bet1p plays an essential role in vesicular transport from the endoplasmic reticulum (ER) to the Golgi in yeast, and it functions as a vesicle soluble N-ethylmaleimide-sensitive factor protein receptor (v-SNARE). A mammalian protein related to Bet1p has been reported previously and was referred to as rbet1. We have now identified a new mammalian protein that is homologous to rbet1 (28% amino acid identity). mRNA for this rbet1 homologue is widely expressed in rat tissues. Affinity-purified polyclonal antibodies raised against recombinant protein specifically recognized a 15-kilodalton integral membrane protein highly enriched in Golgi membranes. Indirect immunofluorescence microscopy revealed that this protein is specifically associated with the Golgi apparatus in diverse cell types. Biochemical characterization established that this protein behaves like a SNARE and was named GS15 (Golgi SNARE with a size of 15 kilodaltons). These properties raise the possibility that GS15 is a novel SNARE mediating a yet to be defined transport event associated with the Golgi apparatus.

Amino Acid Sequence↗
PubMed1998

N-ethylmaleimide-sensitive factor-dependent alpha-SNAP release, an early event in the docking/fusion process, is not regulated by Rab GTPases.

The N-ethylmaleimide-sensitive factor (NSF) is required for multiple intracellular vesicle transport events. In vitro biochemical studies have demonstrated that NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors from a 20 S particle. This complex is disassembled by the ATPase activity of NSF. We have studied particle disassembly in a membrane environment by examining the binding of recombinant SNAPs and NSF to endosomal membranes. We present evidence that alpha-SNAP is released from the membranes in a temperature- and time-dependent manner and that this release is mediated by the ATPase activity of NSF. Our results indicate that NSF mutants in the first ATP binding domain completely abrogate alpha-SNAP release, whereas no inhibitory effect is observed with a mutant in the second ATP binding domain. Interestingly, neither beta-SNAP nor gamma-SNAP are released by the ATPase activity of NSF, indicating that these proteins are retained on the membranes by interactions that differ from those that retain alpha-SNAP. Although the small Rab GTPases are known to play a role in SNARE complex assembly, our results indicate that these GTPases do not regulate the NSF-dependent release of alpha-SNAP.

Adenosine Triphosphatases↗
PubMed2001

Binding of the beta2 adrenergic receptor to N-ethylmaleimide-sensitive factor regulates receptor recycling.

Following agonist stimulation, most G protein-coupled receptors become desensitized and are internalized, either to be degraded or recycled back to the cell surface. What determines the fate of a specific receptor type after it is internalized is poorly understood. Here we show that the rapidly recycling beta2 adrenergic receptor (beta2AR) binds via a determinant including the last three amino acids in its carboxyl-terminal tail to the membrane fusion regulatory protein, N-ethylmaleimide-sensitive factor (NSF). This is documented by in vitro overlay assays and by cellular coimmunoprecipitations. Receptors bearing mutations in any of the last three residues fail to interact with NSF. After stimulation with the agonist isoproterenol, a green fluorescent protein fusion of NSF colocalizes with the wild type beta2AR but not with a tail-mutated beta2AR. The beta2AR-NSF interaction is required for efficient internalization of the receptors and for their recycling to the cell surface. Mutations in the beta2AR tail that ablate NSF binding reduce the efficiency of receptor internalization upon agonist stimulation. Upon subsequent treatment of cells with the antagonist propranolol, wild type receptors return to the cell surface, while tail-mutated receptors remain sequestered. Thus, the direct binding of the beta2AR to NSF demonstrates how, after internalization, the fate of a receptor is reliant on a specific interaction with a component of the cellular membrane-trafficking machinery.

Animals↗
PubMed2003

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.

Adenosine Triphosphate↗
PubMed2002

Abnormal expression of pancreatic islet exocytotic soluble N-ethylmaleimide-sensitive factor attachment protein receptors in Goto-Kakizaki rats is partially restored by phlorizin treatment and accentuated by high glucose treatment.

The role of glucotoxicity in dysregulation of islet exocytotic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex proteins and insulin response was explored in the hyperglycemic Goto-Kakizaki (GK) rat. Syntaxin-1A and vesicle-associated membrane protein isoform 2, which drive insulin granule exocytotic fusion, and the associated nSec1, which modulates the SNARE complex assembly, were diminished in GK pancreatic islets to approximately 40% of the levels in control Wistar rat islets. Phlorizin treatment (12 d) induced normoglycemic control in GK rats, resulting in partial restoration of the insulin response to glucose. Furthermore, islet SNARE complex and nSec1 proteins increased by about 40%. Phlorizin treatment did not affect levels of islet SNARE proteins in controls or on the same SNARE complex proteins in GK rat brain. To examine the role of hyperglycemia per se, GK and control rat islets were exposed for 5 d in culture to 5.5 and 16.7 mM glucose. High glucose treatment greatly increased the levels of synaptosomal-associated membrane protein of 25 kDa and, less markedly, the levels of syntaxin-1A and nSec1 in control islets more than in GK rat islets, whereas levels were reduced in both. This was accompanied by sustained impairment of the insulin response to glucose in GK islets and a normal response in control islets. Thus, GK islets demonstrate dysregulation of SNARE protein expression, and their compensatory increase by high glucose exposure is abrogated. Conversely, normoglycemic control results in partial replenishment of these critical components of the insulin exocytotic machinery and improvement in the insulin response. We propose that dysregulation of SNARE proteins is an important mechanism behind glucotoxicity-mediated impairment of the insulin response to glucose.

Animals↗
PubMed1997

Association of N-ethylmaleimide sensitive fusion (NSF) protein and soluble NSF attachment proteins-alpha and -gamma with glucose transporter-4-containing vesicles in primary rat adipocytes.

To investigate the role of N-ethylmaleimide sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAP)-containing fusion complexes in glucose transporter-4 (GLUT4) membrane trafficking, the subcellular distributions of NSF, alpha-SNAP, and gamma-SNAP in primary rat adipocytes were determined. A large fraction of the NSF and SNAPs were associated with intracellular membranes, distributed between the low-density microsomes (LDM) and high-density microsomes. Very little of the NSF and SNAPs were associated with the plasma membrane fraction. This distribution did not change after insulin stimulation. Approximately 75% of the NSF and SNAPs in the LDM fraction were coimmunoprecipitated with 85% of the GLUT4 and 60% of the vesicle associated membrane proteins (VAMPs; synaptobrevins) VAMP-2 and cellubrevin in anti-GLUT4 immunoadsorptions. In contrast to NSF and the SNAPs, the beta-coatomer protein (beta-COP) found in the LDM fraction was excluded from GLUT4 vesicles. When LDM fractions were solubilized with Thesit (octaethylene glycol dodecyl ether) or Triton X-100, approximately 40% of the alpha-SNAP was colocalized with NSF on glycerol gradients in large (approximately 20S), ATP-sensitive complexes. VAMP-2 and cellubrevin are concentrated in the LDM fractions and in GLUT4 vesicles; both were excluded from these complexes. These data suggest that the steady state association of NSF and the SNAPs with GLUT4 vesicles and cell membranes is independent of the formation of fusion complexes.

Adipocytes↗
PubMed2004

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.

Alleles↗
PubMed2003

Mast cell degranulation requires N-ethylmaleimide-sensitive factor-mediated SNARE disassembly.

Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators. A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules with the plasma membrane of mast cells. In addition to the SNAREs themselves, it is likely that the SNARE accessory protein, N-ethylmaleimide-sensitive factor (NSF), affects the composition and structure of the SNARE complex. NSF is a cytoplasmic ATPase that disassembles the SNARE complexes. To investigate the role of NSF in mast cell degranulation, we developed an assay to measure secretion from transiently transfected RBL (rat basophilic leukemia)-2H3 mast cells (a tumor analog of mucosal mast cells). RBL-2H3 cells were cotransfected with a plasmid encoding a human growth hormone secretion reporter along with either wild-type NSF or an NSF mutant that lacks ATPase activity. Human growth hormone was targeted to and released from secretory granules in RBL-2H3 cells, and coexpression with mutant NSF dramatically inhibited regulated exocytosis from the transfected cells. Biochemical analysis of SNARE complexes in these cells revealed that overexpression of the NSF mutant decreased disassembly and resulted in an accumulation of SNARE complexes. These data reveal a role for NSF in mast cell exocytosis and highlight the importance of SNARE disassembly, or priming, in regulated exocytosis from mast cells.

Adenosine Triphosphatases↗
PubMed1994

The ATPase activity of N-ethylmaleimide-sensitive fusion protein (NSF) is regulated by soluble NSF attachment proteins.

N-Ethylmaleimide-sensitive fusion protein (NSF) is an ATPase required in multiple stages of the secretory and endocytic pathways. NSF requires other proteins for its action in vesicular transport including the soluble NSF attachment proteins (SNAPs), which act to bind NSF to integral membrane proteins. We have investigated the ATPase activity of NSF and its modulation by alpha- and gamma-SNAPs using His6-tagged recombinant proteins. His6-NSF possessed ATPase activity, which was enhanced in a dose-dependent manner by immobilized (i.e. plastic-adsorbed) but not soluble His6-tagged SNAPs. NSF displayed complex enzyme kinetics consistent with the possession of two ATPase domains with different affinities for ATP. SNAPs apparently enhanced NSF ATPase activity primarily by decreasing the Km of its low affinity site 100-fold. In vivo this effect would be predicted to sensitize the low affinity site to physiological ATP concentrations. Thus SNAPs could act as a molecular switch by "turning on" the normally dormant low affinity ATPase site of NSF.

Adenosine Triphosphatases↗
PubMed1997

n-ethylmaleimide and ethacrynic acid inhibit kinesin binding to microtubules in a motility assay.

Treatment of proteins in vitro with sulfhydryl (SH)-reactive compounds has been used successfully to determine protein regions critical for normal function. To probe structure-function relationships in the microtubule (MT) motor kinesin, the motor was treated with two SH reactive compounds, n-ethylmaleimide and ethacrynic acid, and its function was assayed by motility and co-sedimentation techniques. In the motility assay, treatment of kinesin either before or after adsorption to the glass surfaces of a flow cell was found to inhibit the ability of coverslip-bound kinesin to bind to MTs. Inactivation of MT binding was slow, required high molar excess of the SH-reactive drug, and was very sensitive to temperature. Inhibition of MT binding occurred well after complete modification of kinesin light chain, but paralleled modification of the kinesin heavy chain. The results point to a model in which one critical cysteine per kinesin heavy chain is relatively inaccessible to solvent. Surprisingly, when the interaction between modified kinesin and MTs was examined by a co-sedimentation assay, kinesin retained the ability to bind MTs. These contrasting results may be due to conformational differences in the kinesin molecule that exist in the two assays.

Animals↗
PubMedDate not supplied

The effects of N-ethylmaleimide on extracellularly and intracellularly generated chemiluminescence in neutrophils indicate that the rate of deactivation of NADPH-oxidase is higher when the oxidase system is localized on the plasma membrane than when it is localized on the phagosomal membrane.

Sustained generation of reactive oxygen metabolites following respiratory burst activation in neutrophils is a result of continued replenishment of a pool of active NADPH-oxidase. The sulphydryl-modifying reagent N-ethylmaleimide (NEM) has been shown to be without effect on the turnover of activated NADPH-oxidase but to inhibit the replenishment of active oxidase molecules (Akard et al., 1988). NEM was thus used to determine the rate of deactivation of extracellularly and intracellularly generated chemiluminescence in human neutrophils. We have shown that deactivation is more rapid when activation leads to a release of oxygen metabolites (extracellular chemiluminescence) than when the metabolites are generated intracellularly. The results indicate that the rate of deactivation of NADPH-oxidase is higher when the oxidase system is localized on the plasma membrane than when it is localized on the phagosomal membrane.

Adult↗