comatose and NEM-sensitive fusion protein 2
Homotypic vacuole fusion in yeast requires Sec18p (NSF), Sec17p (soluble NSF attachment protein [alpha-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). Vacuolar v- and t-SNAREs are mainly found with Sec17p as v-t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v-t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, since the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (1) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release; (2) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (3) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v-t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, alpha-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes (Ungermann, 1998).
SNARE proteins are required for many fusion processes, and recent studies of isolated SNARE proteins reveal that they are inherently capable of fusing lipid bilayers. Cis-SNARE complexes (formed when vesicle SNAREs [v-SNAREs] and target membrane SNAREs [t-SNAREs] combine in the same membrane) are disrupted by the action of the abundant cytoplasmic ATPase NSF, which is necessary to maintain a supply of uncombined v- and t-SNAREs for fusion in cells. Fusion is mediated by these same SNARE proteins, forming trans-SNARE complexes between membranes. This raises an important question: why doesn't NSF disrupt these SNARE complexes as well, preventing fusion from occurring at all? Several lines of evidence demonstrate that SNAREpins (trans-SNARE complexes) are in fact functionally resistant to NSF, and they become so at the moment they form and commit to fusion. This elegant design allows fusion to proceed locally in the face of an overall environment that massively favors SNARE disruption (Weber, 2000).
Homotypic vacuole fusion occurs in ordered stages of priming, docking, and fusion. Priming, which prepares vacuoles for productive association, requires Sec17p (the yeast homolog of alpha-SNAP), Sec18p (the yeast NSF, an ATP-driven chaperone), and ATP. Sec17p is initially an integral part of the cis-SNARE complex together with vacuolar SNARE proteins and Sec18p (NSF). Previous studies have shown that Sec17p is rapidly released from the vacuole membrane during priming as the cis-SNARE complex is disassembled, but the order and causal relationship of these subreactions has not been known. Addition of excess recombinant his(6)-Sec17p to primed vacuoles can block subsequent docking. This inhibition is reversible by Sec18p, but the reaction cannot proceed to the tethering and trans-SNARE pairing steps of docking while the Sec17p block is in place. Once docking has occurred, excess Sec17p does not inhibit membrane fusion per se. Incubation of cells with thermosensitive Sec17-1p at nonpermissive temperature causes SNARE complex disassembly. These data suggest that Sec17p can stabilize vacuolar cis-SNARE complexes and that the release of Sec17p by Sec18p and ATP allows disassembly of this complex and activates its components for docking (Wang, 2000).
In yeast, assembly of exocytic SNARE complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs (Ssop and Sec9p) occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane (Grote, 2000).
N-ethylmaleimide-sensitive fusion protein (NSF) and alpha-SNAP play key roles in vesicular traffic through the secretory pathway. NSF is able to associate with Golgi membranes in an ATP-dependent fashion. This association is dependent on three peripheral membrane proteins, termed soluble NSF attachment proteins (SNAPs). alpha-SNAP and NSF are associated in a 20S complex with three membrane proteins: syntaxin, SNAP-25 (synaptosomal associated protein of 25 kD) and vesicle-associate membrane protein (VAMP), collectively termed SNAP receptors (SNAREs). In this study, NH2- and COOH-terminal truncation mutants of alpha-SNAP were assayed for the ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH2-terminal amino acids has little effect on the ability of alpha-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH-terminal amino acids results in a marked decrease. Both NH2-terminal (1-160) and COOH-terminal (160-295) fragments of alpha-SNAP are able to bind to NSF, suggesting that alpha-SNAP contains distinct NH2- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs reveals only leucine 294 to be conserved in the final 10 amino acids of alpha-SNAP. Mutation of leucine 294 to alanine [alpha-SNAP(L294A)] results in a decrease in the ability to stimulate NSF ATPase activity but has no effect on the ability of this mutant to bind NSF. alpha-SNAP (1-285) and alpha-SNAP (L294A) are unable to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells. In addition, alpha-SNAP (1-285) and alpha-SNAP (L294A) are able to inhibit the stimulation of exocytosis by exogenous alpha-SNAP. alpha-SNAP, alpha-SNAP (1-285), and alpha-SNAP (L294A) are all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, alpha-SNAP (1-285) and alpha-SNAP (L294A) are unable to fully disassemble the 20S complex and do not allow vesicle-associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that alpha-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the alpha-SNAP stimulation of exocytosis (Barnard, 1997).
Critical to SNARE protein function in neurotransmission are the accessory proteins, soluble NSF attachment protein (SNAP), and NSF, that play a role in activation of the SNAREs for membrane fusion. The depolarization-induced, calcium-dependent phosphorylation of NSF in rat synaptosomes is reported. Phosphorylation of NSF is coincident with neurotransmitter release and requires an influx of external calcium. Phosphoamino acid analysis of the radiolabeled NSF indicates a role for a serine/threonine-specific kinase. Synaptosomal phosphorylation of NSF is stimulated by phorbol esters and is inhibited by staurosporine, chelerythrine, bisindolylmaleimide I, calphostin C, and Ro31-8220 but not the calmodulin kinase II inhibitor, Kn-93, suggesting a role for protein kinase C (PKC). Indeed, NSF is phosphorylated by PKC in vitro at Ser-237 of the catalytic D1 domain. Mutation of this residue to glutamic acid or to alanine eliminates in vitro phosphorylation. Molecular modeling studies suggest that Ser-237 is adjacent to an inter-subunit interface at a position where its phosphorylation could affect NSF activity. Consistently, mutation of Ser-237 to Glu, to mimic phosphorylation, results in a hexameric form of NSF that does not bind to SNAP-SNARE complexes, whereas the S237A mutant does form complex. These data suggest a negative regulatory role for PKC phosphorylation of NSF (Matveeva, 2001).
An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum-Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure-function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr(394)-->Pro). Thr(394) is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast alpha-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts (Steel, 2000).
Specific interaction has been demonstrated between the GluR2 (AMPA) receptor subunit C-terminal peptide (see Drosophila Glutamate receptor IIA and Glutamate receptor IIB), an ATPase N-ethylmaleimide-sensitive fusion protein (NSF), and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as the dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. This paper provides evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. A model is proposed in which NSF functions as a chaperone in the molecular processing of the AMPA receptor (Osten, 1998).
The findings that NSF and alpha- and beta-SNAPs interact with GluR2 in a complex, which in several respects resembles the interaction of NSF and SNAP at the SNARE, can be interpreted to support a functional model of the GluR2-NSF-SNAP binding. In this model, the NSF-SNAP complex is required in chaperone-like priming of the AMPA receptors during a continuous process required for receptor function. This process could involve receptor recycling between the postsynaptic membrane and a cytoplasmic pool. As has been proposed for NSF function at the SNARE complexes, the interaction of NSF and SNAP with the AMPA receptor could involve the disruption of multiprotein complexes, such as those formed between the membrane-inserted receptor and the proteins of the postsynaptic density (such as GRIP). NSF-driven disassembly of these complexes could be required for the proper sorting of these proteins at specific times during development, as for example, prior to a new cycle of insertion and anchoring, or in the processing of newly synthesized receptors (Osten, 1998 and references).
NSF interacts directly and selectively with the intracellular C-terminal domain of the GluR2 subunit of AMPA receptors. The interaction requires all three domains of NSF but occurs between residues Lys-844 and Gln-853 of rat GluR2, with Asn-851 playing a critical role. Loading of decapeptides corresponding to the NSF-binding domain of GluR2 into rat hippocampal CA1 pyramidal neurons results in a marked, progressive decrement of AMPA receptor-mediated synaptic transmission. This reduction in synaptic transmission is also observed when an anti-NSF monoclonal antibody (mAb) is loaded into CA1 neurons. These results demonstrate a previously unsuspected direct interaction in the postsynaptic neuron between two major proteins involved in synaptic transmission and suggest a rapid NSF-dependent modulation of AMPA receptor function (Nishimune, 1998).
NSF specifically interacts with the C terminus of the GluR2 and GluR4c subunits of AMPA receptors in vitro and in vivo. Moreover, intracellular perfusion of neurons with a synthetic peptide that competes with the interaction of NSF and AMPA receptor subunits rapidly decreases the amplitude of miniature excitatory postsynaptic currents (mEPSCs), suggesting that NSF regulates AMPA receptor function (Song, 1998).
Disruption of NSF-GluR2 interaction by infusion into cultured hippocampal neurons of a blocking peptide (pep2m) causes a rapid decrease in the frequency but no change in the amplitude of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs). NMDA receptor-mediated mEPSCs were not changed. Viral expression of pep2m reduces the surface expression of AMPA receptors, whereas NMDA receptor surface expression in the same living cells is unchanged. In permeabilized neurons, the total amount of GluR2 immunoreactivity is unchanged, and a punctate distribution of GluR2 is observed throughout the dendritic tree. These data suggest that the NSF-GluR2 interaction is required for the surface expression of GluR2-containing AMPA receptors and that disruption of the interaction leads to the functional elimination of AMPA receptors at synapses. Based on these findings and the known properties of NSF, a model is favored in which the interaction between NSF and GluR2 is involved in the part of the cycling process that is necessary for the insertion and/or stabilization of AMPA receptors at the postsynaptic membrane. By analogy with its known presynaptic functions, NSF could act at the AMPA receptor complex by stripping the receptors of associated proteins. Candidate proteins interacting with GluR2 include the PDZ-containing proteins GRIP, ABP, and PICK1. Removal of associated proteins could prime or "reset" the AMPA receptor complex to a naive state, thereby allowing insertion into the postsynaptic membrane. If the action of NSF is prevented, for example, by peptide block, the receptors cannot be appropriately processed, and insertion/reinsertion of the reconfigured receptors into the postsynaptic membrane cannot occur (Noel, 1999).
Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with NSF causes a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis causes a gradual increase in the AMPAR EPSC. These manipulations have no effect on the NMDAR EPSC but prevent the subsequent induction of LTD. These results suggest that AMPARs, but not NMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process (Luscher, 1999).
An investigation was carried out to see whether the interaction between the N-ethyl-maleimide-sensitive fusion protein (NSF) and the AMPA receptor (AMPAR) subunit GluR2 is involved in synaptic plasticity in the CA1 region of the hippocampus. Blockade of the NSF-GluR2 interaction by a specific peptide (pep2m) introduced into neurons prevents homosynaptic, de novo long-term depression (LTD). Moreover, saturation of LTD prevents the pep2m-induced reduction in AMPAR-mediated excitatory postsynaptic currents (EPSCs). Minimal stimulation experiments indicated that both pep2m action and LTD are due to changes in quantal size and quantal content but are not associated with changes in AMPAR single-channel conductance or EPSC kinetics. These results suggest that there is a pool of AMPARs dependent on the NSF-GluR2 interaction and that LTD expression involves the removal of these receptors from synapses (Luthi, 1999).
AMPA receptor (AMPAR) trafficking is crucial for synaptic plasticity, which may be important for learning and memory. NSF and PICK1 bind the AMPAR GluR2 subunit and are involved in trafficking of AMPARs. GluR2, PICK1, NSF, and alpha-/beta-SNAPs form a complex in the presence of ATPgammaS. Similar to SNARE complex disassembly, NSF ATPase activity disrupts PICK1-GluR2 interactions in this complex. Alpha- and beta-SNAP have differential effects on this reaction. SNAP overexpression in hippocampal neurons leads to corresponding changes in AMPAR trafficking by acting on GluR2-PICK1 complexes. This demonstrates that the previously reported synaptic stabilization of AMPARs by NSF involves disruption of GluR2-PICK1 interactions (Hanley, 2002).
AMPAR trafficking is thought to involve constitutive cycling of receptors by endocytosis/exocytosis, as well as regulated events as part of LTD (endocytosis) and LTP (exocytosis). AMPAR endocytosis during some forms of LTD is dependent upon GluR2 phosphorylation and regulation of accessory protein binding. The NSF-mediated disassembly of the GluR2-PICK1 complex described in this study is therefore likely to be crucial in limiting endocytosis of AMPARs to maintain constitutive cycling at a constant rate and hence maintain a constant level of receptors at the synaptic membrane. From this baseline, LTD could be induced (in conjunction with phosphorylation events) by reducing the activity of NSF, possibly by modulation of SNAP-PICK1 binding, to stabilize GluR2-PICK1 interactions, and consequently enhance receptor endocytosis. This study has identified the molecular mechanism for the activity of NSF in AMPA receptor trafficking, and has demonstrated that NSF can function as a disassembling molecular chaperone in a protein complex other than the 20S SNARE complex. As additional NSF binding partners are identified, it is possible that this ATPase, previously thought to be faithful to the SNARE complex, will show more promiscuous chaperone behavior (Hanley, 2002).
The precise biochemical role of N-ethylmaleimide-sensitive factor (NSF) in membrane fusion mediated by SNARE proteins is unclear. To provide further insight into the function of NSF, a mutation was introduced into mammalian NSF that, in Drosophila NSF-1, leads to temperature-sensitive neuroparalysis. This mutation is like the comatose mutation and renders the mammalian NSF temperature sensitive for fusion of postmitotic Golgi vesicles and tubules into intact cisternae. Unexpectedly, at the temperature that is permissive for membrane fusion, this mutant NSF binds to, but cannot disassemble, SNARE complexes and exhibits almost no ATPase activity. A well-charaterized NSF mutant containing an inactivating point mutation in the catalytic site of its ATPase domain is equally active in the Golgi-reassembly assay. These data indicate that the need for NSF during postmitotic Golgi membrane fusion may be distinct from its ATPase-dependent ability to break up SNARE pairs (Muller, 1999).
Results obtained with the comatose-like mutant were substantiated by using a distinct mutant, NSF(E329Q), that is defective in ATPase activity and SNARE disassembly. This mutation results in a ~75% reduction in ATPase activity, which completely abolishes its ability to stimulate fusion in an intra-Golgi transport assay. These results indicate a positive correlation between the membrane-fusion-promoting function of NSF and its ATPase activity. In contrast, the ATPase-defective NSF (E329Q), like NSF(G274E), is capable of promoting cisternal regrowth to ~80% of the level of wild-type NSF. Together, data obtained using the mutant NSF proteins indicate that NSF's ATPase activity may not be directly linked to postmitotic Golgi membrane fusion (Muller, 1999).
Since NSF(G274E) and NSF(E329Q) both lack the ability to break up SNARE complexes, NSF-dependent SNARE disassembly seems to be uncoupled from membrane fusion of postmitotic Golgi fragments. The break-up of SNARE complexes is thought to be essential for the recycling of these proteins for further rounds of fusion; thus, these data indicate that recycling may not be needed for Golgi reassembly in the cell-free assay. On the basis of current models, it is therefore predicted that there is an abundant source of disassembled SNAREs on mitotic Golgi fragments before reassembly. If true, this could help to explain the discrepancy in the requirement for NSF's ATPase activity in other published membrane-fusion assays but not during mitotic Golgi reassembly (Muller, 1999).
However, this leaves open the nature of the distinct role for NSF during the membrane-fusion process. Interestingly, assembly of synaptic 20S complexes is temperature sensitive in the presence of the NSF mutant, which indicates that the presence of NSF in a SNARE complex might be critical. One possibility is that NSF is needed to prime (for example, by folding or assisting in accessory-factor recruitment) the SNAREs on MGFs in preparation for fusion. Although priming of vacuole and, perhaps, Golgi SNAREs correlates with the presence of Mg-ATP, it is not clear that ATP hydrolysis is needed for this process. Another possibility is that a checkpoint exists to ensure that NSF is recruited to the fusion site in preparation for its later function in breaking up SNARE complexes. This would certainly explain why NSF has been found on synaptic and clathrin-coated vesicles that still have to dock and fuse. Such recruitment would target NSF to the site at which its action will be needed. A final possibility is that NSF takes part in the actual fusion process itself. It is, therefore, interesting that NSF-SNAPs can directly fuse liposomes together in an ATP-dependent manner. Furthermore, this happens most efficiently when the NSF-SNAP complex has the lowest ATPase activity, the key feature of the NSF mutant (Muller, 1999).
In conclusion, these studies of the Drosophila comatose analog in mammalian NSF provide clear evidence that NSF has a role in membrane fusion that is divorced from its ability to break up SNARE complexes. The likelihood is that NSF has multiple roles and further structure/function studies should provide the means for their dissection (Muller, 1999).
Characterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants reveals an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that is essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments reveals that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/alpha-SNAP catalyzes the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. It is suggested that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function (Muller, 2002).
Myelinated axons are essential for rapid conduction of action potentials in the vertebrate nervous system. Of particular importance are the nodes of Ranvier, sites of voltage-gated sodium channel clustering that allow action potentials to be propagated along myelinated axons by saltatory conduction. Despite their critical role in the function of myelinated axons, little is known about the mechanisms that organize the nodes of Ranvier. Starting with a forward genetic screen in zebrafish, an essential requirement for nsf (N-ethylmaleimide sensitive factor) was identified in the organization of myelinated axons. Previous work has shown that NSF is essential for membrane fusion in eukaryotes and has a critical role in vesicle fusion at chemical synapses. Zebrafish nsf mutants are paralyzed and have impaired response to light, reflecting disrupted nsf function in synaptic transmission and neural activity. In addition, nsf mutants exhibit defects in Myelin basic protein expression and in localization of sodium channel proteins at nodes of Ranvier. Analysis of chimeric larvae indicates that nsf functions autonomously in neurons, such that sodium channel clusters are evident in wild-type neurons transplanted into the nsf mutant hosts. Through pharmacological analyses, it has been shown that neural activity and function of chemical synapses are not required for sodium channel clustering and myelination in the larval nervous system. It is concluded that zebrafish nsf mutants provide a novel vertebrate system to investigate Nsf function in vivo. These results reveal a previously unknown role for nsf, independent of its function in synaptic vesicle fusion, in the formation of the nodes of Ranvier in the vertebrate nervous system (Woods, 2006).
Microarray expression profiling of prefrontal cortex from matched pairs of schizophrenic and control subjects and hierarchical data analysis reveals that transcripts encoding proteins involved in the regulation of presynaptic function (PSYN) were decreased in all subjects with schizophrenia. Genes of the PSYN group showed a different combination of decreased expression across subjects. Over 250 other gene groups did not show altered expression. Selected PSYN microarray observations were verified by in situ hybridization. Two of the most consistently changed transcripts in the PSYN functional gene group, N-ethylmaleimide sensitive factor and synapsin II, were decreased in ten of ten and nine of ten subjects with schizophrenia, respectively. The combined data suggest that subjects with schizophrenia share a common abnormality in presynaptic function (Mirnics, 2000).
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