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Axel T Brunger - One of the best experts on this subject based on the ideXlab platform.
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recent advances in deciphering the structure and molecular mechanism of the aaa atpase n ethylmaleimide Sensitive Factor nsf
Journal of Molecular Biology, 2016Co-Authors: Minglei Zhao, Axel T BrungerAbstract:N-ethylmaleimide-Sensitive Factor (NSF), first discovered in 1988, is a key Factor for eukaryotic trafficking, including protein and hormone secretion and neurotransmitter release. It is a member of the AAA+ family (ATPases associated with diverse cellular activities). NSF disassembles soluble N-ethylmaleimide-Sensitive Factor attachment protein receptor (SNARE) complexes in conjunction with soluble N-ethylmaleimide-Sensitive Factor attachment protein (SNAP). Structural studies of NSF and its complex with SNAREs and SNAPs (known as 20S supercomplex) started about 20years ago. Crystal structures of individual N and D2 domains of NSF and low-resolution electron microscopy structures of full-length NSF and 20S supercomplex have been reported over the years. Nevertheless, the molecular architecture of the 20S supercomplex and the molecular mechanism of NSF-mediated SNARE complex disassembly remained unclear until recently. Here we review recent atomic-resolution or near-atomic resolution structures of NSF and of the 20S supercomplex, as well as recent insights into the molecular mechanism and energy requirements of NSF. We also compare NSF with other known AAA+ family members.
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disassembly of all snare complexes by n ethylmaleimide Sensitive Factor nsf is initiated by a conserved 1 1 interaction between α soluble nsf attachment protein snap and snare complex
Journal of Biological Chemistry, 2013Co-Authors: Sandro Vivona, Daniel J Cipriano, Sean Oleary, Timothy D Fenn, Axel T BrungerAbstract:Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-Sensitive Factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.
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processive atp driven substrate disassembly by the n ethylmaleimide Sensitive Factor nsf molecular machine
Journal of Biological Chemistry, 2013Co-Authors: Daniel J Cipriano, Axel T Brunger, Sandro Vivona, Timothy D Fenn, Jaemyeong Jung, Zev BryantAbstract:Abstract SNARE proteins promote membrane fusion by forming a four-stranded parallel helical bundle that brings the membranes into close proximity. Post-fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide-Sensitive Factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ∼26-fold. One SNARE complex takes an average of ∼5 s to disassemble in a process that consumes ∼50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled-coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.
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processive atp driven substrate disassembly by the n ethylmaleimide Sensitive Factor nsf molecular machine
Journal of Biological Chemistry, 2013Co-Authors: Axel T Brunger, Sandro Vivona, Daniel J Cipriano, Timothy D Fenn, Jaemyeong Jung, Zev BryantAbstract:SNARE proteins promote membrane fusion by forming a four-stranded parallel helical bundle that brings the membranes into close proximity. Post-fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide-Sensitive Factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ∼26-fold. One SNARE complex takes an average of ∼5 s to disassemble in a process that consumes ∼50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled-coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule. Background: NSF is an AAA+ protein that recycles the post-fusion SNARE complex during the membrane fusion cycle. Results: NSF disassembles the SNARE complex processively in vitro and consumes dozens of ATP molecules per SNARE. Conclusion: NSF is a processive motor that progressively unwinds the SNARE complex. Significance: The physical mechanism of this poorly understood machine is illuminated using new assays and new measurements of ATP coupling ratios and processivity.
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single molecule observation of liposome bilayer fusion thermally induced by soluble n ethyl maleimide Sensitive Factor attachment protein receptors snares
Biophysical Journal, 2004Co-Authors: Mark E Bowen, Axel T Brunger, Keith Weninger, Steven ChuAbstract:A single molecule fluorescence assay is presented for studying the mechanism of soluble N-ethyl maleimide Sensitive-Factor attachment protein receptors (SNAREs)-mediated liposome fusion to supported lipid bilayers. The three neuronal SNAREs syntaxin-1A, synaptobrevin-II (VAMP), and SNAP-25A were expressed separately, and various dye-labeled combinations of the SNAREs were tested for their ability to dock liposomes and induce fusion. Syntaxin and synaptobrevin in opposing membranes were both necessary and sufficient to dock liposomes to supported bilayers and to induce thermally activated fusion. As little as one SNARE interaction was sufficient for liposome docking. Fusion of docked liposomes with the supported bilayer was monitored by the dequenching of soluble fluorophores entrapped within the liposomes. Fusion was stimulated by illumination with laser light, and the fusion probability was enhanced by raising the ambient temperature from 22 to 37°C, suggesting a thermally activated process. Surprisingly, SNAP-25 had little effect on docking efficiency or the probability of thermally induced fusion. Interprotein fluorescence resonance energy transfer experiments suggest the presence of other conformational states of the syntaxin•synaptobrevin interaction in addition to those observed in the crystal structure of the SNARE complex. Furthermore, although SNARE complexes involved in liposome docking preferentially assemble into a parallel configuration, both parallel and antiparallel configurations were observed.
Sidney W Whiteheart - One of the best experts on this subject based on the ideXlab platform.
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nucleotide dependent conformational changes in the n ethylmaleimide Sensitive Factor nsf and their potential role in snare complex disassembly
Journal of Structural Biology, 2012Co-Authors: Arne Moeller, Chunxia Zhao, Michael Fried, Elizabeth M Wilsonkubalek, Bridget Carragher, Sidney W WhiteheartAbstract:Homohexameric, N-Ethylmaleimide Sensitive Factor (NSF) disassembles Soluble NSF Attachment Protein Receptor (SNARE) complexes after membrane fusion, an essential step in vesicular trafficking. NSF contains three domains (NSF-N, NSF-D1, and NSF-D2), each contributing to activity. We combined electron microscopic (EM) analysis, analytical ultracentrifugation (AU) and functional mutagenesis to visualize NSF’s ATPase cycle. 3D density maps show that NSF-D2 remains stable, whereas NSF-N undergoes large conformational changes. NSF-Ns splay out perpendicular to the ADP-bound hexamer and twist upwards upon ATP binding, producing a more compact structure. These conformations were confirmed by hydrodynamic, AU measurements: NSF-ATP sediments faster with a lower frictional ratio (f/f0). Hydrodynamic analyses of NSF mutants, with specific functional defects, define the structures underlying these conformational changes. Mapping mutations onto our 3D models allows interpretation of the domain movement and suggests a mechanism for NSF binding to and disassembly of SNARE complexes.
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requirements for the catalytic cycle of the n ethylmaleimide Sensitive Factor nsf
Biochimica et Biophysica Acta, 2012Co-Authors: Chunxia Zhao, Everett Clinton Smith, Sidney W WhiteheartAbstract:Abstract The N-ethylmaleimide-Sensitive Factor (NSF) was one of the initial members of the ATPases Associated with various cellular Activities Plus (AAA+) family. In this review, we discuss what is known about the mechanism of NSF action and how that relates to the mechanisms of other AAA+ proteins. Like other family members, NSF binds to a protein complex (i.e., SNAP–SNARE complex) and utilizes ATP hydrolysis to affect the conformations of that complex. SNAP–SNARE complex disassembly is essential for SNARE recycling and sustained membrane trafficking. NSF is a homo-hexamer; each protomer is composed of an N-terminal domain, NSF-N, and two adjacent AAA-domains, NSF-D1 and NSF-D2. Mutagenesis analysis has established specific roles for many of the structural elements of NSF-D1, the catalytic ATPase domain, and NSF-N, the SNAP–SNARE binding domain. Hydrodynamic analysis of NSF, labeled with (Ni2+-NTA)2-Cy3, detected conformational differences in NSF, in which the ATP-bound conformation appears more compact than the ADP-bound form. This indicates that NSF undergoes significant conformational changes as it progresses through its ATP-hydrolysis cycle. Incorporating these data, we propose a sequential mechanism by which NSF uses NSF-N and NSF-D1 to disassemble SNAP–SNARE complexes. We also illustrate how analytical centrifugation might be used to study other AAA+ proteins. This article is part of a Special Issue entitled: AAA ATPases: structure and function.
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dissecting the n ethylmaleimide Sensitive Factor required elements of the n and d1 domains
Journal of Biological Chemistry, 2010Co-Authors: Chunxia Zhao, Elena A Matveeva, Qiansheng Ren, Sidney W WhiteheartAbstract:N-Ethylmaleimide-Sensitive Factor (NSF) is a homo-hexameric member of the AAA+ (ATPases associated with various cellular activities plus) family. It plays an essential role in most intracellular membrane trafficking through its binding to and disassembly of soluble NSF attachment protein (SNAP) receptor (SNARE) complexes. Each NSF protomer contains an N-terminal domain (NSF-N) and two AAA domains, a catalytic NSF-D1 and a structural NSF-D2. This study presents detailed mutagenesis analyses of NSF-N and NSF-D1, dissecting their roles in ATP hydrolysis, SNAP·SNARE binding, and complex disassembly. Our results show that a positively charged surface on NSF-N, bounded by Arg67 and Lys105, and the conserved residues in the central pore of NSF-D1 (Tyr296 and Gly298) are involved in SNAP·SNARE binding but not basal ATP hydrolysis. Mutagenesis of Sensor 1 (Thr373–Arg375), Sensor 2 (Glu440–Glu442), and Arginine Fingers (Arg385 and Arg388) in NSF-D1 shows that each region plays a discrete role. Sensor 1 is important for basal ATPase activity and nucleotide binding. Sensor 2 plays a role in ATP- and SNAP-dependent SNARE complex binding and disassembly but does so in cis and not through inter-protomer interactions. Arginine Fingers are important for SNAP·SNARE complex-stimulated ATPase activity and complex disassembly. Mutants at these residues have a dominant-negative phenotype in cells, suggesting that Arginine Fingers function in trans via inter-protomer interactions. Taken together, these data establish functional roles for many of the structural elements of the N domain and of the D1 ATP-binding site of NSF.
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type i pdz ligands are sufficient to promote rapid recycling of g protein coupled receptors independent of binding to n ethylmaleimide Sensitive Factor
Journal of Biological Chemistry, 2005Co-Authors: Robert M Gage, Elena A Matveeva, Sidney W Whiteheart, Mark Von ZastrowAbstract:Abstract Molecular sorting of G protein-coupled receptors (GPCRs) between divergent recycling and lysosomal pathways determines the functional consequences of agonist-induced endocytosis. The carboxyl-terminal cytoplasmic domain of the β2 adrenergic receptor (β2AR) mediates both PDZ binding to Na+/H+ exchanger regulatory Factor/ezrin/radixin/moesin-binding phosphoprotein of 50 kDa (NHERF/EBP50) family proteins and non-PDZ binding to the N-ethylmaleimide-Sensitive Factor (NSF). We have investigated whether PDZ interaction(s) are actually sufficient to promote rapid recycling of endocytosed receptors and, if so, whether PDZ-mediated sorting is restricted to the β2AR tail or to sequences that bind NHERF/EBP50. The trafficking effects of short (10 residue) sequences differing in PDZ and NSF binding properties were examined using chimeric mutant receptors. The recycling activity of the β2AR-derived tail sequence was not blocked by a point mutation that selectively disrupts binding to NSF, and naturally occurring PDZ ligand sequences were identified that do not bind detectably to NSF yet function as strong recycling signals. The carboxyl-terminal cytoplasmic domain of the β1-adrenergic receptor, which does not bind either to NSF or NHERF/EBP50 and interacts selectively with a distinct group of PDZ proteins, promoted rapid recycling of chimeric mutant receptors with efficiency similarly high as that of the β2AR tail. These results indicate that PDZ domain-mediated protein interactions are sufficient to promote rapid recycling of GPCRs, independent of binding to NSF. They also suggest that PDZ-directed recycling is a rather general mechanism of GPCR regulation, which is not restricted to a single GPCR, and may involve additional PDZ domain-containing protein(s) besides NHERF/EBP50.
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multiple binding proteins suggest diverse functions for the n ethylmaleimide Sensitive Factor
Journal of Structural Biology, 2004Co-Authors: Sidney W Whiteheart, Elena A MatveevaAbstract: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.
Heidi E Hamm - One of the best experts on this subject based on the ideXlab platform.
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G␥ Interferes with Ca 2ϩ -Dependent Binding of Synaptotagmin to the Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor (SNARE) Complex
2020Co-Authors: □ Eun-ja S Yoon, Simon Alford, Tatyana Gerachshenko, Bryan D Spiegelberg, Heidi E HammAbstract:ABSTRACT Presynaptic inhibitory G protein-coupled receptors (GPCRs) can decrease neurotransmission by inducing interaction of G␥ with the soluble N-ethylmaleimide-Sensitive Factor attachment protein receptor (SNARE) complex. We have shown that this action of G␥ requires the carboxyl terminus of the 25-kDa synaptosomeassociated protein (SNAP25) and is downstream of the well known inhibition of Ca 2ϩ entry through voltage-gated calcium channels. We propose a mechanism in which G␥ and synaptotagmin compete for binding to the SNARE complex. Here, we characterized the G␥ interaction sites on syntaxin1A and SNAP25 and demonstrated an overlap of the G␥-and synaptotagmin I -binding regions on each member of the SNARE complex. Synaptotagmin competes in a Ca 2ϩ -Sensitive manner with binding of G␥ to SNAP25, syntaxin1A, and the assembled SNARE complex. We predict, based on these findings, that at high intracellular Ca 2ϩ concentrations, Ca 2ϩ -synaptotagmin I can displace G␥ binding and the G␥-dependent inhibition of exocytosis can be blocked. We tested this hypothesis in giant synapses of the lamprey spinal cord, where 5-HT works via G␥ to inhibit neurotransmission . We showed that increased presynaptic Ca 2ϩ suppresses the 5-HT-and G␥-dependent inhibition of exocytosis. We suggest that this effect may be due to Ca 2
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gβγ inhibits exocytosis via interaction with critical residues on soluble n ethylmaleimide Sensitive Factor attachment protein 25
Molecular Pharmacology, 2012Co-Authors: Christopher A Wells, Zack Zurawski, Katherine M Betke, Yun Young Yim, Karren Hyde, Shelagh Rodriguez, Simon Alford, Heidi E HammAbstract:Spatial and temporal regulation of neurotransmitter release is a complex process accomplished by the exocytotic machinery working in tandem with numerous regulatory proteins. G-protein βγ dimers regulate the core process of exocytosis by interacting with the soluble N-ethylmaleimide-Sensitive Factor attachment protein receptor (SNARE) proteins soluble N-ethylmaleimide-Sensitive Factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. Gβγ binding to ternary SNAREs overlaps with calcium-dependent binding of synaptotagmin, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of Gβγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gβγ binding. Peptides that bound Gβγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gβγ-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gβγ with SNAP-25. A peptide from the C terminus, residues 193 to 206, significantly inhibited the interaction. In addition, Ala mutants of SNAP-25 residues from the C terminus of SNAP-25, as well as from the amino-terminal region decreased binding to Gβ1γ1. When SNAP-25 with eight residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for Gβγ, but it still interacted with synaptotagmin-1 in a Ca2+-dependent manner and reconstituted evoked exocytosis in botulinum neurotoxin E-treated neurons. However, the mutant SNAP-25 could no longer support 5-hydroxytryptamine-mediated inhibition of exocytosis.
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gβγ interferes with ca2 dependent binding of synaptotagmin to the soluble n ethylmaleimide Sensitive Factor attachment protein receptor snare complex
Molecular Pharmacology, 2007Co-Authors: Eun Ja Yoon, Simon Alford, Tatyana Gerachshenko, Bryan D Spiegelberg, Heidi E HammAbstract:Presynaptic inhibitory G protein-coupled receptors (GPCRs) can decrease neurotransmission by inducing interaction of Gbetagamma with the soluble N-ethylmaleimide-Sensitive Factor attachment protein receptor (SNARE) complex. We have shown that this action of Gbetagamma requires the carboxyl terminus of the 25-kDa synaptosome-associated protein (SNAP25) and is downstream of the well known inhibition of Ca2+ entry through voltage-gated calcium channels. We propose a mechanism in which Gbetagamma and synaptotagmin compete for binding to the SNARE complex. Here, we characterized the Gbetagamma interaction sites on syntaxin1A and SNAP25 and demonstrated an overlap of the Gbetagamma- and synaptotagmin I -binding regions on each member of the SNARE complex. Synaptotagmin competes in a Ca2+-Sensitive manner with binding of Gbetagamma to SNAP25, syntaxin1A, and the assembled SNARE complex. We predict, based on these findings, that at high intracellular Ca2+ concentrations, Ca2+-synaptotagmin I can displace Gbetagamma binding and the Gbetagamma-dependent inhibition of exocytosis can be blocked. We tested this hypothesis in giant synapses of the lamprey spinal cord, where 5-HT works via Gbetagamma to inhibit neurotransmission (Blackmer et al., 2001). We showed that increased presynaptic Ca2+ suppresses the 5-HT- and Gbetagamma-dependent inhibition of exocytosis. We suggest that this effect may be due to Ca2+-dependent competition between Gbetagamma and synaptotagmin I for SNARE binding. This type of dynamic regulation may represent a novel mechanism for modifying transmitter release in a graded manner based on the history of action potentials that increase intracellular Ca2+ concentrations and of inhibitory signals through G(i)-coupled GPCRs.
Richard L Huganir - One of the best experts on this subject based on the ideXlab platform.
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s nitrosylation of n ethylmaleimide Sensitive Factor mediates surface expression of ampa receptors
Neuron, 2005Co-Authors: Yunfei Huang, Charles J. Lowenstein, Richard L Huganir, Hengye Man, Yoko Sekineaizawa, Yefei Han, Krishna R Juluri, Hongbo R Luo, Jaime Cheah, Solomon H SnyderAbstract:Postsynaptic AMPA receptor (AMPAR) trafficking mediates some forms of synaptic plasticity that are modulated by NMDA receptor (NMDAR) activation and N-ethylmaleimide Sensitive Factor (NSF). We report that NSF is physiologically S-nitrosylated by endogenous, neuronally derived nitric oxide (NO). S-nitrosylation of NSF augments its binding to the AMPAR GluR2 subunit. Surface insertion of GluR2 in response to activation of synaptic NMDARs requires endogenous NO, acting selectively upon the binding of NSF to GluR2. Thus, AMPAR recycling elicited by NMDA neurotransmission is mediated by a cascade involving NMDA activation of neuronal NO synthase to form NO, leading to S-nitrosylation of NSF which is thereby activated, enabling it to bind to GluR2 and promote the receptor's surface expression.
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n ethylmaleimide Sensitive Factor is required for the synaptic incorporation and removal of ampa receptors during cerebellar long term depression
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Jordan P Steinberg, Richard L Huganir, David J LindenAbstract:Cerebellar long-term depression (LTD) is a persistent attenuation of synaptic transmission at the parallel fiber–Purkinje cell synapse mediated by the removal of GluR2 subunit-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The removal of AMPA receptors requires protein kinase C phosphorylation of the GluR2 subunit within its carboxyl-terminal PSD-95/Discs Large/Zona Occludens-1 (PDZ) ligand and binding of the PDZ domain-containing protein, PICK1. The sequence of the GluR2 subunit is similar to that of the GluR3 and GluR4c subunits, which also contain PDZ ligands and protein kinase C consensus sites. Although GluR3 and GluR4c are also expressed in Purkinje cells, we have previously shown that cerebellar LTD is absent in GluR2–/– mice, suggesting that these subunits are unable to substitute functionally for GluR2. Here, we examine the apparent difference in the regulation of these AMPA receptor subunits by attempting to rescue LTD in GluR2–/– Purkinje cells with WT and mutant GluR2 and GluR3 subunits. Our results show that the selective interaction of the GluR2 subunit with the N-ethylmaleimide-Sensitive Factor protein is required for synaptic, but not extrasynaptic, incorporation of AMPA receptors as well as for their competence to undergo LTD. In addition, perfusion of a synthetic peptide that acutely disrupts the interaction of GluR2 with N-ethylmaleimide-Sensitive Factor selectively depletes GluR2-containing receptors from synapses and occludes LTD. These findings demonstrate that interaction of AMPA receptors with N-ethylmaleimide-Sensitive Factor plays a critical role in incorporation of AMPA receptors into synapses and for their subsequent removal during cerebellar LTD.
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interaction of the n ethylmaleimide Sensitive Factor with ampa receptors
Neuron, 1998Co-Authors: Insuk Song, Sunjeev K Kamboj, Jun Xia, Hualing Dong, Dezhi Liao, Richard L HuganirAbstract:Glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system (CNS) and play important roles in synaptic plasticity and neuronal development. Recently, protein-protein interactions with the C-terminal domain of glutamate receptor subunits have been shown to be involved in the modulation of receptor function and clustering at excitatory synapses. In this paper, we have found that the N-ethylmaleimide-Sensitive Factor (NSF), a protein involved in membrane fusion events, 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.
Daniel J Cipriano - One of the best experts on this subject based on the ideXlab platform.
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disassembly of all snare complexes by n ethylmaleimide Sensitive Factor nsf is initiated by a conserved 1 1 interaction between α soluble nsf attachment protein snap and snare complex
Journal of Biological Chemistry, 2013Co-Authors: Sandro Vivona, Daniel J Cipriano, Sean Oleary, Timothy D Fenn, Axel T BrungerAbstract:Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-Sensitive Factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.
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processive atp driven substrate disassembly by the n ethylmaleimide Sensitive Factor nsf molecular machine
Journal of Biological Chemistry, 2013Co-Authors: Daniel J Cipriano, Axel T Brunger, Sandro Vivona, Timothy D Fenn, Jaemyeong Jung, Zev BryantAbstract:Abstract SNARE proteins promote membrane fusion by forming a four-stranded parallel helical bundle that brings the membranes into close proximity. Post-fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide-Sensitive Factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ∼26-fold. One SNARE complex takes an average of ∼5 s to disassemble in a process that consumes ∼50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled-coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.
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processive atp driven substrate disassembly by the n ethylmaleimide Sensitive Factor nsf molecular machine
Journal of Biological Chemistry, 2013Co-Authors: Axel T Brunger, Sandro Vivona, Daniel J Cipriano, Timothy D Fenn, Jaemyeong Jung, Zev BryantAbstract:SNARE proteins promote membrane fusion by forming a four-stranded parallel helical bundle that brings the membranes into close proximity. Post-fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide-Sensitive Factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ∼26-fold. One SNARE complex takes an average of ∼5 s to disassemble in a process that consumes ∼50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled-coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule. Background: NSF is an AAA+ protein that recycles the post-fusion SNARE complex during the membrane fusion cycle. Results: NSF disassembles the SNARE complex processively in vitro and consumes dozens of ATP molecules per SNARE. Conclusion: NSF is a processive motor that progressively unwinds the SNARE complex. Significance: The physical mechanism of this poorly understood machine is illuminated using new assays and new measurements of ATP coupling ratios and processivity.
