Identification of NSF as a beta-arrestin1-binding protein. Implications for beta2-adrenergic receptor regulation.
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Previous studies have demonstrated that beta-arrestin1 serves to target G protein-coupled receptors for internalization via clathrin-coated pits and that its endocytic function is regulated by dephosphorylation at the plasma membrane. Using the yeast two-hybrid system, we have identified a novel beta-arrestin1-binding protein, NSF (N-ethylmaleimide-sensitive fusion protein), an ATPase essential for many intracellular transport reactions. We demonstrate that purified recombinant beta-arrestin1 and NSF interact in vitro and that these proteins can be coimmunoprecipitated from cells. beta-Arrestin1-NSF complex formation exhibits a conformational dependence with beta-arrestin1 preferentially interacting with the ATP bound form of NSF. In contrast to the beta-arrestin1-clathrin interaction, however, the phosphorylation state of beta-arrestin1 does not affect NSF binding. Functionally, overexpression of NSF in HEK 293 cells significantly enhances agonist-mediated beta2-adrenergic receptor (beta2-AR) internalization. Furthermore, when coexpressed with a beta-arrestin1 mutant (betaarr1S412D) that mimics a constitutively phosphorylated form of beta-arrestin1 and that acts as a dominant negative with regards to beta2-AR internalization, NSF rescues the betaarr1S412D-mediated inhibition of beta2-AR internalization. The demonstration of beta-arrestin1-NSF complex formation and the functional consequences of NSF overexpression suggest a hitherto unappreciated role for NSF in facilitating clathrin coat-mediated G protein-coupled receptor internalization. (+info)
Early requirement for alpha-SNAP and NSF in the secretory cascade in chromaffin cells.
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NSF and alpha-SNAP have been shown to be required for SNARE complex disassembly and exocytosis. However, the exact requirement for NSF and alpha-SNAP in vesicular traffic through the secretory pathway remains controversial. We performed a study on the kinetics of exocytosis from bovine chromaffin cells using high time resolution capacitance measurement and electrochemical amperometry, combined with flash photolysis of caged Ca2+ as a fast stimulus. alpha-SNAP, a C-terminal mutant of alpha-SNAP, and NEM were assayed for their effects on secretion kinetics. Two kinetically distinct components of catecholamine release can be observed upon fast step-like elevation of [Ca2+]i. One is the exocytotic burst, thought to represent the readily releasable pool of vesicles. Following the exocytotic burst, secretion proceeds slowly at maintained high [Ca2+]i, which may represent vesicle maturation/recruitment, i.e. some priming steps after docking. alpha-SNAP increased the amplitude of both the exocytotic burst and the slow component but did not change their kinetics, which we examined with millisecond time resolution. In addition, NEM only partially inhibited the slow component without altering the exocytotic burst, fusion kinetics and the rate of endocytosis. These results suggest a role for alpha-SNAP/NSF in priming granules for release at an early step, but not modifying the fusion of readily releasable granules. (+info)
Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion.
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Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p. (+info)
Surface expression of AMPA receptors in hippocampal neurons is regulated by an NSF-dependent mechanism.
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Here, we show that disruption of N-ethylmaleimide-sensitive fusion protein- (NSF-) GluR2 interaction by infusion into cultured hippocampal neurons of a blocking peptide (pep2m) caused a rapid decrease in the frequency but no change in the amplitude of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs). N-methyl-D-aspartate (NMDA) receptor-mediated mEPSCs were not changed. Viral expression of pep2m reduced the surface expression of alpha-amino-3-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptors, whereas NMDA receptor surface expression in the same living cells was unchanged. In permeabilized neurons, the total amount of GluR2 immunoreactivity was unchanged, and a punctate distribution of GluR2 was 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. (+info)
The Drosophila NSF protein, dNSF1, plays a similar role at neuromuscular and some central synapses.
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The N-ethylmaleimide sensitive fusion protein (NSF) was originally identified as a cytosolic factor required for constitutive vesicular transport and later implicated in synaptic vesicle trafficking as well. Our previous work at neuromuscular synapses in the temperature-sensitive NSF mutant, comatose (comt), has shown that the comt gene product, dNSF1, functions after synaptic vesicle docking in the priming of vesicles for fast calcium-triggered fusion. Here we investigate whether dNSF1 performs a similar function at central synapses associated with the well-characterized giant fiber neural pathway. These include a synapse within the giant fiber pathway, made by the peripherally synapsing interneuron (PSI), as well as synapses providing input to the giant fiber pathway. The latency (delay) between stimulation and a resulting muscle action potential was used to assess the function of each class of synapses. Repetitive stimulation of the giant fiber pathway in comt produced wild-type responses at both 20 and 36 degrees C, exhibiting a characteristic and constant latency between stimulation and the muscle response. In contrast, stimulation of presynaptic inputs to the giant fiber (referred to as the "long latency pathway") revealed a striking difference between wild type and comt at 36 degrees C. Repetitive stimulation of the long latency pathway led to a progressive, activity-dependent increase in the response latency in comt, but not in wild type. Thus the giant fiber pathway, including the PSI synapse, appears to function normally in comt, whereas the presynaptic inputs to the giant fiber pathway are disrupted. Several aspects of the progressive latency increase observed in the long latency pathway can be understood in the context of the activity-dependent reduction in neurotransmitter release we observed previously at neuromuscular synapses. These results suggest that repetitive stimulation causes a progressive reduction in neurotransmitter release by presynaptic inputs to the giant fiber neuron, resulting in an increased latency preceding a giant fiber action potential. Thus synapses presynaptic to the giant fiber appear to utilize dNSF1 in a manner similar to the neuromuscular synapse, whereas the PSI chemical synapse may differ with respect to the expression or activity of dNSF1. (+info)
A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling Golgi cisternae in a cell-free system.
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During telophase, Golgi cisternae are regenerated and stacked from a heterogeneous population of tubulovesicular clusters. A cell-free system that reconstructs these events has revealed that cisternal regrowth requires interplay between soluble factors and soluble N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) via two intersecting pathways controlled by the ATPases, p97 and NSF. Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process. NSF-mediated cisternal regrowth requires a vesicle tethering protein, p115, which we now show operates through its two Golgi receptors, GM130 and giantin. p97-mediated cisternal regrowth is p115-independent, but we now demonstrate a role for p115, in conjunction with its receptors, in stacking p97 generated cisternae. Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking. These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation. (+info)
A critical role for N-ethylmaleimide-sensitive fusion protein (NSF) in platelet granule secretion.
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The molecular mechanisms that regulate membrane targeting/fusion during platelet granule secretion are not yet understood. N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (SNAP receptors) are elements of a conserved molecular machinery for membrane targeting/fusion that have been detected in platelets. We examined whether NSF, an ATPase that has been shown to play a critical role in membrane targeting/fusion in many cell types, is necessary for platelet granule secretion. Peptides that mimic NSF sequence motifs inhibited both alpha-granule and dense-granule secretion in permeabilized human platelets. This inhibitory effect was sequence-specific, because neither proteinase K-digested peptides nor peptides containing similar amino acids in a scrambled sequence inhibited platelet secretion. The peptides that inhibited platelet granule secretion also inhibited the human recombinant alpha-SNAP-stimulated ATPase activity of recombinant NSF. It was also found that anti-NSF antibodies, which inhibited recombinant alpha-SNAP-stimulated ATPase activity of NSF, inhibited platelet granule secretion in permeabilized cells. The inhibition by anti-NSF antibodies was abolished by the addition of recombinant NSF. These data provide the first functional evidence that NSF plays an important role in platelet granule secretion. (+info)
NSF N-terminal domain crystal structure: models of NSF function.
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N-ethylmaleimide-sensitive factor (NSF) is a hexameric ATPase essential for eukaryotic vesicle fusion. Along with SNAP proteins, it disassembles cis-SNARE complexes upon ATP hydrolysis, preparing SNAREs for trans complex formation. We have determined the crystal structure of the N-terminal domain of NSF (N) to 1.9 A resolution. N contains two subdomains which form a groove that is a likely SNAP interaction site. Unexpectedly, both N subdomains are structurally similar to domains in EF-Tu. Based on this similarity, we propose a model for a large conformational change in NSF that drives SNARE complex disassembly. (+info)