Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Anti-synaptobrevin 2 immunoprecipitates obtained from freshly prepared Triton X-100 extracts of rat synaptosomes contained, in addition to synaptophysin, a 10-kDa band, which we identified by peptide sequencing and Western blotting as the c subunit of the vacuolar proton pump (V-ATPase) also called ductin or mediatophore. Ac39 and Ac116, two other transmembrane subunits of the V0 sector of the V-ATPase, were also found by Western blotting to be enriched in the immunoprecipitates. None of these V-ATPase subunits, or synaptophysin, was present in anti-synaptobrevin 2 immunoprecipitates obtained from frozen-thawed Triton X-100 extracts, which were greatly enriched, instead, in SNAP-25 and syntaxin 1. Accordingly, V-ATPase subunit c was found in anti-synaptophysin immunoprecipitates. Thus, the two complexes appear to be mutually exclusive. Subcellular fractionation of rat brain demonstrated that V-ATPase subunit c is localized with synaptobrevin 2 and synaptophysin in synaptic vesicles. The coprecipitation of V-ATPase subunit c with the synaptobrevin-synaptophysin complex suggests that this interaction may play a role in recruiting the proton pump into synaptic vesicles. Freeze-thawing, which involves a mild denaturing step, may produce a conformational change which dissociates the complex and mimics a change which occurs in vivo as a prerequisite to SNARE complex formation.
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PMID:The V0 sector of the V-ATPase, synaptobrevin, and synaptophysin are associated on synaptic vesicles in a Triton X-100-resistant, freeze-thawing sensitive, complex. 856 78

Discovery of the protein machinery of vesicle transport has followed the reconstitution of transport in a cell-free system, and established general mechanisms that underlie a broad variety of physiological processes, including cell surface growth, the biogenesis of distinct intracellular organelles, endocytosis, and the controlled release of hormones and neurotransmitters. Transport vesicles are formed as coat proteins assemble on membranes, are targetted by SNARE proteins, and fuse using a general mechanism involving SNAP proteins and the ATPase NSF.
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PMID:Felix Hoppe-Seyler Lecture 1996. Mechanisms of intracellular protein transport. 892 73

Soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE) mechanisms are thought to be involved in two important processes in axonal growth cones: (1) membrane expansion for axonal growth and (2) vesicular membrane fusion for mature synaptic transmission. We investigated the localization and interactions among the proteins involved in SNARE complex formation in isolated growth cone particles (GCP) from forebrain. We demonstrated that the SNARE complex is present in GCPs morphologically without synaptic vesicles (SVs) and associated with growth cone vesicles. However, the apparently SV-free GCP was lacking in the regulatory mechanisms inhibiting SNARE complex formation proposed in SV fusion, i.e., the association of synaptotagmin with the SNARE complex, and vesicle-associated membrane protein (VAMP)-synaptophysin complex formation. The core components of the SNARE complex (syntaxin, SNAP-25, and VAMP) accumulated for several days before postnatal day 7, when SVs first appeared, and preceded the accumulation of marker proteins such as synaptophysin, SV2, and V-ATPase. Our present results suggest that the SNARE mechanism for vesicular transmitter release is not fully functional in growth cones before the appearance of SVs, but the SNARE mechanism is working for membrane expansion in growth cones, which supports our recent report. We concluded that the regulation of the SNARE complex in growth cones is different from that in mature presynaptic terminals and that this switching may be one of the key steps in development from the growth cone to the presynaptic terminal.
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PMID:The soluble N-ethylmaleimide-sensitive factor attached protein receptor complex in growth cones: molecular aspects of the axon terminal development. 900 87

Exocytosis in neurons requires proteins known as SNAREs, membrane proteins that have now been implicated in many intracellular fusion events. SNAREs assemble into stable ternary complexes that are dissociated by the ATPase NSF (N-ethylmaleimide-sensitive factor), working together with SNAPs (soluble NSF attachment proteins). Recent results have shed new light on the mechanisms underlying SNARE (SNAP receptor) complex assembly and disassembly, and suggest changes in models that relate these reactions to vesicle docking and fusion.
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PMID:Neurotransmitter release - four years of SNARE complexes. 923 12

Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) clear the amine from extracellular spaces in the CNS and periphery as a mechanism for transmitter inactivation and recycling. Although it is known that SERTs are preferentially expressed on basolateral domains in transfected epithelial cells, details of the transporter's membrane localization in vivo are lacking. 5HT and 5HT receptors have been identified in the rodent adrenal gland. Using SERT antagonist autoradiography, we establish the presence of antidepressant-sensitive transport sites in the rat adrenal medulla. Immunofluorescence experiments using antibodies specific for the SERT COOH and NH2 termini, for 5HT, or for catecholamine biosynthetic enzymes suggest that SERT mediates intra-cellular 5HT accumulation by epinephrine-secreting chromaffin cells. Using confocal microscopy, we establish that SERT expression is nonuniformly distributed along the plasma membrane of chromaffin cells. Notably, SERT immunoreactivity is largely absent from plasma membranes bordering smooth muscle that surrounds vascular sinusoids. Rather, SERT is highly expressed in membranes adjoining other chromaffin cells, consistent with a role for 5HT and SERT in autocrine or paracrine control of chromaffin cell physiology. SNAP-25, a t-SNARE protein implicated in neurotransmitter release, was found to colocalize with SERT. In contrast, Na,K ATPase and NCAM are uniformly distributed along the entire perimeter of chromaffin cell membranes. These findings underscore a role for 5HT and SERT in adrenal physiology, reveal unrecognized polarity of chromaffin cell plasma membranes, and warrant a consideration of common targeting mechanisms localizing amine transporters near release sites.
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PMID:Polarized expression of the antidepressant-sensitive serotonin transporter in epinephrine-synthesizing chromaffin cells of the rat adrenal gland. 924

Using quick-freeze/deep-etch electron microscopy of recombinant proteins adsorbed to mica, we show that NSF, the oligomeric ATPase involved in membrane fusion, is a hollow 10 x 16 nm cylinder whose conformation depends upon nucleotide binding. Depleted of nucleotide, NSF converts to a "splayed" protease-sensitive conformation that reveals its subunit composition. NSF's synaptic membrane substrate, the ternary SNARE complex containing syntaxin, SNAP-25, and synaptobrevin, is a 4 x 14 nm rod with a "tail" at one end, corresponding to the N-terminus of syntaxin. Using epitope tags, antibodies, and maltose-binding protein markers, we find that syntaxin and synaptobrevin are aligned in parallel in the complex, with their membrane anchors located at the same end of the rod. This SNARE rod binds with alpha-SNAP to one end of the NSF cylinder to form an asymmetric "20S" complex. Together, these images suggest how NSF could dissociate the SNARE complex and how association and dissociation of the complex could be related to membrane fusion.
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PMID:Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy. 926 32

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.
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PMID:N-ethylmaleimide-sensitive factor-dependent alpha-SNAP release, an early event in the docking/fusion process, is not regulated by Rab GTPases. 943 Jun 66

The fusion of endoplasmic reticulum (ER) membranes in yeast does not require Sec18p/NSF and Sec17p, two proteins needed for docking of vesicles with their target membrane. Instead, ER membranes require a NSF-related ATPase, Cdc48p. Since both vesicular and organelle fusion events use related ATPases, we investigated whether both fusion events are also SNARE mediated. We present evidence that the fusion of ER membranes requires Ufe1p, a t-SNARE that localizes to the ER, but no known v-SNAREs. We propose that the Ufe1 protein acts in the dual capacity of an organelle membrane fusion-associated SNARE by undergoing direct t-t-SNARE and Cdc48p interactions during organelle membrane fusion as well as a t-SNARE for vesicular traffic.
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PMID:Organelle membrane fusion: a novel function for the syntaxin homolog Ufe1p in ER membrane fusion. 950 16

Vacuole fusion requires Sec18p (NSF), Sec17p (alpha-SNAP), Ypt7p (GTP binding protein), Vam3p (t-SNARE), Nyv1p (v-SNARE), and LMA1 (low Mr activity 1, a heterodimer of thioredoxin and I(B)2). LMA1 requires Sec18p for saturable, high-affinity binding to vacuoles, and Sec18p "priming" ATPase requires both Sec17p and LMA1. Either the sec18-1 mutation and deletion of I(B)2, or deletion of both I(B)2 and p13 (an I(B)2 homolog) causes a striking synthetic vacuole fragmentation phenotype. Upon Sec18p ATP hydrolysis, LMA1 transfers to (and stabilizes) a Vam3p complex. LMA1 is released from vacuoles in a phosphatase-regulated reaction. This LMA1 cycle explains how priming by Sec18p is coupled to t-SNARE stabilization and to fusion.
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PMID:LMA1 binds to vacuoles at Sec18p (NSF), transfers upon ATP hydrolysis to a t-SNARE (Vam3p) complex, and is released during fusion. 965 46

Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle-derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.
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PMID:Formation and turnover of NSF- and SNAP-containing "fusion" complexes occur on undocked, clathrin-coated vesicle-derived membranes. 965 60


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