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PubMed1996

N-Ethylmaleimide-sensitive factor acts at a prefusion ATP-dependent step in Ca2+-activated exocytosis.

An ATP-dependent activity of NSF (N-ethylmaleimide-sensitive factor) that rearranges soluble NSF attachment protein (SNAP) receptor (SNARE) protein complexes was proposed to be the driving force for membrane fusion. The Ca2+-activated fusion of secretory vesicles with the plasma membrane in permeable PC12 cells requires ATP; however, the ATP requirement is for a priming step that precedes the Ca2+-triggered fusion reaction. While phosphoinositide phosphorylation is a key reaction required for priming, additional ATP-dependent reactions are also necessary. Here we report that the NSF-catalyzed rearrangement of SNARE protein complexes occurs during ATP-dependent priming. NSF with alpha-SNAP (soluble NSF attachment protein) were required for ATP-dependent priming but not Ca2+-triggered fusion, indicating that NSF acts at an ATP-dependent prefusion step rather than at fusion itself. NSF-catalyzed activation of SNARE proteins may reorganize membranes to generate a vesicle-plasma membrane prefusion intermediate that is poised for conversion to full fusion by Ca2+-dependent mechanisms.

Adenosine Triphosphate↗
PubMed1996

Identification and characterization of the N-ethylmaleimide-sensitive site in lambda-integrase.

Integrase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and a type I topoisomerase. Upon modification with N-ethylmaleimide (NEM), a sulfhydryl-directed reagent, Int loses its capacity to bind "arm-type" DNA sequences and, consequently, to carry out recombination; however, its ability to bind "core-type" sequences and its topoisomerase activity are unaffected. In this report, the NEM-sensitive site was identified by modifying Int with [14C]NEM. Following cleavage by formic acid, which cleaves Asp-Pro bonds, and fractionation on a Fractogel HW-50 (F) sizing column, the fragment containing the primary site of [14C]NEM incorporation was subjected to amino acid sequencing. The results indicate that the primary site of [14C]NEM incorporation is in the peptide-spanning amino acid residues 1-28, which contains a cysteine at position 25. To confirm that Cys-25 is the target of NEM reactivity, site-directed mutagenesis was used to change this cysteine to alanine or serine. The mutant protein is not chemically modified by NEM and shows no loss of activity after NEM treatment. The fact that C25A and C25S both retain full recombination activity indicates that the SH group of Cys-25 does not provide any critical contacts, either with arm-type DNA or with other parts of the Int protein to form the arm-type recognition pocket. The loss of arm-type DNA binding and the concomitant loss of recombination function as a result of NEM modification must be due to the presence of the maleimide moiety and not due to loss of a critical cysteine contact.

Amino Acid Sequence↗
PubMed1996

N-Ethylmaleimide inactivates a nucleotide-free Hsp70 molecular chaperone.

Hsp70 molecular chaperones are ATPases that bind to hydrophobic regions of proteins and guide their folding, assembly, and translocation across membranes. The ability of purified Hsp70s to uncoat clathrin-coated vesicles or to stimulate the post-translational translocation of precursor proteins into the endoplasmic reticulum, mitochondria, and the nucleus was previously shown not to be sensitive to the sulfhydryl-modifying reagent N-ethylmaleimide (NEM). During purification of factors required for protein folding in the cytosol, we found that the ATP-agarose binding activity of the yeast Hsp70 Ssa1p in postribosomal supernatants was inhibited by NEM. We also found that completely removing nucleotides from purified Ssa1p rendered its ATP-agarose binding activity, ATPase activity, and post-translational translocation-stimulating activity sensitive to NEM. We modified nucleotide-free Ssa1p with [14C]NEM and then digested it with proteases. Purification and sequencing of the radiolabeled proteolytic fragments revealed that each of Ssa1p's three cysteine residues (Cys-15, Cys-264, and Cys-303) was modified with [14C]NEM. ADP protected each of the cysteine residues from modification and protected Ssa1p from inactivation. The cysteine residues are the reactive centers of three NEM-reactive sites (NRS1-3). A comparison of Ssa1p's NRSs to sequences of other Hsp70s and actin revealed that Cys-15 of NRS1 is highly conserved and that sensitivity to NEM may be a property of many Hsp70s. Based on the three-dimensional structure of Hsc70, the predicted locations of Ssa1p's cysteine residues suggest that NEM may disrupt the conformation of Ssa1p or interfere with its ability to bind nucleotides. Together the results demonstrate that Ssa1p is an NEM-sensitive factor in cytosolic extracts from yeast that stimulates post-translational translocation of proteins into organelles.

Adenosine Triphosphatases↗
PubMed1997

Reactive cysteines of the yeast plasma-membrane H+-ATPase (PMA1). Mapping the sites of inactivation by N-ethylmaleimide.

We have taken advantage of cysteine mutants described previously (Petrov, V. V., and Slayman, C. W. (1995) J. Biol. Chem. 270, 28535-28540) to map the sites at which N-ethylmaleimide (NEM) reacts with the plasma-membrane H+ATPase (PMA)1 of Saccharomyces cerevisiae. When membrane vesicles containing the ATPase were incubated with NEM, six of nine mutants with single cysteine substitutions showed sensitivity similar to the wild-type enzyme. By contrast, C221A and C532A were inactivated more slowly than the wild-type control, and the C221, 532A double mutant was completely resistant, indicating that Cys-221 and Cys-532 are NEM-reactive residues. In the presence of 10 mM MgADP, the wild-type ATPase was partially protected against NEM; parallel experiments with the C221A and C532A mutants showed that the protection occurred at Cys-532, located in or near the nucleotide-binding site. Unexpectedly, the inactivation of the C409A ATPase was approximately 4-fold more rapid than in the case of the wild-type enzyme. Experiments with double mutants made it clear that this resulted from an acidic shift in pKa and a consequent acceleration of the reaction rate at Cys-532. One simple interpretation is that substitution of Cys-409 leads to a local conformational change within the central hydrophilic domain. Consistent with this idea, the reaction of fluorescein 5'-isothiocyanate at Lys-474 was also stimulated approximately 3. 5-fold by the C409A mutation. Taken together, the results of this study provide new information about the reactivity of individual Cys residues within the ATPase and pave the way to tag specific sites for structural and functional studies of the enzyme.

Cell Membrane↗
PubMed1997

N-Ethylmaleimide-sensitive fusion protein contains high and low affinity ATP-binding sites that are functionally distinct.

N-Ethylmaleimide-sensitive factor (NSF) has been shown to be involved in numerous intracellular membrane fusion events of both the regulated and constitutive secretory pathways. Sequence analysis indicates that the NSF subunit contains two nucleotide-binding sites, both with the classical Walker A and B motifs. In this report, we examine the nucleotide binding properties of NSF. The homotrimer contains three high affinity ATP-binding sites with Kd = 30-40 nM for ATP and Kd = 2 microM for ADP. This class of binding sites did not bind AMP, adenine, or GTP. A second class of lower affinity nucleotide binding sites with a Kd = 15-20 microM was also detected. Using various mutant forms of NSF, the high affinity nucleotide-binding sites were localized to the D2 domains and the low affinity sites were localized to the D1 domains. Functionally it is these lower affinity sites in D1 that are crucial for NSF activity. Nucleotide concentration greatly affected the ability of NSF to interact with alpha-SNAP.SNARE (soluble NSF attachment protein-SNAP receptor) complex, suggesting that only when the D1 domain ATP-binding sites are occupied does NSF bind to the alpha-SNAP.SNARE complex.

Adenosine Triphosphate↗
PubMed1997

A novel assay reveals a role for soluble N-ethylmaleimide-sensitive fusion attachment protein in mannose 6-phosphate receptor transport from endosomes to the trans Golgi network.

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (alpha-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events. Although NSF and alpha-SNAP have been shown to be required for many membrane transport processes, their role in the transport of mannose 6-phosphate receptors from endosomes to the trans Golgi network was not established. We present here a novel in vitro assay that monitors the transport of cation-dependent mannose 6-phosphate receptors between endosomes and the trans Golgi network. The assay relies on the trans Golgi network localization of tyrosine sulfotransferase and monitors transport of mannose 6-phosphate receptors engineered to contain a consensus sequence for modification by this enzyme. Using this new assay we show that alpha-SNAP strongly stimulates transport in reactions containing limiting amounts of cytosol. Together with alpha-SNAP, NSF can increase the extent of transport. These data show that alpha-SNAP, a soluble component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.

Amino Acid Sequence↗
PubMed1998

A revised model for the oligomeric state of the N-ethylmaleimide-sensitive fusion protein, NSF.

The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking. Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions. However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers. We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical techniques. Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer. Sedimentation equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains. The sedimentation coefficient, s20,w0, of 13.4 (+/-0. 1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape. The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions.

Adenosine Triphosphatases↗
PubMed1999

alpha-soluble N-ethylmaleimide-sensitive factor attachment protein is expressed in pancreatic beta cells and functions in insulin but not gamma-aminobutyric acid secretion.

The function of soluble N-ethylmaleimide-sensitive attachment protein-alpha (alpha-SNAP) in exocytosis still remains obscure. This study was conducted to determine the physiological role of alpha-SNAP in the secretion of insulin and gamma-aminobutryric acid (GABA) from pancreatic beta cells. Reverse transcriptase-polymerase chain reaction analysis of total RNA isolated from rat islets disclosed alpha-SNAP, but not beta-SNAP, mRNA expression, and an immunofluorescence study of rat pancreas showed that alpha-SNAP was present predominantly in the cytoplasm of the islets of Langerhans. alpha-SNAP overexpression in rat islets enhanced insulin release relative to the control levels. An in vitro binding study showed that both wild-type alpha-SNAP and C-terminal-deleted alpha-SNAP mutant (1-285) can bind to syntaxin 1A. alpha-SNAP mutant (1-285) was overexpressed to evaluate its activity as dominant-negative effector on insulin release. Overexpression of alpha-SNAP mutant (1-285) in rat islets and MIN6 cells decreased glucose-stimulated insulin release to about 50% of the control levels. Suppression of endogeneous alpha-SNAP in MIN6 cells by treatment with an antisense phosphorothioate oligonucleotide resulted in inhibition of insulin release. In order to examine if alpha-SNAP functions in exocytosis from synaptic-like microvesicles in pancreatic beta cells, the functional role of alpha-SNAP in GABA release from MIN6 cells was studied. The data showed no effect of alpha-SNAP mutant (1-285) overexpression on GABA release. We conclude that 1) alpha-SNAP plays a crucial role in insulin exocytosis via large dense core vesicles, but not GABA released via synaptic-like microvesicles, in pancreatic beta cells; and 2) the interaction of alpha-SNAP and syntaxin 1A may play an important role in the insulin exocytotic process.

Animals↗
PubMed1999

The mitochondrial p55 accessory subunit of human DNA polymerase gamma enhances DNA binding, promotes processive DNA synthesis, and confers N-ethylmaleimide resistance.

Human DNA polymerase gamma is composed of a 140-kDa catalytic subunit and a smaller accessory protein variously reported to be 43-54 kDa. Immunoblot analysis of the purified, heterodimeric native human polymerase gamma complex identified the accessory subunit as 55 kDa. We isolated the full-length cDNA encoding a 55-kDa polypeptide, expressed the cDNA in Escherichia coli and purified the 55-kDa protein to homogeneity. Recombinant Hp55 forms a high affinity, salt-stable complex with Hp140 during protein affinity chromatography. Immunoprecipitation, gel filtration, and sedimentation analyses revealed a 190-kDa complex indicative of a native heterodimer. Reconstitution of Hp140.Hp55 raises the salt optimum of Hp140, stimulates the polymerase and exonuclease activities, and increases the processivity of the enzyme by several 100-fold. Similar to Hp140, isolated Hp55 binds DNA with moderate strength and was a specificity for double-stranded primer-template DNA. However, Hp140.Hp55 has a surprisingly high affinity for DNA, and kinetic analyses indicate Hp55 enhances the affinity of Hp140 for primer termini by 2 orders of magnitude. Thus the enhanced DNA binding caused by Hp55 is the basis for the salt tolerance and high processivity characteristic of DNA polymerase gamma. Observation of native DNA polymerase gamma both as an Hp140 monomer and as a heterodimer with Hp55 supports the notion that the two forms act in mitochondrial DNA repair and replication. Additionally, association of Hp55 with Hp140 protects the polymerase from inhibition by N-ethylmaleimide.

Base Sequence↗
PubMed2000

Low N-ethylmaleimide concentrations activate ryanodine receptors by a reversible interaction, not an alkylation of critical thiols.

Previous studies proposed that N-ethylmaleimide (NEM) alkylates 3 classes of thiols on skeletal muscle ryanodine receptors (RyRs) producing 3 phases of channel modification, as function of time and concentration. NEM (5 mm) decreased, increased, and then decreased the open probability (P(o)) of the channel by thiol alkylation, a reaction not reversed by reducing agents. We now show that low NEM concentrations (20-200 microm) elicit Ca(2+) release from sarcoplasmic reticulum (SR) vesicles, but contrary to expectations, the effect was fully reversed by reducing agents or by washing SR vesicles. In bilayers, NEM (0.2 mm) increased P(o) of RyRs within seconds when added to the cis (not trans) side, and dithiothreitol (DTT; 1 mm) decreased P(o) in seconds. High (5 mm) NEM concentrations elicited SR Ca(2+) release that was not reversed by DTT, as expected for an alkylation reaction. A non-sulfhydryl reagent structurally related to NEM, N-ethylsuccinimide (0.1-0.5 mm), also elicited SR Ca(2+) release that was not reversed by DTT (1 mm). Other alkylating agents elicited SR Ca(2+) release, which was fully (N-methylmaleimide) or partially (iodoacetic acid) reversed by DTT and inhibited by ruthenium red. Nitric oxide (NO) donors at concentrations that did not activate RyRs inhibited NEM-induced Ca(2+) release, most likely by an interaction of NO with NEM rather than an inactivation of RyRs by NO. Thus, at low concentrations, NEM does not act as a selective thiol reagent and activates RyRs without alkylating critical thiols indicating that the multiple phases of ryanodine binding are unrelated to RyR activity or to NEM alkylation of RyRs.

Alkylation↗
PubMed2000

Identification of a cysteine residue in the active site of nitroalkane oxidase by modification with N-ethylmaleimide.

The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrification of primary or secondary nitroalkanes to the corresponding aldehydes or ketones with production of hydrogen peroxide and nitrite. The enzyme is irreversibly inactivated by treatment with N-ethylmaleimide at pH 7. The inactivation is time-dependent and shows first-order kinetics for three half-lives. The second-order rate constant for inactivation is 3.4 +/- 0.06 m(-)(1) min(-)(1). The competitive inhibitor valerate protects the enzyme from inactivation, indicating an active site-directed modification. Comparison of tryptic maps of enzyme treated with N-[ethyl-1-(14)C]maleimide in the absence and presence of valerate shows a single radioactive peptide differentially labeled in the unprotected enzyme. The sequence of this peptide was determined to be LLNEVMCYPLFDGGNIGLR using Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cysteine residue was identified as the site of alkylation by ion trap mass spectrometry.

Amino Acid Sequence↗
PubMed2001

Phosphorylation of the N-ethylmaleimide-sensitive factor is associated with depolarization-dependent neurotransmitter release from synaptosomes.

Critical to SNARE protein function in neurotransmission are the accessory proteins, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP), and NSF, that play a role in activation of the SNAREs for membrane fusion. In this report, we demonstrate the depolarization-induced, calcium-dependent phosphorylation of NSF in rat synaptosomes. 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.

Amino Acid Sequence↗
PubMed2000

Requirement for N-ethylmaleimide-sensitive factor activity at different stages of bacterial invasion and phagocytosis.

Bacterial invasion, like the process of phagocytosis, involves extensive and localized protrusion of the host cell plasma membrane. To examine the molecular mechanisms of the membrane remodeling that accompanies bacterial invasion, soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic was studied in cultured cells during infection by Salmonella typhimurium. A green fluorescent protein-tagged chimera of VAMP3, a SNARE characteristic of recycling endosomes, was found to accumulate at sites of Salmonella invasion. To analyze the possible role of SNARE-mediated membrane traffic in bacterial infection, invasion was measured in cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (NSF), an essential regulator of membrane fusion. Inhibition of NSF activity did not affect cellular invasion by S. typhimurium nor the associated membrane remodeling. By contrast, Fcgamma receptor-mediated phagocytosis was greatly reduced in the presence of the mutant NSF. Most important, dominant-negative NSF significantly impaired the fusion of Salmonella-containing vacuoles with endomembranes. These observations indicate that the membrane protrusions elicited by Salmonella invasion, unlike those involved in phagocytosis, occur via an NSF-independent mechanism, whereas maturation of Salmonella-containing vacuoles is NSF-dependent.

Animals↗
PubMed2001

A homologue of N-ethylmaleimide-sensitive factor in the malaria parasite Plasmodium falciparum is exported and localized in vesicular structures in the cytoplasm of infected erythrocytes in the brefeldin A-sensitive pathway.

N-Ethylmaleimide-sensitive factor (NSF) and its homologues play a central role in vesicular trafficking in eukaryotic cells. We have identified a NSF homologue in Plasmodium falciparum (PfNSF). The reported PfNSF gene sequence (GenBank accession number CAB10575) indicated that PfNSF comprises 783 amino acids with a calculated molecular weight of 89,133. The overall identities of its gene and amino acid sequences with those of rat NSF are 50.9 and 48.8%, respectively. Reverse transcription-polymerase chain reaction analysis and Northern blotting with total P. falciparum RNA indicated expression of the PfNSF gene. Polyclonal antibodies against a conserved region of NSF specifically recognized an 89-kDa polypeptide in the parasite cells. After homogenization of the parasite cells, approximately 90% of an 89-kDa polypeptide is associated with particulate fraction, suggesting membrane-bound nature of PfNSF. PfNSF was present within both the parasite cells and the vesicular structure outside of the parasite cells. The export of PfNSF outside of the parasite cells appears to occur at the early trophozoite stage and to terminate at the merozoite stage. The export of PfNSF is inhibited by brefeldin A, with 9 microM causing 50% inhibition. Immunoelectromicroscopy indicated that intracellular PfNSF was associated with organelles such as food vacuoles and that extracellular PfNSF was associated with vesicular structures in the erythrocyte cytoplasm. These results indicate that PfNSF expressed in the malaria parasite is exported to the extracellular space and then localized in intraerythrocytic vesicles in a brefeldin A-sensitive manner. It is suggested that a vesicular transport mechanism is involved in protein export targeted to erythrocyte membranes during intraerythrocytic development of the malaria parasite.

Amino Acid Sequence↗
PubMed2002

N-Ethylmaleimide inhibits platelet-derived growth factor BB-stimulated Akt phosphorylation via activation of protein phosphatase 2A.

The redox state plays an important role in gene regulation. Thiols maintain the intracellular redox homeostasis. To understand the role of thiols in redox signaling, we have studied the effect of thiol alkylation on platelet-derived growth factor-BB (PDGF-BB)-induced cell survival events in vascular smooth muscle cells. PDGF-BB stimulated Akt phosphorylation predominantly at Ser-473. N-Ethylmaleimide (NEM), a thiol alkylating agent, blocked PDGF-BB-induced Akt phosphorylation without affecting its upstream phosphatidylinositol 3-kinase (PI3K). On the other hand, LY294002 and wortmannin, specific inhibitors of PI3K, prevented PDGF-BB-induced phosphorylation of Akt and its downstream effector molecules, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. NEM also abrogated the phosphorylation of p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E induced by PDGF-BB, suggesting that thiol alkylation interferes with the PI3K/Akt pathway at the level of Akt. In addition, NEM blocked PDGF-BB-induced phosphorylation of BAD and forkhead transcription factor FKHR-L1, and these events correlated with increased apoptosis. NEM alone and in concert with PDGF-BB increased reactive oxygen species (ROS) production and protein phosphatase 2A (PP2A) activity in VSMC. The inhibition of PDGF-BB-induced Akt phosphorylation by NEM was completely reversed by PP2A inhibitors fostriecin and okadaic acid, ceramide synthase inhibitor fumonisin B1, and ROS scavenger N-acetylcysteine (NAC). NAC also attenuated the apoptosis induced by NEM, alone or in combination with PDGF-BB. Together, these findings demonstrate for the first time that PP2A mediates thiol alkylation-dependent redox regulation of Akt and cell survival.

Animals↗
PubMed2002

Localization and function of soluble N-ethylmaleimide-sensitive factor attachment protein-25 and vesicle-associated membrane protein-2 in functioning gastric parietal cells.

The soluble N-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the gastric parietal cell. Here we examined the cellular localization and functional importance of SNAP-25 in parietal cell cultures. Adenoviral constructs were used to express SNAP-25 tagged with cyan fluorescent protein, VAMP-2 tagged with yellow fluorescent protein, and SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25 Delta181-206). Membrane fractionation experiments and fluorescent imaging showed that SNAP-25 is localized to the apical plasma membrane. The expression of the mutant SNAP-25 Delta181-226 inhibited the acid secretory response of parietal cells. Also, SNAP Delta181-226 bound poorly in vitro with recombinant syntaxin-1 compared with wild type SNAP-25, indicating that pairing between syntaxin-1 and SNAP-25 is required for parietal cell activation. Dual expression of SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with yellow fluorescent protein revealed a dynamic change in distribution associated with acid secretion. In resting cells, SNAP-25 is at the apical plasma membrane and VAMP-2 is associated with cytoplasmic H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins co-localize on the apical plasma membrane. These data demonstrate the functional significance of SNAP-25 as a SNARE protein in the parietal cell and show the dynamic stimulation-associated redistribution of VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane.

Adenoviridae↗
PubMed2004

Type I PDZ ligands are sufficient to promote rapid recycling of G Protein-coupled receptors independent of binding to N-ethylmaleimide-sensitive factor.

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 beta2 adrenergic receptor (beta2AR) 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 beta2AR 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 beta2AR-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 beta1-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 beta2AR 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.

Cell Line↗
PubMed2005

Modulation of N-ethylmaleimide-sensitive factor activity upon amino acid deprivation.

Adaptation of eukaryotic cells to changing environmental conditions entails rapid regulation of protein targeting and transport to specific organelles. Such adaptation is well exemplified in mammalian cells exposed to nitrogen starvation that are triggered to form and transport autophagosomes to lysosomes, thus constituting an inducible intracellular trafficking pathway. Here we investigated the relationship between the general secretory machinery and the autophagic pathway in Chinese hamster ovary cells grown in the absence of amino acid. Utilizing VSVG-YFP (vesicular stomatitis virus G protein fused to yellow fluorescent protein) and norepinephrine as markers for constitutive and regulated exocytosis, respectively, we found that secretion is attenuated in cells grown in media lacking amino acid. Such decrease in exocytosis stems from partial inhibition of N-ethylmaleimide-sensitive factor ATPase activity, which in turn causes an accumulation of SNARE complexes at both the Golgi apparatus and the plasma membrane of the starved cells. These findings expose a novel cellular strategy to attenuate secretion of proteins under conditions of limited amino acid supply.

Adenosine Triphosphatases↗