Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Coated Vesicles”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources
PubMed1983

Clathrin-coated vesicles purified from a human B lymphocyte cell line contain newly synthesized glycoproteins.

Clathrin-coated vesicles were purified from the cultured human B lymphocyte cell line RPMI 1788. These preparations were shown to be substantially free of contaminating membranes by the following criteria: (a) polypeptide composition, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) electron microscopy, (c) lack of binding of intact vesicles to concanavalin A-Sepharose, (d) migration of coated vesicles as a single major peak when applied to a Sephacryl S-1000 gel filtration column. An association between HLA-A, -B, and -DR antigens and immunoglobulins with coated vesicle fractions was demonstrated by lentil lectin affinity chromatography and by immunoprecipitation using antisera directed against these glycoproteins. [35S]Methionine pulse-chase experiments revealed that there was a kinetic correlation between the expression of newly synthesized glycoproteins and their presence in coated vesicles. These results indicate that coated vesicles may be involved in the transport of cell surface and secreted eukaryotic glycoproteins through the cell to their final destinations at the plasma membrane or in the extra-cellular medium.

B-Lymphocytes↗
PubMed1985

Solubilization of proteins from bovine brain coated vesicles by protein perturbants and Triton X-100.

To identify integral and peripheral membrane proteins, highly purified coated vesicles from bovine brain were exposed to solutions of various pH, ionic strength, and concentrations of the nonionic detergent Triton X-100. At pH 10.0 or above most major proteins were liberated, but four minor polypeptides sedimented with the vesicles. From quantitative analysis of phospholipids in the pellet and extract, we determined that at a pH of up to 12 all phospholipids could be recovered in the pellet. Electron microscopic examination of coated vesicles at pH 12.0 showed all vesicles devoid of coat structures. Treatment with high ionic strength solutions (0-1.0 M KCl) at pH 6.5-8.5 also liberated all major proteins, except tubulin, which remained sedimentable. The addition of Triton X-100 to coated vesicles or to stripped vesicles from which 90% of the clathrin had been removed resulted in the release of four distinct polypeptides of approximate Mr 38,000, 29,000, 24,000 and 10,000. The 38,000-D polypeptide (pK approximately 5.0), which represents approximately 50% of the protein liberated by Triton X-100, appears to be a glycoprotein on the basis of its reaction with periodic acid-Schiff reagent. Extraction of 90% of the clathrin followed by extraction of 90% of the phospholipids with Triton X-100 produced a protein residue that remained sedimentable and consisted of structures that appeared to be shrunken stripped vesicles. Together our data indicate that most of the major polypeptides of brain coated vesicles behave as peripheral membrane proteins and at least four polypeptides behave as integral membrane proteins. By use of a monoclonal antibody, we have identified one of these polypeptides (38,000 mol wt) as a marker for a subpopulation of calf brain coated vesicles.

Animals↗
PubMed1988

An antibody against 100- to 116-kDa polypeptides in coated vesicles inhibits triskelion binding.

There is considerable evidence that the 100- to 116-kDa polypeptides in calf brain coated vesicles are involved in the assembly of clathrin triskelions to form coated vesicles. We have raised polyclonal antibodies against these polypeptides. By Western blot analysis, these antibodies bind to a distinct subset of the six polypeptides in the region 100-116 kDa. Whole cell homogenates from calf brain, calf liver, and rat liver also show immunoreactivity in the 100-kDa region with no other cross reactivity. Isolated coated vesicles from calf liver, rat brain, and soybean roots also cross-react. Stripped coated vesicles, which are depleted of clathrin but which retain the 100- to 116-kDa polypeptides, quantitatively rebind 125I-triskelions. This binding is inhibited in a dose-dependent manner by 100- to 116-kDa antibody but not by nonimmune serum or by anti-clathrin polyclonal antibody. These studies indicate that (1) specific sites on the 100- to 116-kDa polypeptides are required for assembly of coated vesicles, and (2) this antibody will be useful in clarifying more precisely the role of the 100- to 116-kDa polypeptides in coated vesicle recycling.

Animals↗
PubMed1990

Bovine brain coated vesicles contain adenosine A1 receptors. Presence of adenylate cyclase coupled to the receptor.

Clathrin-coated vesicles purified from bovine brain express adenosine A1 receptor binding activity. N6-Cyclohexyl[3H]adenosine [( 3H]CHA), an agonist for the A1 receptor, binds specifically to coated vesicles. High and low agonist affinity states of the receptor for the radioligand [3H]CHA with KD values of 0.18 and 4.4 nM, respectively, were detected. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Binding competition experiments using agonists (N6CHA, N-cyclopentyladenosine, 5'-(N-ethylcarboxamido)adenosine, and N6-[(R)- and N6-[(S)-phenylisopropyl]adenosine) and antagonists (theophylline, 3-isobutyl-1-methylxanthine, and caffeine) confirmed the typical adenosine A1 nature of the binding site. This binding site presents stereospecificity for N6-phenylisopropyladenosine, showing 33 times more affinity for N6-[(R)- than for N6-[(S)-phenylisopropyl]adenosine. The specific binding of [3H]CHA in coated vesicles is regulated by guanine nucleotides. [3H]CHA specific binding was decreased by 70% in the presence of the hydrolysis-resistant GTP analogue guanyl-5-yl-imidodiphosphate. Bovine brain coated vesicles present adenylate cyclase activity. This activity was modulated by forskolin and CHA. The results of this study support the evidence that adenosine A1 receptors present in coated vesicles are coupled to adenylate cyclase activity through a Gi protein.

Adenosine↗
PubMed1983

Clathrin-coated vesicles contain an ATP-dependent proton pump.

Clathrin-coated vesicles isolated from calf brain contain an ATP-dependent proton pump. Proton movement was monitored by measuring [14C]methylamine distribution. Addition of Mg2+ and ATP to coated vesicles equilibrated with [14C]methylamine resulted in the generation of a 4- to 5-fold concentration gradient, corresponding to a delta pH of 0.6-0.7 units between the medium and the acidic inside of the coated vesicles. ATP-dependent [14C]methylamine uptake was abolished by the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and partially inhibited by the carboxyl reagent N,N'-dicyclohexylcarbodiimide but was unaffected by the Na+, K+-ATPase inhibitors strophanthidin (100 microM) and vanadate (10 microM) and the mitochondrial ATPase inhibitors oligomycin (10 microgram/ml) and aurovertin (1 microgram/ml). GTP, but not the nonhydrolyzable analog 5'-adenylyl imidodiphosphate, could support [14C]methylamine uptake. Dissipation of the membrane potential with K+ and valinomycin resulted in stimulation of [14C]methylamine uptake, whereas both FCCP and valinomycin stimulated the strophanthidin-resistant ATPase activity. These results are consistent with the existence of an electrogenic, ATP-dependent proton pump in clathrin-coated vesicles. This proton pump may play a role in the acidification events that are essential in receptor-mediated endocytosis.

Adenosine Triphosphatases↗
PubMed1993

Uncoating of clathrin-coated vesicles by uncoating ATPase from developing peas.

A cytosolic ATPase (an enzyme that dissociates clathrin from clathrin-coated vesicles in the presence of ATP) was isolated from developing pea (Pisum sativum L.) cotyledons using chromatography on ATP-agarose. After chromatography on phenyl Sepharose, the fraction with uncoating activity was enriched in a doublet of 70-kD peptides. Using chromatofocusing, it was possible to produce fractions enriched in the upper component of the doublet of 70-kD peptides; these fractions still retained ATP-dependent uncoating activity. In western blot analysis, antibodies against a member of the 70-kD family of heat-shock proteins interacted with the upper component of the doublet of the 70-kD peptides from the phenyl Sepharose-purified fractions. On the basis of these data, it appears that the uncoating ATPase may be a member of the 70-kD family of heat-shock proteins. The uncoating activity removed clathrin from both pea and bovine brain clathrin-coated vesicles. The uncoating ATPase from bovine brain also uncoated coated vesicles from peas. Pea clathrin-coated vesicles that were prepared by three different methods were uncoated to different extents by the plant uncoating ATPase. Different populations of clathrin-coated vesicles from the same preparation showed differential sensitivity to the uncoating ATPase. Limited proteolysis of the clathrin light chains in the protein coat abolished the susceptibility of the clathrin-coated vesicles to the uncoating ATPase. The properties of the uncoating ATPase isolated from developing pea cotyledons are similar to those of uncoating ATPases previously described from mammalian and yeast systems. It appears that despite dissimilarities in composition of the clathrin components of the vesicles from the respective sources, uncoating is achieved by a common mechanism.

Adenosine Triphosphatases↗
PubMed1985

Biochemical and cytochemical evidence indicates that coated vesicles in chick embryo myotubes contain newly synthesized acetylcholinesterase.

We have isolated highly purified coated vesicles from 17-d-old chick embryo skeletal muscle. These isolated coated vesicles contain acetylcholinesterase (AChE) in a latent, membrane-protected form as demonstrated enzymatically and morphologically using the Karnovsky and Roots histochemical procedure (J. Histochem. Cytochem., 1964, 12:219-221). By the use of appropriate inhibitors the cholinesterase activity can be shown to be specific for acetylcholine. It also can be concluded that most of the AChE represents soluble enzyme since it is rendered soluble by repeated freeze-thaw cycles. To determine the origin of the coated vesicle-associated AChE, we have isolated coated vesicles from cultured chick embryo myotubes which have been treated with diisopropylfluorophosphate, an essentially irreversible inhibitor of both intra- and extracellular AChE, and have been allowed to recover for 3 h. This time is not enough to allow any newly synthesized AChE to be secreted. These coated vesicles also contain predominantly soluble AChE. These data are compatible with the hypothesis that coated vesicles are important intermediates in the intracellular transport of newly synthesized AChE.

Acetylcholinesterase↗
PubMed1989

Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack.

We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.

Animals↗
PubMed1987

Rabbit reticulocyte coated vesicles carrying the transferrin-transferrin receptor complex: I. Purification and partial characterization.

Coated vesicles bearing the transferrin-transferrin receptor complex were isolated from rabbit reticulocytes by freeze-thaw cell lysis, followed by differential centrifugation with pelleting of vesicles at 100,000 g. Electronmicroscopy demonstrated the vesicles to have the characteristic morphology of coated vesicles, including the appearance of triskelions. The protein composition of the vesicles as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis included transferrin, transferrin receptor, and proteins of apparent mol wt of approximately 180,000, 140,000, 100,000, and 47,000 daltons. The 180,000 and 100,000 mol wt proteins were identified as clathrin and coated vesicle assembly factor proteins, respectively, by Western blot analyses. The vesicles had a Mg2+-dependent ATPase with a specific activity of approximately 8.5 nmoles ATP converted/min/mg vesicle protein. The vesicles could acidify the intravesicular space, as evidenced by the stimulation of the Mg2+-ATPase by the protonophore FCCP. Reticulocytes appear to be an excellent source of coated vesicles and as such should provide a model for studying the endocytosis of transferrin and the steps of iron uptake that proceed in these vesicles.

Acid-Base Equilibrium↗
PubMed1996

The speed of partial reactions of the uncoating ATPase Hsc70 depends on the source of coated vesicles.

Hsc70 was previously isolated by its ability to catalyse the uncoating of clathrin-coated vesicles from bovine brain. We have recently shown that Hsc70 is more active towards coated vesicles from brain than those from other tissues. In order to gain information on the mechanistic reason for this difference we have examined the ability of brain and placental coated vesicles to stimulate partial reactions during a single round of ATP turnover. The Hsc70-ATP complex is turned over to Hsc70-ADP center dot Pi, from which phosphate is slowly released. The resulting Hsc70-ADP complex exchanges ATP for ADP. Dissociation of ATP or ADP from Hsc70 does not seem to occur under physiological conditions. The hydrolysis of ATP is accelerated by the presence of clathrin-coated vesicles, with vesicles from brain being about twice as effective as vesicles from placenta. Additionally, it appears that brain, but not placental, coated vesicles can also stimulate the exchange of ADP for ATP.

Adenosine Triphosphate↗
PubMed1996

Identification of a 14-kDa subunit associated with the catalytic sector of clathrin-coated vesicle H+-ATPase.

The clathrin-coated vesicle H+-ATPase is composed of a peripheral catalytic sector (VC) and an integral membrane proton channel (VB), both of which are multiple subunit complexes. This study was conducted to determine if subunit F, previously identified in vacuolar proton pumps of tobacco hornworm and yeast, was present in mammalian pumps. Using a polymerase chain reaction-based strategy, we have isolated and sequenced cDNA clones from bovine and rat brain cDNA libraries. A full-length clone from rat brain encodes a 119-amino acid polypeptide with a predicted molecular mass of 13, 370 Da and with approximately 72 and 49% identity to subunit F of tobacco hornworm and yeast, respectively. Southern and Northern blot analyses indicate that the protein is encoded by a single gene. An anti-peptide antibody, directed against deduced protein sequence, was affinity-purified and shown to react with a 14-kDa polypeptide that is present in a highly purified pump prepared from clathrin-coated vesicles and also isolated VC. When stripped clathrin-coated vacuolars and purified chromaffin granule membranes were treated with KI in the presence of ATP, the 14-kDa subunit was released from both membranes, further indicating that it is part of the peripheral catalytic sector. In addition, direct sequencing of this 14-kDa component of the coated vacuolar proton pump confirmed its identity as a subunit F homologue.

Amino Acid Sequence↗
PubMed2002

Regulating the clathrin-coated vesicle cycle by AP2 subunit phosphorylation.

Clathrin-coated pits form from the concerted assembly of many different protein components that interact with each other and with the lipid bilayer. When the coated pit pinches off to form a coated vesicle, the coat is removed to allow fusion of the endocytic vesicle with an early endosome, and the disassembled coat components can then recycle through the cytosol to participate in new rounds of coated pit formation. One potential mechanism to regulate the cycles of coat protein assembly and disassembly is reversible phosphorylation, particularly given that many of the coat components, such as the AP2 adaptor complex, are phosphorylated on multiple sites both in vitro and in vivo. Recent studies have provided particular insight into the role of phosphorylation of the cargo-binding micro2 subunit of AP2.

Adaptor Protein Complex 1↗
PubMed1991

Can coated vesicles bind directly to microtubules?

Using an ultrathin-sectioning electron microscopic procedure, no efficient binding of coated vesicles to microtubules (both purified from brain tissue) could be achieved, independently of the presence of microtubule-associated proteins. Addition of the ATP analogue AMP-PNP or the inorganic tripolyphosphate, known to cause tight associations of (uncoated) vesicles to microtubules by means of specific motor proteins, could not enhance the binding efficiency. Moreover, crude preparations of clathrin, the major protein of the coat, did not affect the turbidity course of microtubule assembly. These results were confirmed by electrophoresis indicating that within the preparations of microtubule protein, obtained by temperature-dependent cycles of disassembly/reassembly, constituents of coated vesicles were not present. Beside this, coated vesicles have never been found among microtubules reconstituted in vitro. Vice versa, preparations of coated vesicles were completely free of microtubules. Our results suggest that further proteins should be involved in the binding of coated vesicles to microtubules, if there is indeed a biologically relevant interaction.

Animals↗
PubMed1996

Association of the fusion protein NSF with clathrin-coated vesicle membranes.

N-ethylmaleimide-sensitive fusion protein (NSF) is a component of intracellular transport reactions. In order to understand the role of NSF during the fusion of endocytic transport vesicles with the endosome, we have investigated the binding of NSF to purified clathrin-coated vesicle components. First, we have examined whether detergent-solubilized coated vesicle membranes will support formation of NSF-containing 'fusion complexes'. Our results show that these membranes are substantially enriched in components capable of driving formation of these complexes, when compared with membranes from other sources. Secondly, we have analysed coated vesicle preparations for their NSF content. Coated vesicle preparations contain significant amounts of NSF. This was shown to be associated with coated vesicles rather than contaminating membranes by a number of criteria, and was found to be bound in an ATP-independent manner. These findings are discussed in the light of current models for vesicle fusion.

Biological Transport↗
PubMed1982

Comparison of calmodulin binding to brain synaptic and coated vesicles.

Several characteristics of calmodulin association with brain synaptic and coated vesicles wer analyzed and compared. Radioimmunoassay revealed that both classes of vesicles contain approx. 1 microgram of calmodulin per mg of vesicle protein. Discontinuous sucrose gradients revealed that coated and synaptic vesicles preparations were homogeneous and had different sedimentation properties. Binding of 125I-labeled calmodulin to sympatic and coated vesicles was Ca2+ dependent and displaced by unlabeled calmodulin but not by troponin-C. Scatchard analysis revealed the presence of two binding sites. In both vesicle types there was one high-affinity, low-binding-capacity site (Kd = 1-39 nM and Bmax = 4-16 pmol/mg) and one low-affinity, high-binding-capacity site (Kd = 102-177 nM and Bmax = 151-202 pmol/mg). (Ca2+ +Mg2+)-ATPase activity was stimulated in both synaptic and coated vesicles by calmodulin. Thus synaptic and coated vesicles may possess similar calmodulin binding sites.

Animals↗
PubMed1994

Identification of a beta-type adaptin in plant clathrin-coated vesicles.

Plant clathrin-coated vesicles (CCV), suitably protected against proteolysis, were isolated from zucchini hypocotyls, and screened for the presence of adaptin-like polypeptides using monoclonal antibodies prepared against alpha, beta(beta') and gamma-adaptins of bovine brain. An immunoreactive polypeptide in plant CCV was only detected in the case of the beta(beta')-adaptin antibody. This polypeptide has a molecular mass of 108 kDa in SDS-PAGE, and gives rise to a major cleavage product of 70 kDa after proteolysis with trypsin. Gel filtration of 0.75 M MgCl2-dissociated coat proteins showed that the plant beta(beta')-type adaptin eluted with other polypeptides in a manner similar to the adaptor complexes of brain CCV. Upon subsequent hydroxyapatite chromatography the immunoreactive polypeptide eluted in fractions corresponding to Golgi (HA-I) rather than plasma membrane (HA-II) brain adaptor complexes. In addition, this polypeptide did not shift to a higher molecular mass when subjected to urea-SDS-PAGE. Confirmation of the presence of a beta-type adaptin in plants was provided by dot and Southern blotting experiments using genomic DNA from zucchini hypocotyls and a beta-adaptin cDNA clone from human fibroblasts.

Adaptor Protein Complex beta Subunits↗
PubMed1995

Neuropeptide-like immunoreactivities and carboxypeptide H activity associated with bovine brain clathrin coated vesicles.

Chromatographically purified clathrin coated vesicles (CCV) from bovine brain gray matter were collected and analyzed for their adaptor protein profile via SDS-PAGE, for their carboxypeptidase H (CPH) activity via radioenzymatic assays and for their NP (NPY1-36, CCK-8 and SS-14) content by radioimmunoassays. The results reveal the expression of CPH and neuropeptide-like immunoreactivities (NP-Li: NPY-Li, CCK-Li and SS-Li) associated with CCV. CPH activity associated with CCV is stimulated by cobalt, inhibited by 5 microM GEMSA and has an acidic pH optimum (4.5-6.5). The Michaelis-Menten kinetics assay of CPH in CCV reveals a Km = 28.9 microM and Vmax = 1.88 nmol/min/mg at pH 4.5 using 3H.Bz-FAR as a substrate. The NP-Li and CPH were mainly associated with the ascending portion of the CCV peak suggesting that these molecules are found in the larger diameter CCV subpopulation associated with CCV of the trans Golgi network (TGN), endocytic or recycling CCV. Analysis of the adaptor protein profile of the material eluting from the Sephacryl S-1000 column demonstrates a wide distribution of the Golgi-associated AP-1 (HA-I) molecules (AP47 and AP19) in relation to the CCV peak, spanning both the ascending and descending portions of the CCV peak. This adaptor protein pattern is consistent with the inherent size microheterogeneity of TGN CCV, large diameter CCV of the regulated secretory pathway and smaller diameter CCV of the lysosomal pathway.

Amino Acid Sequence↗