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 163 records · Page 9Linked to original sources
PubMed1986

Isolation and reconstitution of the clathrin-coated vesicle proton translocating complex.

Clathrin-coated vesicles contain a proton translocating ATPase which is insensitive to azide but inhibited by N-ethylmaleimide. The ATP hydrolytic subunit of this proton pump has been solubilized, partially purified, and reconstituted into H+-ATPase-depleted coated vesicle membranes (Xie, X.-S., Stone, D.K., and Racker, E. (1984) J. Biol. Chem. 259, 11676-11678). In this communication we report that the entire proton transporting complex has been solubilized and purified 200-fold. The complex, when reconstituted into brain lipid liposomes, catalyzes azide-resistant, N-ethylmaleimide-sensitive H+ transport manifested as both generation of a pH gradient and an electrical gradient. The complex has an apparent molecular mass of 530 kDa.

Animals↗
PubMed1986

Cyclic nucleotide phosphodiesterase activity in bovine brain coated vesicles.

Cyclic AMP phosphodiesterase activity in bovine brain coated vesicles displayed a Km of approximately 22 microM for cyclic AMP, a Vmax of 3.2 nmol/min/mg protein, and a Hill coefficient of 1.5, suggesting positive cooperativity. The enzyme activity was stimulated by cyclic GMP with maximal indexes of stimulation ranging between 40 and 300%. Both basal and stimulated phosphodiesterase activities were immunotitrated with polyclonal antibodies against clathrin attached to heat-inactivated, formaldehyde-fixed Staphylococcus aureus cells. The main form of phosphodiesterase activity present in the immunoprecipitated brain coated vesicle preparation also is stimulated by cyclic GMP. The allosteric behavior was modulated by cyclic GMP. All of these properties are typical of type II or cyclic GMP-sensitive phosphodiesterases in addition to their calcium and calmodulin independence. Competition experiments with a series of phosphodiesterase inhibitors, papaverine, 1-methyl-3-isobutylxanthine, and theophylline, showed inhibition of cyclic AMP hydrolysis. Trifluoperazine was inactive at the highest concentration used, 100 microM. These compounds also inhibited the cyclic GMP-stimulated cyclic AMP hydrolysis with trifluoperazine practically inactive. At 5 microM cyclic AMP none of the inhibitors was seen to stimulate the cyclic AMP hydrolytic activity. The presence of an enzyme for the breakdown of cyclic nucleotides in brain coated vesicles may suggest a role for these second messengers in the in vivo functions of this organelle.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

G-protein-coupled A1 adenosine receptors in coated vesicles of mammalian brain: characterization by radioligand binding and photoaffinity labelling.

A1 adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaffinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1,3-[3H]dipropyl-xanthine ([3H]DPCPX) gave a Kd value of 0.7 nM and a Bmax value of 82 +/- 13 fmol/mg protein. For the highly A1-selective agonist 2-chloro-N6-[3H]cyclopentyladenosine ([3H]CCPA) a Kd value of 1.7 nM and a Bmax value of 72 +/- 29 fmol/mg protein was estimated. Competition of agonists for [3H]DPCPX binding gave a pharmacological profile with R-N6-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5'-N-ethylcarboxamidoadenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCPA to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N6-125I-p-hydroxyphenylisopropyladenosine ([125I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A1 receptors labelled in brain membranes. Therefore, coated vesicles contain A1 adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A1 receptors in these vesicles are not a processed receptor form. These results confirm that A1 receptors in coated vesicles are coupled to a G-protein, and it appears that the A1 receptor systems in coated vesicles and in plasma membranes are identical.

Affinity Labels↗
PubMed1978

Coated vesicles: characterization, selective dissociation, and reassembly.

Sodium dodecyl sulfate/polyacrylamide gels of coated vesicles from porcine brain (mean 76% coated vesicles) show three major proteins (180,000, 125,000, and 55,000 daltons) that account for 73% of the total protein. Preparations consisting predominantly of coats (65%) have less of the 55,000-dalton protein. Clathrin (180,000 daltons) comprises 40% of the protein of a coated vesicle. Conditions of 2 M urea, 0.25 M MgCl2, or pH 7.5 disrupt the coat and solubilize clathrin. Solubilized clathrin reforms coat structures after dilution of urea or MgCl2. High-pH-solubilized clathrin reassembles after dialysis against buffer at pH 6.5 containing dithiothreitol (5 mM). Reassembled coats are predominantly clathrin.

Animals↗
PubMed2000

Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells.

Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to auxilin; like auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not auxilin, are uncoated by Hsc70, suggesting that GAK acts as an auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.

Adaptor Proteins, Vesicular Transport↗
PubMed1996

A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding.

Formation of non-clathrin-coated vesicles requires the recruitment of several cytosolic factors to the Golgi membrane. To identify membrane proteins involved in this budding process, a highly abundant type I transmembrane protein (p23) was isolated from mammalian Golgi-derived COPI-coated vesicles, and its cDNA was cloned and sequenced. It belongs to the p24 family of proteins involved in the budding of transport vesicles (Stamnes, M.A., M.W. Craighead, M.H. Hoe, N. Lampen, S. Geromanos, P. Tempst, and J.E. Rothman. 1995. Proc. Natl. Acad. Sci. USA. 92:8011-8015). p23 consists of a large NH2-terminal luminal domain and a short COOH-terminal cytoplasmic tail (-LRRFFKAKKLIE-CO2-) that shows similarity, but not identity, with the sequence motif-KKXX-CO2-, known as a signal for retrieval of escaped ER-resident membrane proteins (Jackson, M.R., T. Nilsson, and P.A. Peterson. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:3153-3162; Nilsson, T., M. Jackson, and P.A. Peterson. 1989. Cell. 58:707-718). The cytoplasmic tail of p23 binds to coatomer with similar efficiency as known KKXX motifs. However, the p23 tail differs from the KKXX motif in having an additional motif needed for binding of coatomer. p23 is localized to Golgi cisternae and, during vesicle formation, it concentrates into COPI-coated buds and vesicles. Biochemical analysis revealed that p23 is enriched in vesicles by a factor of approximately 20, as compared with the donor Golgi fraction, and is present in amounts stoichiometric to the small GTP-binding protein ADP-ribosylation factor (ARF) and coatomer. From these data we conclude that p23 represents a Golgi-specific receptor for coatomer involved in the formation of COPI-coated vesicles.

Amino Acid Sequence↗
PubMed1988

Rapid purification of clathrin-coated vesicles by free-flow electrophoresis.

Free-flow electrophoresis was successfully used as the final step in the purification of clathrin-coated vesicles from bovine brain. Based on biochemical analysis, the material obtained in this way was found to be of equal purity with respect to the protein composition and lipid content as that purified by the previously widely used methods of permeation chromatography on controlled pore glass or Sephacryl S-1000. However, as judged by electron microscopy, the electrophoretically purified coated vesicles contained less smooth membranes than the coated vesicle preparations that had been obtained by permeation chromatography. Free-flow electrophoresis offers considerable advantages in speed of purification, in the total amount of material processed and in flexibility of operation. Analysis of the electrophoretic mobility of purified coated vesicles showed that this is governed by the coat proteins rather than by the vesicle contained therein. A shift in electrophoretic mobility of purified coated vesicles was obtained by the binding of coat protein specific monoclonal antibodies. This raises the possibility of purifying subpopulations of coated vesicles with respect to coat protein composition.

Animals↗
PubMed1980

Immunocytochemical localization of coated vesicle protein in rodent nervous system.

Immunocytochemistry has been used to study the distribution of the major 180,000-mol wt protein of coated vesicles in rodent cerebellum. An antibody to the coat protein was prepared in rabbits and characterized by immunodiffusion and immunofixation of polyacrylamide gels. At the light microscope level the protein was primarily localized in punctate profiles surrounding Purkinje cells and within the cerebellar glomeruli. At the electron microscope level the punctate distribution was confined to presynaptic terminals of basket and Golgi II neurons as well as mossy fiber terminals of the glomeruli. This label was heaviest on the lattice coat of coated vesicles but, in addition, label was found within the presynaptic axoplasm and along the cytoplasmic surface of the plasmalemma. Coated vesicles in cell somata were labeled as well as the cytosol around groupings of these vesicles. These data suggest that there may be two forms (or more) of coated vesicle protein in neurons, a lattice form associated with coated vesicles and a soluble form associated with the cytoplasmic matrix.

Animals↗
PubMed1995

Comparative biochemical study of coated vesicles purified from cock brains.

Cock brain coated vesicles (CBCVs) were purified and compared with porcine brain coated vesicles (PBCVs) from several biochemical aspects. Clathrin heavy and light chains of CBCVs were immunologically similar to those of PBCVs. Coat proteins (CPs) of CBCVs behaved in almost the same manner as those of PBCVs for limited proteolysis. Dissociation of CPs from CBCVs by treatment with 10 mM Tris-Cl, pH 8.5, or 2M urea resembled that of CPs from PBCVs. pH dependency of dissociation of CPs from CBCVs was slightly different from those of PBCVs.

Animals↗
PubMed2006

Evolution of intraflagellar transport from coated vesicles and autogenous origin of the eukaryotic cilium.

The cilium/flagellum is a sensory-motile organelle ancestrally present in eukaryotic cells. For assembly cilia universally rely on intraflagellar transport (IFT), a specialised bidirectional transport process mediated by the ancestral and conserved IFT complex. Based on the homology of IFT complex proteins to components of coat protein I (COPI) and clathrin-coated vesicles, we propose that the non- vesicular, membrane-bound IFT evolved as a specialised form of coated vesicle transport from a protocoatomer complex. IFT thus shares common ancestry with all protocoatomer derivatives, including all vesicle coats and the nuclear pore complex (NPC). This has major implications for the evolutionary origin of the cilium. First, it reinforces the tenet that duplication and divergence of pre-existing structures, rather than symbiosis, were the major themes during cilium evolution. Second, it suggests that the initial step in the autogenous origin of the cilium was the establishment of a membrane patch with transmembrane proteins transported by the ancestral vesicle-coating IFT complex. We propose a scenario for how the initial membrane patch gradually protruded to enhance exposure to the environment, then started to move, and finally compartmentalised to render receptor signalling and ciliary beating more efficient.

Animals↗
PubMed1981

The role of clathrin-coated vesicles in acrosome formation.

Ultrastructural and cytochemical observations are presented which suggest that coated vesicles originating from the trans (distal) aspect of the rat spermatid Golgi apparatus transport lysosomal enzymes from the Golgi to the developing acrosome. Using an affinity purified antibody against the clathrin coats of pig brain coated vesicles, immunofluorescence and immuno-electron microscopic studies were carried out on frozen sections of rat seminiferous tubules. The results showed a specific labelling of the Golgi-acrosome region which corresponded precisely with the location of the coated vesicles in Epon sections. We conclude tht a least some of the coated vesicles in this region have clathrin coats.

Acid Phosphatase↗
PubMed1993

Isolation of a protein activator of the clathrin-coated vesicle proton pump.

An activator of the clathrin-coated vesicle proton translocating ATPase has been purified 1600-fold from bovine brain. The activator, which requires detergent (polyoxyethylene 9-lauryl ether) for release from clathrin-coated vesicles, is heat-stable, trypsin-sensitive, and has an apparent molecular mass of about 6 kDa as determined by high performance liquid chromatography. The activator stimulates the purified H(+)-ATPase of coated vesicles over 50-fold under acidic conditions. Similarly, the activator stimulates proton pumping catalyzed by the reconstituted proton pump. Importantly, this stimulation of proton pumping is observed only when the activator is reconstituted into the interior of the proteoliposomes. Moreover, the activator protein is demonstrated to protect, and co-sediment with, purified proton pump during glycerol gradient centrifugation performed in the presence of ATP. These observations support the notion that this activator serves to determine the pH set point of acidic endomembranes through interactions with the transmembranous sectors of the proton pump.

Animals↗
PubMed1977

Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum.

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.

Adenosine Triphosphatases↗
PubMed1997

Direct measurement of clathrin-coated vesicle formation using a cell-free assay.

Factors controlling the last stages of clathrin-coated vesicle formation were investigated using an assay allowing direct measurement of the detachment of these vesicles from the plasma membrane. Plasma membranes from cultured cells surface-labelled with 125I-alpha2-macroglobulin (a ligand that preferentially associates with clathrin-coated pits) were isolated by sonication of cells attached to a poly-L-lysine-coated substratum and incubated in the presence of nucleotide(s) +/- cytosol. A significant proportion of the membrane-associated radioactivity was released into the incubation medium in sedimentable form (14x10(6)g). The nucleotide and ligand specificities of this process together with the results of a series of biochemical, morphological and gradient analyses, led to the conclusion that measurement of the released sedimentable radioactivity provides a direct estimate of the formation of clathrin-coated vesicles from clathrin-coated pits. A morphological analysis of quick-frozen replicas of these membranes indicated that only the last stages of clathrin-coated vesicle formation were studied in the assay. Taking advantage of this cell-free system, we demonstrate that membrane-associated cytosolic factors and GTP-binding proteins, noteably dynamin, play a crucial role. Moreover, although these events can occur in the absence of ATP and Ca2+, optimal conditions for the formation of clathrin-coated vesicles require the presence of ATP, GTP and cytosol.

3T3 Cells↗
PubMed1982

Instability of coated vesicles in concentrated sucrose solutions.

A method of preparing homogeneous coated vesicles that eliminates the high sucrose concentrations heretofore used is presented. It is shown that sucrose at high concentrations dissociates the coat from coated vesicles. This reaction can explain the presence of empty coats observed with preparations obtained with high concentrations of sucrose. The protein and membrane lipid components have been analyzed by the intrinsic tryptophan and extrinsic diphenylhexatriene fluorescence, respectively. Analysis of mixtures of coated vesicles and baskets resolved the contributions of the two species to the fluorescence curves.

Animals↗
PubMed1997

Clathrin-coated vesicle formation and protein sorting: an integrated process.

Clathrin-coated vesicles were the first discovered and remain the most extensively characterized transport vesicles. They mediate endocytosis of transmembrane receptors and transport of newly synthesized lysosomal hydrolases from the trans-Golgi network to the lysosome. Cell-free assays for coat assembly, membrane binding, and coated vesicle budding have provided detailed functional and structural information about how the major coat constituents, clathrin and the adaptor protein complexes, interact with each other, with membranes, and with the sorting signals found on cargo molecules. Coat constituents not only serve to shape the budding vesicle, but also play a direct role in the packaging of cargo, suggesting that protein sorting and vesicle budding are functionally integrated. The functional interplay between the coated vesicle machinery and its cargo could ensure sorting fidelity and packaging efficiency and might enable modulation of vesicular trafficking in response to demand.

Adaptor Protein Complex alpha Subunits↗
PubMed1986

Detection and characterization of beta-adrenergic receptors and adenylate cyclase in coated vesicles isolated from bovine brain.

To assess whether internalization of beta-adrenergic receptor occurs in the CNS, we have isolated clathrin-coated vesicles from bovine forebrain and examined them for the presence of beta-adrenergic receptor binding and adenylate cyclase activities. A coated vesicle enriched preparation isolated by successive D2O-Ficoll density gradient centrifugations was applied to a glass bead permeation column to achieve further purification. Two major peaks of protein were eluted from the column and monitored by electron microscopy and SDS-PAGE. Peak II contained almost exclusively coated vesicles (98%), whereas peak I, which appeared in the void volume, contained larger smooth vesicles and few coated vesicles. beta-Adrenergic receptor binding to peaks I and II was measured with 125I-cyanopindolol (CYP) as ligand in Sepharose 4B column assays. 125I-CYP was found to bind specifically and saturably to both peaks I and II with a Bmax of 28 +/- 4 and 32 +/- 3 fmol/mg protein, respectively. 3H-CGP 12177, a hydrophilic beta-adrenergic receptor ligand, did not label receptors present in peak II, but it specifically bound to synaptic plasma membranes (SPM) prepared from bovine hippocampus and, to a lesser extent, to peak I. These results suggest that receptors present in coated vesicles are cryptic in nature. In the displacement of 125I-CYP binding by (-)-isoproterenol, addition of 50 microM GppNHp caused a significant "right shift" with SPM and peak I but not the peak II preparation. Adenylate cyclase activities could also be detected in both peaks I and II (specific activities, 21 +/- 0.6 and 24 +/- 0.5 pmol cAMP/mg protein/min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1993

Structure of clathrin-coated vesicles from small-angle scattering experiments.

Previously published small-angle neutron and X-ray scattering data from coated vesicles, reassembled coats, and stripped vesicles have been analyzed in terms of one common model. The neutron data sets include contrast variation measurements at three different D2O solvent concentrations. The model used for interpreting the data has spherical symmetry and explicitly takes into account polydispersity, which is described by a Gaussian distribution. A constant thickness of the clathrin coats is assumed. The fitting of the model shows that the coated vesicles consist of a low-density outer protein shell (clathrin) and a central protein shell (accessory polypeptides and receptors) of approximately six times higher density. For the X-ray scattering and neutron contrast variation data, the polydispersity of the samples is of the order of 90 A (full-width-at-half-maximum value) and the average outer radius is approximately 400 A. The inner high-density shell has inner and outer radii of 115 and 190 A, respectively. A simultaneous fit to the three neutron contrast variation data sets identifies the lipid membrane with a thickness of 40 A and an outer radius of 196 A. Thus, the membrane and the high-density protein shell overlap in space, which shows that the lipid membrane contains protein. The molecular mass of the average particle is 27 x 10(6) Da. The coated vesicles consist, on average, of approximately 85% protein and 15% lipids. About 40% of the protein mass is situated in the central high-density shell, which gives a large amount of protein in the lipid membrane. The densities of the central shell and the lipid membrane show that the hydration is small in the central region. A comparison of the total mass, the mass distribution, and the structure of the average-size particles with the barrel structure shows that the accessory polypeptides are incorporated in the lipid membrane. The results from the neutron data for the reassembled coats show that the structure of these particles is very similar to the structure of the native coats. The main difference is a higher density of the central protein shell, which shows that the membrane is replaced by protein in the reassembled coats.

Clathrin↗