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At least 253 records · Page 14Linked to original sources
PubMed1997

A role for the hinge/ear domain of the beta chains in the incorporation of AP complexes into clathrin-coated pits and coated vesicles.

The clathrin-associated adaptor protein (AP) complexes drive the polymerization of clathrin in coated pits to form coated vesicles. It has previously been shown that the carboxyl-terminal hinge/ear domain of the beta 2 chain contains a binding site for clathrin and that removal of this domain from APs or from isolated beta 2 chains abrogates their ability to form clathrin coats in vitro. We show here that the hinge/ear domain is necessary for efficient incorporation of AP complexes into coated pits and coated vesicles in cells, a result that is consistent with the view that the beta chains indeed provide an important interaction between the AP complexes and clathrin.

Adaptor Proteins, Vesicular Transport↗
PubMed1991

Uncoating of coated vesicles by yeast hsp70 proteins.

The ability of hsp70 isoenzymes from wild-type and mutant yeast strains to uncoat bovine brain coated vesicles was analyzed and compared with that of the brain uncoating ATPase. Results show that, among the four major cytoplasmic isoenzymes produced in wild-type yeast, almost all of the activity is associated with the SSA1 and SSA2 isoenzymes. The SSB1 and SSB2 isoenzymes have almost no uncoating activity and are not found in the clathrin-hsp70 complexes formed during the uncoating reaction. Using hsp70 mutant yeast strains we find a marked difference in uncoating activity between the SSA1 and SSA2 isoenzymes, although there is only a 3% difference between their amino acid sequences. The SSA4 isoenzyme, which is produced only under stress conditions, has an uncoating activity intermediate between SSA1 and SSA2. These results suggest that the ability of hsp70 isoenzymes to uncoat clathrin-coated vesicles is restricted to certain members of the hsp70 family and can be affected by subtle changes in amino acid sequence. We also investigated the uncoating activity of mixtures of isoenzymes and find that the isoenzyme with lower uncoating activity reduces the activity of the isoenzyme with higher uncoating activity possibly by occupying binding sites on coated vesicles.

Adenosine Triphosphatases↗
PubMed1986

In vivo biosynthesis of clathrin and other coated vesicle proteins from rat liver.

A biosynthetic study of rat liver coated vesicle (CV) proteins was undertaken by using in vivo labeling with L-[35S]methionine. CVs were isolated and purified by using standard procedures and characterized by electron microscopy, sedimentation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography, or by gel slicing and liquid scintillation counting. After 5 1/2 min of labeling (the earliest time examined), incorporation of radioactive clathrin heavy-chain (180-kD (kilodalton] subunits as well as a 90-kD CV-associated protein into purified CVs was demonstrated. The level of labeled 180-kD clathrin in coated vesicles increased rapidly during the first 2 hr of labeling and then continued to rise at a slower rate between 4 and 16 hr. This slow accumulation of labeled clathrin heavy chains in the CV pool may reflect early compartmental sequestration of a fraction of newly synthesized clathrin with delayed assembly into free CVs. By 16 hr of labeling, clathrin 180-kD chains and the 90-kD CV-associated protein accounted for approximately 48 and 26%, respectively, of the radioactivity in all CV proteins. Two proteins of MWa 68 kD and 53 kD showed marked declines in cpm/unit protein between 30 min and 4 hr, raising the possibility that these species may be transferred out of CVs during or after transport without loss of the other CV proteins. The possibility is also raised that clathrin heavy chains may be recycled during CV formation. Possible heterogeneity within individual CV preparations with respect to protein composition and derivation from both plasma membrane and Golgi regions are proposed.

Animals↗
PubMed2003

Clathrin- and dynamin-dependent coated vesicle formation from isolated plasma membranes.

We have developed a new rapid cell-free assay for endocytic clathrin-coated vesicle formation using highly purified rat liver plasma membrane sheets. After incubation in the presence of cytosol and nucleotides, released vesicles were collected by high-speed centrifugation and incorporated cargo receptors were detected by Western blotting. Three different cargo receptors were internalized into vesicles while a receptor, known to be excluded from coated pits, was not. The recruitment of cargo receptors into the vesicle fraction was cytosol, ATP and temperature-dependent and was enhanced by addition of GTP. Vesicle formation in this assay was confirmed by subcellular fractionation and EM analysis. Plasma membranes stripped of their endogenous coat proteins with 0.5 m Tris retained vesicle formation activity, which was highly dependent on clathrin and dynamin. Coat proteins and dynamin were not sufficient for clathrin-coated vesicle formation, and other peripheral membrane proteins recruited from the cytosol are required. The nonhydrolyzable ATP analogue, AMPPNP did not support clathrin-coated vesicle formation; however, surprisingly, GTP gamma S was as effective as GTP. This assay will provide a powerful tool to dissect the minimum machinery and to probe the hierarchy of events involved in cargo selection and endocytic clathrin-coated vesicle formation.

Adaptor Protein Complex 2↗
PubMed1999

SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation.

Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions remain undefined. Here we report the efficient reconstitution of ATP-, GTP-, cytosol- and dynamin-dependent formation of clathrin-coated vesicles in permeabilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndapin I and amphiphysin II inhibit coated-vesicle formation in vitro through interactions with membrane-associated proteins. Most of the SH3 domains tested selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interactions between SH3 domains and their partners function sequentially in endocytic coated-vesicle formation.

3T3 Cells↗
PubMed1984

Accumulation of coated vesicles bearing mannose 6-phosphate receptors for lysosomal enzymes in the Golgi region of I-cell fibroblasts.

The distribution of mannose 6-phosphate (Man6P) receptors for lysosomal enzymes was investigated in human normal and mucolipidosis II (I-cell) disease fibroblasts by light and electron microscopic immunocytochemistry. In normal fibroblasts whose lysosomal enzymes contain Man6P recognition markers required for binding to the receptor, intracellular immunoreactive receptors were concentrated in one or two cisternae located on one side of the Golgi stack, in coated vesicles, and in lysosomes and endosomes. Presumably, this distribution delineates the Man6P-dependent transport pathway for newly synthesized lysosomal enzymes. In I-cell fibroblasts whose lysosomal enzymes lack the Man6P recognition marker, intracellular receptors were found almost exclusively in one or two Golgi cisternae and in numerous coated vesicles located near the immunoreactive cisternae but were rarely detected in lysosomes and endosomes. The results obtained on I-cell fibroblasts suggest that in the absence of endogenous ligands (lysosomal enzymes bearing Man6P residues), Man6P receptors do not constitutively shuttle-via coated vesicles-between the Golgi complex and lysosomes or endosomes. Rather, they accumulate in coated vesicles concentrated at the presumptive sorting site in the Golgi complex. The findings suggest that a specific subpopulation of coated vesicles (primary lysosomes) is involved in Man6P receptor transport of lysosomal enzymes from the Golgi complex to lysosomes and that their relocation is triggered by ligand binding.

Cells, Cultured↗
PubMed2001

Characterization of coated vesicles that participate in endocytic recycling.

While the recycling pathway of endocytosis has been shown to participate in many cellular functions, little is known regarding the transport carriers that mediate this pathway. In this study, we overexpressed a point mutant of ADP-ribosylation factor 6 (ARF6), that perturbs its GTPase cycle, to accumulate endosome-derived coated vesicles. Characterization by their purification revealed that, upon cell homogenization, these vesicles were mostly aggregated with larger noncoated membranes, and could be released with high-salt treatment. Equilibrium centrifugation revealed that these vesicles had buoyant density similar to the COP-coated vesicles. To purify the ARF6-regulated vesicles to homogeneity, enriched fractions from equilibrium centrifugation were subjected to immunoisolation through the hemagglutinin (HA) epitope of the mutant ARF6, by using a newly developed, high-affinity, anti-HA monoclonal antibody. Surface iodination of the purified vesicles revealed multiple prominent proteins. Immunoblotting with antibodies against subunits of the currently known coat proteins suggested that these vesicles have a novel coat complex. These vesicles are carriers for endocytic recycling, because they are enriched for transferrin receptor and also the v-SNARE cellubrevin that functions in transport from the recycling endosome to the plasma membrane. Thus, we have characterized transport vesicles that participate in endocytic recycling.

ADP-Ribosylation Factor 6↗
PubMed1985

Coated vesicles contain a phosphatidylinositol kinase.

When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. We have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of our CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism.

1-Phosphatidylinositol 4-Kinase↗
PubMed1993

Regulatory factors for polymerization of melanin monomers within coated vesicles and premelanosomes in melanoma cells.

Coated vesicles (CV) and premelanosomes (PMS) differ in the amount of melanin monomers produced from 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) in vitro. CV convert dopachrome preferentially to 5,6-dihydroxy-indole-2-carboxylic acid (DHI2C) and PMS mainly to 5,6-dihydroxyindole (DHI). We examined the effect of CV and PMS on in vitro spontaneous polymerization of DHI and DHI2C. The polymerization of DHI is accelerated by addition of extracts from both CV and PMS, while polymerization of DHI2C is suppressed. Factors associated with these two regulatory activities have been separated on concanavalin A-Sepharose. The ConA-adhesive glycoproteins isolated from PMS enhance polymerization of not only DHI but also DHI2C. The latter effect is stimulated by dopa. Both accelerating activities can be inhibited by phenylthiourea (PTU). In contrast to glycoproteins, the non-glycoprotein subfraction suppresses spontaneous polymerization of both melanin monomers. Our data suggest that the absence of in vivo melanin polymer formation within CV can be explained by formation by these organelles of relatively stable DHI2C rather than easily polymerizing DHI.

Animals↗
PubMed1994

Dopamine D1 and D2 receptors and their signal system present in coated vesicles prepared from bovine striatal tissue.

Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D1 and D2 receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D1 receptor antagonist, [3H]SCH 23390, and a dopamine D2 receptor antagonist, [3H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant (KD) of 0.65 and 0.5 nM, respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [3H]SCH 23390 and [3H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D1 or D2 receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [32P]NAD demonstrated predominant labeling of bands of M(r) 47,000-52,000, 42,000-45,000, and 40,000-39,000, which corresponded to the known molecular weights of the alpha subunits of Gs and Gi proteins. The presence of alpha and beta subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D2 receptor agonists, was present in the CVs. These findings suggest that the dopamine D1 and D2 receptors in the CVs couple with adenylate cyclase via Gs/Gi protein.

Adenylyl Cyclases↗
PubMed1981

Isolation and biochemical characterization of tyrosinase-rich GERL and coated vesicle in melanin synthesizing cells.

The site of condensation and activation of tyrosinase prior to melanosome melanogenesis has been investigated using subcellular fractionation and enzymic analysis of so-called Golgi apparatus of melanoma cells. Two different functional units have been found in the so-called Golgi fraction, the tyrosinase-rich GERL-coated vesicle unit and the TPPase-rich Golgi unit. The tyrosinase-rich unit consists of a tubular structure with a cisternal portion corresponding to the three-dimensional structural characteristics of GERL, and spheroid vesicles, corresponding to the currently accepted criteria for coated vesicles. This fact suggests, in agreement with electron microscopic observations, that the GERL-coated vesicle unit is a specialized portion of smooth endoplasmic reticulum having the function of activating and condensing the tyrosinase synthesized by ribosomes.

Animals↗
PubMed1981

The binding of clathrin triskelions to membranes from coated vesicles.

We have developed a quantitative method to study the interaction of radiolabeled clathrin triskelions with membranes from brain coated vesicles. Clathrin triskellions do not bind to coated vesicles unless these are stripped of their clathrin coat. The binding of triskelions is of high affinity (KD congruent to 2 x 10(-9) M), is saturable and depends on vesicle concentration. Triskelions bind to protein-sensitive structures of the stripped vesicles. Polypeptides of about 110,000 daltons appear to be involved in the binding structure. The association of triskelions to stripped vesicles was confirmed by electron microscopy.

Cell-Free System↗
PubMed1992

Evaluation of mucoadhesive polymers in ocular drug delivery. II. Polymer-coated vesicles.

Association of Carbopol 934P and Carbopol 1342 (a hydrophobic modified Carbopol resin) with phospholipid vesicles was assessed by photon correlation spectroscopy and microelectrophoresis at pH 7.4 and 5. The precorneal clearance of the polymer-coated vesicles was compared to that of uncoated vesicles by lacrimal dacryoscintigraphy in the rabbit. The mucoadhesive polymer-coated vesicles demonstrated significantly enhanced precorneal retention compared to noncoated vesicles only at pH 5 (P less than 0.005). The entrapment and subsequent release of tropicamide from Carbopol 1342-coated and uncoated liposomes were determined in vitro together with an in vivo evaluation of the vesicles formulated at the lower pH. Mucoadhesive polymer-coated vesicles failed to increase significantly the bioavailability of the entrapped tropicamide compared to uncoated vesicles and aqueous solution.

Acrylic Resins↗
PubMed1990

Novel proteins mediate an interaction between clathrin-coated vesicles and polymerizing actin filaments.

A monoclonal antibody, A-7C11, was generated which reacts with two polypeptides of 40 kDa and 80 kDa associated with the coat proteins of purified brain clathirn-coated vesicles. The 40-kDa antigen was purified and found to display actin-binding properties. Negative-staining electron microscopy showed that one of the antigens reactive with A-7C11 appears to mediate the association of isolated clathrin-coated vesicles with assembling actin filaments in vitro. Immunofluorescence microscopy of cultured fibroblasts with A-7C11 revealed the antigens aligned with both actin filaments and as punctate structures near the plasma membrane. The data suggest that the interaction between clathrin-coated vesicles and the actin cytoskeleton is mediated by antigens identified by monoclonal antibody A-7C11.

Actins↗
PubMed1995

Targeting signals and subunit interactions in coated vesicle adaptor complexes.

There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN. The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19. To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains. We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting. Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19. These observations suggest that these other subunits may play an important role in targeting. In contrast, beta- and beta'-adaptins are clearly not involved in this event. Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal. We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system. Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma-adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19. These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane.

Adaptor Protein Complex 1↗
PubMed1985

Coat formation in coated vesicles.

The proteins of Mr 100 000-110 000 present in the protein coat of coated vesicles have been shown to facilitate formation of a homogeneous small-size basket (coat) when added to clathrin [Zaremba, S., & Keen, J.H. (1983) J. Cell Biol. 97, 1339]. We have prepared this protein of coat proteins by two different methods and shown that they are very important for the binding of clathrin to uncoated vesicles to form coated vesicles. By labeling the three components (clathrin, 100 000-110 000 proteins, and uncoated vesicles) with different fluorescent markers and analyzing their distribution on sucrose gradients, we have been able to determine the composition of the products formed. In the presence of the 100 000-100 000 fraction of coat proteins, not only does the size distribution of the clathrin basket become uniform but also the rate of polymerization is strongly increased.

Animals↗
PubMed1988

Partial resolution and reconstitution of the subunits of the clathrin-coated vesicle proton ATPase responsible for Ca2+-activated ATP hydrolysis.

The clathrin-coated vesicle proton-translocating complex is composed of a maximum of eight major polypeptides. Of these potential subunits, only the 17-kDa component, which is a proton pore, has been defined functionally (Sun, S.Z., Xie, X. S., and Stone, D. K. (1987) J. Biol. Chem. 262, 14790-14794). ATPase-and proton-pumping activities of the 200-fold purified proton-translocating complex are supported by Mg2+, whereas Ca2+ will only activate ATP hydrolysis. Like Mg2+-activated ATPase activity, Ca2+-supported ATP hydrolysis is inhibited by N-ethylmaleimide, NO3-, and an inhibitory antibody and is stimulated by Cl- and phosphatidylserine. Thus, Ca2+ prevents coupling of ATPase activity to vectoral proton movement, and Ca2+-activated ATPase activity is a partial reaction useful for analyzing the subunit structure required for ATP hydrolysis. The 530-kDa holoenzyme was dissociated with 3 M urea and subcomplexes, and isolated subunits were partially resolved by glycerol gradient centrifugation. No combination of these components yielded Mg2+-activated ATPase or proton pumping. Ca2+-activated ATP hydrolysis was not catalyzed by a subcomplex containing the 70- and 58-kDa subunits but was restored by recombination of the 70-, 58-, 40-, and 33-kDa polypeptides, indicating that these are subunits of the clathrin-coated vesicle proton pump which are necessary for ATP hydrolysis.

Adenosine Triphosphate↗
PubMed2003

Dsl1p, an essential component of the Golgi-endoplasmic reticulum retrieval system in yeast, uses the same sequence motif to interact with different subunits of the COPI vesicle coat.

Dsl1p is required for Golgi-endoplasmic reticulum (ER) retrograde transport in yeast. It interacts with the ER resident protein Tip20p and with delta-COP, a subunit of coatomer, the coat complex of COPI vesicles. To test the significance of these interactions, we mapped the different binding sites and created mutant versions of Dsl1p and delta-COP, which are unable to bind directly to each other. Three domains were identified in Dsl1p: a Tip20p binding region within the N-terminal 200 residues, a highly acidic region in the center of Dsl1p containing crucial tryptophan residues that is required for binding to delta-COP and essential for viability, and an evolutionarily well conserved domain at the C terminus. Most importantly, Dsl1p uses the same central acidic domain to interact not only with delta-COP but also with alpha-COP. Strong interaction with alpha-COP requires the presence of comparable amounts of epsilon-COP or beta' -COP. Thus, the binding characteristics of Dsl1p resemble those of many accessory factors of the clathrin coat. They interact with different layers of the vesicle coat by using tandemly arranged sequence motifs, some of which have dual specificity.

Amino Acid Motifs↗