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PubMed1984

Coated pits and coated vesicles in unicellulars (Tetrahymena, Crithidia) and in Hydra.

Coated pits and coated vesicles were observed in the ciliated unicellular Tetrahymena, in the flagellated unicellular Crithidia, and in Hydra. In the unicellulars the coated structures localized for the most part near to the origin of cilia or flagella, and many were present around the Golgi complex, whereas in Hydra they occurred in nonspecific random locations. Since the membrane receptors of the unicellulars serve originally as food receptors, the coated pits and vesicles are presumably involved in food selection, and represent as such the primitive form of the receptor-mediated ligand internalization mechanism operative in higher organisms.

Animals↗

PubMed1987

Female sterile (1) yolkless: a recessive female sterile mutation in Drosophila melanogaster with depressed numbers of coated pits and coated vesicles within the developing oocytes.

Ultrastructural analysis of developing oocytes produced by the recessive female sterile mutant, yolkless (yl), in Drosophila melanogaster shows that yl+ gene activity is necessary for coated pit and coated vesicle formation within these oocytes. 29 alleles of the mutation are known to exist, and they fall either within a strongly affected class or a weakly affected class. Analysis of oocytes produced by females homozygous for the strongly affected class of alleles shows a greater than 90% reduction in the numbers of coated pits and coated vesicles. These oocytes have very little proteinaceous yolk, and the females accumulate vitellogenin (the yolk protein precursor) within their hemolymph. Moreover, females homozygous or hemizygous for a given strong allele produce mature oocytes that are flaccid. Alternatively, females homozygous or hemizygous for weak alleles produce yolk-filled oocytes, but the number of coated pits and coated vesicles within these oocytes is 50% of that found in the oocytes of wild-type females. Despite the presence of yolk within these oocytes, females homozygous for weak yl- alleles remain sterile, and their mature oviposited eggs collapse with time.

Alleles↗

PubMed1991

Immunolocalization of the vacuolar-type (H+)-ATPase from clathrin-coated vesicles.

Proton-translocating ATPases of the vacuolar class (V-ATPases) are found in a variety of animal cell compartments that participate in vesicular membrane transport, including clathrin-coated vesicles, endosomes, the Golgi apparatus, and lysosomes. The exact structural relationship that exists among the V-ATPases of these intracellular compartments is not currently known. In the present study, we have localized the V-ATPase by light and electron microscopy, using monoclonal antibodies that recognize the V-ATPase present in clathrin-coated vesicles. Localization using light microscopy and fluorescently labeled antibodies reveals that the V-ATPase is concentrated in the juxtanuclear region, where extensive colocalization with the Golgi marker wheat germ agglutinin is observed. The V-ATPase is also present in approximately 60% of endosomes and lysosomes fluorescently labeled using alpha 2-macroglobulin as a marker for the receptor-mediated endocytic pathway. Localization using transmission electron microscopy and colloidal gold-labeled antibodies reveals that the V-ATPase is present at and near the plasma membrane, alone or in association with clathrin. These results provide evidence that the V-ATPases of plasma membrane, endosomes, lysosomes, and the Golgi apparatus are immunologically related to the V-ATPase of clathrin-coated vesicles.

Animals↗

PubMed2001

Live stripping of clathrin-coated vesicles.

Vesicle budding requires recruitment of a coat, which must then be removed to allow fusion with the target compartment. In vitro assays have implicated Hsc70 and auxilin family members as key players in clathrin-coated vesicle uncoating. New in vivo studies now show that this is indeed the case and reveal additional functions of the Hsc70/auxilin complex.

Adaptor Proteins, Vesicular Transport↗

PubMed1992

Clathrin-associated proteins of bovine brain coated vesicles. An analysis of their number and assembly-promoting activity.

In search of hitherto undiscovered coat proteins, clathrin-associated proteins (APs) from coated vesicles of bovine brain were affinity-purified by one cycle of coat assembly and fractionated by gel filtration on Superose 6. Immunochemical and gel electrophoretic analysis of the fractions revealed, besides AP180, auxilin, HA1, and HA2, a component with M(r) approximately 140,000. This protein (p140) is present in coated vesicles in about equimolar proportion to auxilin. The contribution of HA1, HA2, AP180, and auxilin to the total assembly activity in the Tris-soluble coat protein fraction were quantitatively analyzed by measuring the reduction in activity when each protein was removed from the mixture by immunoaffinity chromatography. It was found that AP180 accounts for 61% and HA2 for 33% of the activity, whereas auxilin, HA1, and p140 made a negligible contribution. Based on the relative molar concentration of APs in the coat protein fraction, AP180 is about 4 times more active in promoting clathrin assembly than are HA2 or the other APs.

Adaptor Proteins, Vesicular Transport↗

PubMed2001

Biological basket weaving: formation and function of clathrin-coated vesicles.

There has recently been considerable progress in understanding the regulation of clathrin-coated vesicle (CCV) formation and function. These advances are due to the determination of the structure of a number of CCV coat components at molecular resolution and the identification of novel regulatory proteins that control CCV formation in the cell. In addition, pathways of (a) phosphorylation, (b) receptor signaling, and (c) lipid modification that influence CCV formation, as well as the interaction between the cytoskeleton and CCV transport pathways are becoming better defined. It is evident that although clathrin coat assembly drives CCV formation, this fundamental reaction is modified by different regulatory proteins, depending on where CCVs are forming in the cell. This regulatory difference likely reflects the distinct biological roles of CCVs at the plasma membrane and trans-Golgi network, as well as the distinct properties of these membranes themselves. Tissue-specific functions of CCVs require even more-specialized regulation and defects in these pathways can now be correlated with human diseases.

Adaptor Proteins, Vesicular Transport↗

PubMed1990

Dissociation, cross-linking, and glycosylation of the coated vesicle proton pump.

In order to refine further our structural model of the coated vesicle (H+)-ATPase (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802), we have extended our structural analysis to identify peripheral and glycosylated subunits of the pump as well as to identify subunits which are in close proximity in the native (H+)-ATPase complex. Treatment of the purified, reconstituted (H+)-ATPase with 0.30 M KI in the presence or absence of ATP or MgATP results in the release of the 73-, 58-, 40-, 34-, and 33-kDa subunits, leaving behind the 100-, 38-, 19-, and 17-kDa subunits in the membrane. Because the former group of polypeptides is released from the membrane in the absence of detergent, they correspond to peripheral membrane proteins. To determine which subunits are in close proximity, cross-linking of the purified (H+)-ATPase was carried out using the cleavable, bifunctional amino reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by two-dimensional gel electrophoresis. These studies indicate that contact regions exist between the 73- and 58-kDa subunits as well as between the 17-kDa subunit and the 40-, 34-, and 33-kDa subunits. To test for glycosylation of the (H+)-ATPase, the detergent-solubilized complex was treated with neuraminidase followed by electrophoresis and blotting using a peanut lectin/horseradish peroxidase conjugate. Galactose-inhibitable staining of the 100-kDa subunit, together with affinity chromatography of the intact (H+)-ATPase on peanut lectin agarose, indicates that the 100-kDa subunit is glycosylated, most likely at a site exposed on the luminal side of the membrane. These results, together with those presented in the preceding paper (Adachi, I., Arai, H., Pimental, R., and Forgac, M. (1990) J. Biol. Chem. 265, 960-966), were used in the construction of a refined model of the coated vesicle (H+)-ATPase.

Biological Transport, Active↗

PubMed1990

Analysis of 100-180-kDa phosphoproteins in clathrin-coated vesicles from bovine brain.

Protein kinases which co-purify with clathrin-coated vesicles are known to phosphorylate in vitro the 50-kDa subunit of the HA-II adaptor complex and upon inclusion of polylysine the beta-light chain of clathrin and polypeptides above 100 kDa. Here we relate the high molecular mass phosphoproteins to the known subunits of the adaptor protein complexes and to other clathrin-associated proteins by means of immunoprecipitation with monoclonal antibodies, two-dimensional electrophoresis, or electrophoresis in urea-sodium dodecyl sulfate-polyacrylamide gels. Our results show that some of the labeling of the 100-120-kDa region is accounted for by the beta'- and gamma-subunits of the HA-I adaptor complex, the alpha a-, and, to a lesser extent, by the beta-subunits of the HA-II adaptor complex. In addition, we found the assembly protein AP 180 and a hitherto undescribed 110-kDa coat polypeptide to be heavily phosphorylated upon release of these proteins from the coated vesicle membrane. In all cases, labeling was confined to serine residues.

Amino Acids↗

PubMed1984

The association of clathrin fragments with coated vesicle membranes.

The association between clathrin triskelions and the clathrin-stripped membranes of coated vesicles has been investigated using a filter assay to separate bound from unbound clathrin. The filter assay is more sensitive and less cumbersome than a sedimentation assay used previously (1). While confirming the high affinity interaction between clathrin and the vesicle membrane, our results yield Scatchard plots that are curvilinear and consistent with a positively cooperative interaction between clathrin and the vesicle membranes. Controlled digestion with trypsin removes the distal portions of the triskelion legs leaving the proximal 31 nm portions that form the hub of the triskelions. These hubs are trimers of large 112,000- and 124,000-dalton fragments of clathrin heavy chains. They competitively inhibit the binding of 125I-labeled intact triskelions to stripped vesicles with a KI identical to the KD for the association of 125I-labeled intact triskelions to stripped vesicles. Furthermore, these large fragment trimers bind to stripped vesicles with approximately the same high affinity as do intact triskelions and also show evidence of a positively cooperative interaction. It is concluded that clathrin binds to coated vesicles by an interaction that is mediated by the proximal 112,000-dalton fragment of the clathrin heavy chains.

Animals↗

PubMed1988

In vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes.

We have studied the in vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes. The major 32P-labeled polypeptides of clathrin-coated vesicles isolated from metabolically labeled cells were the the 165-, 100-110-, and 50-kDa polypeptides of the assembly protein, the clathrin beta-light chain, and to a lesser extent the clathrin alpha-light chain. The phosphorylation of the assembled (particulate) and unassembled (soluble) pools of clathrin and assembly protein was compared by immunoprecipitating the respective protein complexes from particulate and soluble cell fractions. Although all the phosphorylated polypeptides were present in both fractions, the extent of labeling was protein and fraction specific: the apparent specific activities of the assembly protein 50-kDa polypeptide and clathrin light chain were higher in the unassembled pool, whereas those of the 100-110-kDa polypeptides were higher in the assembled pool. The amino acids and polypeptide fragments labeled in vivo appeared similar to those labeled in vitro.

Animals↗

PubMed1996

Reconstitution of ATPase activity from individual subunits of the clathrin-coated vesicle proton pump. The requirement and effect of three small subunits.

The vacuolar-type proton pump of clathrin-coated vesicles is composed of two general domains, a peripheral, catalytic sector (VC) and a transmembranous proton channel (VB). In its native form, the enzyme can hydrolyze both MgATP and CaATP, whereas VC, when separated from VB, loses its MgATPase activity and switches to a state that can hydrolyze only CaATP. Further dissociation of VC results in subcomplexes that are depleted of one or more subunits and lack ATPase activity altogether. Reconstitution of recombinant subunits to these biochemically prepared subcomplexes has demonstrated the necessity of polypeptides of 70, 58, 40, and 33 kDa (subunits A, B, C, and E, respectively) for CaATPase activity of the VC complex. The current studies demonstrate that mixtures of these four recombinant subunits cannot support CaATPase activity in the absence of a biochemically prepared subcomplex. Investigation of the other components required for ATPase activity has led to the identification of three additional polypeptides present in preparations of VC, with apparent molecular masses of 15, 14, and 10 kDa. Each of these proteins was found to activate ATPase activity of mixtures of subunits A, B, C, and E. In addition, ATPase reconstituted from these individual subunits hydrolyses ATP, not only in the presence of Ca2+ but also in the presence of Mg2+. Investigation of the individual properties of these three subunits revealed that the 10-kDa polypeptide is subunit F, as determined by immunoblot analysis. This subunit had no effect on MgATPase activity of VC but stimulated CaATPase activity 6-fold in the presence of subunit D. Under optimal conditions the 14-kDa component resulted in a 10-fold stimulation and the 15-kDa component a 20-fold stimulation of MgATPase activity; based on this observation, the 14- and 15-kDa polypeptides were named subunits G and H, respectively. In addition, proton pumping activity was reconstituted through the reassembly of subunits A-H with VB and SFD, a previously described pump component composed of polypeptides of 50 and 57 kDa (Xie, X.-S, Crider, B.P., Ma, Y. M., and Stone, D. K. (1994) J. Biol. Chem. 269, 25809-25815). Together, these experiments completely define the catalytic center of the vacuolar proton pump of clathrin-coated vesicles.

Animals↗

PubMed1993

Beta very low density lipoprotein and clathrin-coated vesicles co-localize to microvilli in pigeon monocyte-derived macrophages.

Macrophages derived from blood monocytes are key in the development of atherosclerosis, as monocyte migration into the intima and accumulation of cholesterol leads to foam cell formation. To investigate the relationship between lipoprotein binding and the distribution of clathrin-coated endocytic vesicles, monocyte-derived macrophages were exposed in vitro to beta very low density lipoprotein (beta VLDL), conjugated to colloidal gold, and later were processed for immuno-electron microscopy to localize clathrin-coated vesicles. The immunolocalization was done in conjunction with either cryosectioning or whole mount intermediate voltage electron microscopy. Preferential binding of beta VLDL on small membrane ruffles and microvilli was quantitatively verified. Clathrin-coated vesicles were distributed throughout the cell; however, clusters of microvilli were associated with both a high concentration of coated vesicles and lipoprotein. Small membrane ruffles were not associated with clathrin-coated vesicles. These data support our hypothesis that endocytosis of beta VLDL near microvilli involves coated vesicles, whereas endocytosis of beta VLDL near ruffles is not mediated by coated endocytic vesicles. Furthermore, the association of coated vesicles with microvilli but not membrane ruffles may be important in understanding ligand trafficking within the cell. Given the distribution of coated vesicles within the cell, it is possible that the site of lipoprotein binding may determine the mechanism of entry into the cell and the metabolic effects of the internalized ligand.

Animals↗

PubMed1993

Redistribution of clathrin-coated vesicle adaptor complexes during adipocytic differentiation of 3T3-L1 cells.

Mechanisms for intracellular retention of proteins are induced during adipocytic differentiation of 3T3-L1 cells. To investigate the potential role of clathrin lattices in these retention processes, we performed a morphological and biochemical analysis of coated vesicle components in 3T3-L1 cells. Optical sectioning and image restoration revealed a marked increase in the staining of clathrin and beta adaptins in the perinuclear region of cells with differentiation. In addition, predominance of beta (subunit of the AP-2, plasma membrane adaptor) over beta' (subunit of the AP-1, Golgi adaptor) adaptin was observed in immunoblots of clathrin-coated vesicles purified from nondifferentiated fibroblasts, and this ratio was reversed in coated vesicles purified from differentiated adipocytes. These results indicate that the relative abundance of TGN-derived clathrin lattices increases markedly during adipocytic differentiation. Subcellular fractionation indicated that cytosolic AP-1 and AP-2 adaptors comprised approximately 70% of the total cellular adaptor pool. Interestingly, neither the concentration nor the relative ratio of cytosolic AP-1 to AP-2 adaptors increased significantly during differentiation. These data suggest that the increase in TGN-derived lattices results from differentiation-induced mechanisms for enhanced assembly or stabilization of adaptors on Golgi membranes. Interestingly, double-immunofluorescence microscopy also revealed that whereas extensive colocalization between clathrin and beta adaptins occurred both in fibroblasts and adipocytes, structures stained only with anti-adaptin antibody could be detected. Taken together these results suggest that membranes coated with adaptors, but not clathrin, can exist in these cells.

3T3 Cells↗

PubMed1989

Detection and characterization of 3H-clonidine binding sites in coated vesicles isolated from bovine brain.

The binding of 3H-clonidine to bovine brain coated vesicles was studied. The binding was reversible and saturable. Saturation studies revealed a one-site interaction: Kd was 8.7 nM and Bmax was 24.5 fmol/mg protein. Noradrenaline, yohimbine and phenoxybenzamine displaced 3H-clonidine binding from the binding sites at concentrations of 10(-7)-10(-4) M. The results indicate that the coated vesicles from the bovine cerebral cortex contain 3H-clonidine binding sites comparable to the low affinity sites of alpha 2-adrenergic receptors in bovine brain membranes.

Adrenergic alpha-Agonists↗

PubMed1988

Melanin monomers within coated vesicles and premelanosomes in melanin synthesizing cells.

We have found substantial amounts (6.6-143 and 0.5-13 micrograms/mg. protein, respectively) of 5,6-dihydroxyindole (5,6-DHI) and 5,6-dihydroxyindole-2-carboxylic acid (5,6-DHI2C), which are key intermediate monomers for the formation of the eumelanin polymer, within coated vesicle fraction of pigment cells. In addition, the amounts of these eumelanin monomers have been found to decrease along with the process of eumelanin polymer formation from coated vesicles to premelanosomes and finally to melanosomes among melanogenic subcellular compartments. Our present findings seem to indicate that coated vesicles transfer not only highly glycosylated T1-tyrosinase but also eumelanin monomers into premelanosomes.

Animals↗

PubMed2002

CK2 and GAK/auxilin2 are major protein kinases in clathrin-coated vesicles.

Several peripheral membrane proteins associated with clathrin-coated vesicles (CCVs) are reversibly phosphorylated, but it is not clear precisely which protein kinases are involved. In order to address this question directly, we have isolated highly purified CCVs from porcine brain. The peripheral membrane proteins have been removed and assayed for kinase activity using the CCV peripheral membrane proteins as substrate. The major kinase activity identified has a molecular mass of 40 kDa, is inhibited by known specific inhibitors of the protein kinase CK2 and is recognised by an antibody specific to CK2. We show that CK2 is responsible for the phosphorylation of the majority of CCV-associated proteins that are subject to phosphorylation. Intriguingly, CK2 is inactive when associated with CCVs but becomes active once the clathrin coat has been removed. The medium subunit of the AP2 adaptor complex (mu2) is not a substrate for CK2, but is phosphorylated by a second kinase that we show to be cyclin G-associated kinase (GAK/auxilin2). Unlike the situation for the CK2 substrates, mu2 is a substrate for GAK/auxilin2, both in intact CCVs and in solution. In addition, we show that the 'stripped' CCV membranes that remain once the peripheral membrane proteins have been removed from CCVs inhibit CK2 but not GAK/auxilin2 activity.

Animals↗

PubMed2005

Clathrin-coated vesicle formation from isolated plasma membranes.

Endocytic clathrin-coated vesicle (CCV) formation is a complex process involving a large number of proteins and lipids. The minimum machinery and the hierarchy of the events involved in CCV formation have yet to be defined. Here we describe an in vitro assay for CCV formation from highly purified rat liver plasma membranes. This rapid and easy assay can be used to quantitatively evaluate the different protein requirements for different endocytic receptors.

Animals↗

PubMed2000

The use of liposomes to study COPII- and COPI-coated vesicle formation and membrane protein sorting.

We have established systems that reconstitute the biogenesis of coated transport vesicles with liposomes made of pure lipids and purified coat proteins. Optimization of the lipid composition in the liposomes allowed the efficient binding of both coat protein I and coat protein II (COPII) coat subunits. Coated vesicles of approximately the size generated from biomembranes were detected and characterized by centrifugation analysis and electron microscopy. A variation of this budding reaction allowed us to measure the sorting of v-SNARE proteins into synthetic COPII vesicles. We developed a novel system to tether glutathione S-transferase (GST)-hybrid proteins to the surface of liposomes formulated with a glutathione-derivatized phospholipid. This system allowed us to detect the positive role of cytoplasmic domains of two v-SNARE proteins that are packaged into COPII vesicles. Therefore, both generation of coated vesicles and protein sorting into the vesicles can be reproduced with liposomes and purified proteins.

Biological Transport↗