Structural characterization of labeled clathrin and coated vesicles

Clathrin (8 S) and coated vesicles have been covalently labeled by using the sulfhydryl-labeling fluorescent probe N-(1-anilinonaphthalene)maleimide. A large increase in energy transfer from Trp to anilinonaphthalene (AN) residues was observed in clathrin in the pH range approximately 6.5-6.0, where the rate of clathrin self-association increased rapidly. The change in energy transfer was indicative of a conformational rearrangement, which could be responsible for the initiation of the clathrin self-association reaction to form coat structure. The AN label was found in both the coat and membrane proteins after dissociation of coated vesicles at pH 8.5. The labeled coat and membrane proteins readily recombined to form coated vesicles after reducing the pH to 6.5, indicating that the labeling did not interfere with the ability of clathrin to self-associate and interact with uncoated vesicles to form coat structure. A comparison of the AN fluorescence with the Coomassie blue pattern after electrophoresis in sodium dodecyl sulfate-gels revealed that a 180,000-Da protein (clathrin) was mainly labeled in coated vesicles, while a 110,000-Da protein was also strongly labeled in uncoated vesicles. AN-labeled baskets and coated vesicles have been prepared. Trypsin digestion reduced the sedimentation rate of baskets from 150 S to 120 S and of coated vesicles from 200 S to 150 S. Gel electrophoresis of baskets and coated vesicles showed extensive conversion of clathrin (Mr 180,000) to a product of Mr approximately equal to 110,000, suggesting equivalent structural organization of the coat in coated vesicles as in baskets. In both cases, the peptide(s) released from the vesicles by digestion were essentially free of fluorescent label. In the case of the uncoated vesicles, tryptic digestion released most of the proteins remaining after coat removal.     

Lipid bilayer dynamics in plasma and coated vesicle membranes from bovine adrenal cortex. Evidence of two types of coated vesicle involved in the LDL receptor traffic

Pure coated vesicles have been prepared from the bovine adrenal cortex and two homogeneous populations have been separated, one of large diameter (100 nm) and one of small diameter (70 nm). The chemical composition in lipids and proteins of coated vesicles has been compared with that of partially purified plasma membranes and evidences a higher protein/lipid ratio and a higher concentration in phosphatidylethanolamine and unsaturated fatty acids. Evaluation of the lateral diffusion of pyrene in the lipid bilayer of coated vesicles as compared to uncoated vesicles evidences a slowing-down effect of clathrin. Measurements of lipids' rotational diffusion by time-resolved fluorescence indicate a decrease in the order parameter of the lipids in the coated vesicles due to clathrin. A hypothesis is proposed for a possible role of the clathrin coat in the concerted motion of lipids and proteins toward coated pits and in the mechanism of formation of coated vesicles. Separation of the large from the small coated vesicles made it possible to reveal different protein components in the two types of vesicle by electrophoresis and autoradiograms of the [γ-³²P]adenosine triphosphate- (ATP-) treated vesicles. Visualisation of the low-density lipoprotein receptor by ligand blotting and enzyme-linked immunosorbent assay (ELISA) techniques indicates an increased low-density lipoprotein receptor binding capacity in small coated vesicles as compared to large ones and plasma membranes.     

Preparation of a coated vesicle-enriched fraction from plant cells

A fraction rich in coated vesicles has been prepared from suspension-cultured cells of tobacco (Nicotiana tabacum L.) by sucrose gradient centrifugation. Isolated, negatively-stained plant coated vesicles are approx. 100 nm in diameter, and show the characteristic basket-like structure of the clathrin coat previously reported for both plant [2–5] and animal [1, 6–9] coated vesicles. Analysis of the various plant subcellular fractions by SDS polyacrylamide gel electrophoresis demonstrates that a polypeptide of 190 000 D is enriched in parallel with the morphologically identifiable coated vesicles. It is postulated that this polypeptide is plant clathrin with a molecular weight about 10 000 D greater than that previously reported for animal clathrin [1, 6].     

Purification and properties of a new clathrin assembly protein.

A clathrin assembly protein (AP180) has been purified and characterized from coated vesicles of bovine brain. This protein has hitherto escaped detection because in SDS-gel electrophoresis it is obscured by the 180 kd heavy chain of clathrin. Despite the similarity in electrophoretic mobility, AP180 differs from clathrin in both its subunit and native mol. wt, as well as hydrodynamic properties, surface charge and tryptic peptide composition. It also appears immunologically distinct from clathrin, since neither a polyclonal antiserum nor a monoclonal antibody, that have been shown to be specific for AP180, cross-react with the heavy chain of clathrin. AP180 binds to clathrin triskelia and thereby promotes clathrin assembly into regular polyhedral structures of narrow size-distribution (60-90 nm), reminiscent of the surface coat of coated vesicles. In this respect AP180 bears a functional resemblance to the 100-110 kd clathrin assembly polypeptides that have been previously described.     

Isolation and partial characterization of clathrin-coated vesicles from pea (Pisum sativum L.) cotyledons

Summary Clathrin-coated vesicles have been isolated from cotyledons of both developing and germinating pea seeds using differential centrifugation, ribonuclease treatment, discontinuous sucrose gradients, and isopycnic centrifugation on a linear D₂O-Ficoll gradient. The yield of coated vesicles from developing pea cotyledons was exceptional, being 1.6 × higher than the yield from hog and bovine brain, 5.3 × higher than the yield from carrot suspension cultures, and 13 × the yield from cotyledons of germinating pea seeds. The pea coated vesicles are similar to other plant coated vesicles in size (approximately 80 nm in diameter) and in having a clathrin heavy chain of 190,000 M_r. The lipid phosphorus to protein ratio, 190–250 nmol P per mg protein, of the coated vesicles from plants is comparable to that reported for highly purified coated vesicles from animals. The nondenatured pea clathrin reacted weakly with an antiserum to bovine brain clathrin, but pea clathrin denatured by sodium dodecyl sulfate did not.Abbreviations CLC Clathrin light chain - CHC clathrin heavy chain - CV coated vesicle - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffered saline     

A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack

Isolated Golgi membranes incubated in the presence of ATP and a cytosolic protein fraction form a population of coated buds or vesicles from the Golgi cisternae. The coats do not have the characteristic hexagonal-pentagonal basketwork of clathrin, and do not react with anti-clathrin polyclonal antibody. The conditions that produce these apparently nonclathrin-coated buds also reconstitute protein transport between compartments of the Golgi stack. The membrane of the buds contains the glycoprotein in transit through these Golgi stacks (VSV-encoded G protein). This suggests that protein transport through the Golgi stack is mediated by a new type of coated vesicle that does not contain clathrin. The concentration of G protein in the coated buds reflects the local concentration of G protein in the cisternae, raising the possibility that the Golgi coated vesicles may be "bulk" membrane carriers.     

Phosphorylation of Brain Synaptic and Coated Vesicle Proteins by Endogenous Ca2+/Calmodulin- and cAMP-Dependent Protein Kinases

Phosphorylation of brain synaptic and coated vesicle proteins was stimulated by Ca2+ and calmodulin. As determined by 5-15% sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE), molecular weights (Mr) of the major phosphorylated proteins were 55,000 and 53,000 in synaptic vesicles and 175,000 and 55,000 in coated vesicles. In synaptic vesicles, phosphorylation was inhibited by affinity-purified antibodies raised against a 30,000 Mr protein doublet endogenous to synaptic and coated vesicles. When this doublet, along with clathrin, was extracted from coated vesicles, phosphorylation did not take place, implying that the protein doublet may be closely associated with Ca2+/calmodulin-dependent protein kinase. Affinity-purified antibodies, raised against clathrin used as a control antibody, failed to inhibit Ca2+/calmodulin-dependent phosphorylation in either synaptic or coated vesicles. Immunoelectron cytochemistry revealed that this protein doublet was present in axon terminal synaptic and coated vesicles. Synaptic vesicles also displayed cAMP-dependent kinase activity; coated vesicles did not. The molecular weights of phosphorylated synaptic vesicle proteins in the presence of Mg2+ and cAMP were: 175,000, 100,000, 80,000, 57,000, 55,000, 53,000, 40,000, and 30,000. Based on the different phosphorylation patterns observed in synaptic and coated vesicles, we propose that brain vesicle protein kinase activities may be involved in the regulation of exocytosis and in retrieval of synaptic membrane in presynaptic axon terminals.     

Characterization of the coated vesicle uncoating ATPase: tissue distribution, association with and activity on intact coated vesicles

We have analyzed the uncoating process of clathrin-coated vesicles (CV) performed by an ATPase (UA; apparent molecular mass 70 kDa) prepared from various mammalian tissues. Our data show that this enzyme removes the clathrin coat from isolated, intact coated vesicles, as seen by sedimentation analysis on gels and also by electron microscopy. The isolated UA does not discriminate between CV from homologous or heterologous tissues. This finding implies that the brain-specific insertion in clathrin light chains cannot be essential for the binding of brain UA to target vesicles. Polyclonal antibodies were raised against UA and were found to inhibit UA activity. Immunoblotting of purified CV and immunoblotting of CV in situ indicate that a subpopulation of CV contains bound UA. However, most of the uncoating enzyme is not associated with coated structures in mammalian tissue culture cells. Our data support the hypothesis that the 70 kDa uncoating ATPase is responsible for the in vivo uncoating of coated vesicles.     

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.     

Homology between antigenic determinants of bovine clathrin and clathrin from the brown alga Laminaria digitata

Coated vesicles, essential organelles of intracellular membrane traffic, have been extensively studied in animal and higher plant cells. In the algae, cytological studies only have been performed which demonstrate the presence of such coated vesicles with their surrounding clathrin lattice. The present work has been carried out on coated vesicles isolated for the first time from the brown algae Laminaria digitata. For comparison of the antigenic characteristics of clathrin prepared from the Bovine brain or adrenocortical cells and the clathrin prepared from algae, polyclonal antibodies have been raised to a purified Bovine brain clathrin in Goat and to Bovine adrenocortical clathrin in Rabbit. The positive immunological responses of the coated vesicles and the clathrin from Algae to these antibodies, evidence an homology between antigenic determinants of clathrin from animal and vegetal cells.     

Reversibility of coated vesicle dissociation

The dissociation of the coated vesicles to clathrin and uncoated vesicles and their reassociation have been studied under various conditions. The extent of reassociation is pH dependent and increases slightly with increasing concentrations of the components. Unlike the self-association of clathrin which is strongly salt dependent, the reassociation of clathrin and uncoated vesicles is practically independent of salt concentration. The coated vesicle gradually loses its coat with increasing pH, and the dissociation process is not an all or none reaction. Ca2+ inhibits dissociation of the coated vesicles and enhances the reassociation of clathrin and uncoated vesicles. Our results show that, although many conditions result in reassociation of protein and lipid vesicle, few conditions result in vesicles of both the same size and composition as native coated vesicles.     

An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells

Clathrin provides an external scaffold to form small 50-100-nm transport vesicles. In contrast, formation of much larger dense-cored secretory granules is driven by selective aggregation of internal cargo at the trans-Golgi network; the only known role of clathrin in dense-cored secretory granules formation is to remove missorted proteins by small, coated vesicles during maturation of these spherical organelles. The formation of Weibel-Palade bodies (WPBs) is also cargo driven, but these are cigar-shaped organelles up to 5 mum long. We hypothesized that a cytoplasmic coat might be required to make these very different structures, and we found that new and forming WPBs are extensively, sometimes completely, coated. Overexpression of an AP-180 truncation mutant that prevents clathrin coat formation or reduced AP-1 expression by small interfering RNA both block WPB formation. We propose that, in contrast to other secretory granules, cargo aggregation alone is not sufficient to form immature WPBs and that an external scaffold that contains AP-1 and clathrin is essential.     

Phosphorylation of the medium chain subunit of the AP-2 adaptor complex does not influence its interaction with the tyrosine based internalisation motif of TGN38

Tyrosine based motifs conforming to the consensus YXXphi (where phi represents a bulky hydrophobic residue) have been shown to interact with the medium chain subunit of clathrin adaptor complexes. These medium chains are targets for phosphorylation by a kinase activity associated with clathrin coated vesicles. We have used the clathrin coated vesicle associated kinase activity to specifically phosphorylate a soluble recombinant fusion protein of mu2, the medium chain subunit of the plasma membrane associated adaptor protein complex AP-2. We have tested whether this phosphorylation has any effect on the interaction of mu2 with the tyrosine based motif containing protein, TGN38, that has previously been shown to interact with mu2. Phosphorylation of mu2 was shown to have no significant effect on the in vitro interaction of mu2 with the cytosolic domain of TGN38, indicating that reversible phosphorylation of mu2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs. In addition, although a casein kinase II-like activity has been shown to be associated with clathrin coated vesicles, we show that mu2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coated vesicles. Furthermore the kinase activity associated with clathrin coated vesicles was shown to be capable of phosphorylating dynamin 1. Phosphorylation of dynamin 1 has previously been shown to regulate its interaction with other proteins involved in clathrin mediated endocytosis.     

Identification of the Plasma Membrane Proteolipid Protein as a Constituent of Brain Coated Vesicles and Synaptic Plasma Membrane

We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.     

Using quantum dots to visualize clathrin associations

Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.     

Light and electron microscopic immunocytochemical localization of clathrin in rat cerebellum and kidney

The precise cellular and subcellular locations of coated vesicle protein, clathrin, in rat kidney and cerebellum have been visualized by immunocytochemical techniques. In the renal tubular epithelia, clathrin-positive products were found on both free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No clathrin was observed in the cisternae of RER or the Golgi apparatus. Clathrin-positive reaction products could also be seen on coated pits, coated vesicles, Golgi-associated vesicles, basolateral cell membrane, the ground substance, and in the autophagic vacuoles. In cerebellar Purkinje and granule cell bodies, reaction products were seen localized on coated vesicles, on the budding areas from the Golgi-associated membrane and Golgi-associated vesicles. Furthermore, the membrane of the multivesicular body, the bound-ribosomes, and the ground substance were also stained. In the myelinated axon, the clathrin appeared to be concentrated on certain segments and seemed to fill in the space between neurotubules and some vesicles. In certain synaptic terminals clathrin was often seen attached to presynaptic vesicles, presynaptic membrane, and post-synaptic membrane. However, in most mossy fibers, some synaptic vesicles were not stained. These observations suggest that clathrin is synthesized on bound and free ribosomes and discharged into the cytosol where it becomes associated with a variety of ground substances and assembles on coated pits, coated vesicles, Golgi-associated vesicles, presynaptic vesicles, and pre- and postsynaptic membranes. Clathrin may be finally degraded in autophagic vacuoles.     

Association between coated vesicles and microtubules

In this study, a possible functional association between microtubules and coated vesicles is described. We have found that our preparations of microtubules contained coated vesicles in quantities of usually above 10%. These coated vesicles were identified both by immunological methods using anticoat antibodies and by electron microscopy of negatively stained specimens. In the immune replica, two components of coated vesicles, i.e., heavy (clathrin) and light chains, were recognized as constituents of the preparations. In the electron microscope, it was found that coated vesicles were attached predominantly along the length of microtubules. Furthermore, projections from the microtubules to the triskelion centers of the clathrin lattice were identified and thus seem to serve as linkers between the cytoskeletal structure of the organelle. A similar type of association was detected in tissue culture cells; bridges between coated vesicles and microtubules were clearly identified by electron microscopy of thin sections.     

Purification and characterization of a molecular weight 100,000 coat protein from coated vesicles obtained from bovine brain

A protein designated as a 100-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis was purified from coated vesicles obtained from bovine brain, with uncoated vesicles as starting material. Two gel filtration steps, one involving 0.5 M tris(hydroxymethyl)aminomethane, pH 8.0, buffer, and the other 0.01 M tris(hydroxymethyl)aminomethane, pH 8.0, and 3 M urea buffer, were employed. The purified protein has a native molecular weight of 114,000 as determined by sedimentation equilibrium analysis. Circular dichroism data showed that the protein has 28% helical structure, 29% beta-structure, and 15% beta-turns, and the rest is random coil. Addition of the purified protein to clathrin results in the polymerization of clathrin to homogeneous size baskets of sedimentation velocity 150 S. A scan of the Coomassie Blue stained electrophoresis gels of the polymerized baskets shows that, for every clathrin trimer, there is approximately one 100-kDa protein molecule.     

Purification and Characterization of Clathrin from Bovine Brain

Abstract: Clathrin has been purified to electrophoretic homogeneity by initial extraction of clathrin from purified coated vesicle fraction, followed by column chromatographies with gel filtration. DEAE-cellulose, and hydroxylapatite and finally by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Antibody specific to clathrin has also been obtained. Two forms of native clathrin, fast and slow components, have been prepared to about 95% purity by hydroxylapatite column chromatography. Both fast and slow components are believed to represent two different aggregates of clathrin subunit because they comigrate in agarose electrophoresis. pH 7.4, and also migrate as clathrin subunit on SDS-PAGE with a molecular weight of 175,000. Furthermore, both components cross-react with antibody against purified clathrin and compete for antibody binding site with labeled fast component. The fast component can also be converted to the slow component. In addition to clathrin, two proteins of about 38,000 and 35,000 M.W. that consistently co-purified with native clathrin are probably also intrinsic to coated vesicle.     

A monoclonal antibody to the heavy chain of clathrin.

Monoclonal antibodies have been raised to pig brain triskelions and one clone, DC41, was found to recognize the clathrin heavy chain by immunoblotting. However, both by immunofluorescence and immunoelectron microscopy, and in complete contrast to polyclonal anti-clathrin antibodies, monoclonal DC41 did not label either coated pits or coated vesicles anywhere in the cell. Instead it appeared to label the cell cytoplasm. These data suggest that DC41 recognizes a cytoplasmic form of clathrin, perhaps that form produced by uncoating of coated vesicles which is then ready to re-build another coated pit.