Coated Vesicle Fraction Research Articles

Purification of clathrin assembly protein from rat liver.

Recently, the gene encoding clathrin assembly protein of lymphoid myeloid leukemia (CALM), which is homologous to the AP180, was cloned from rat brain, and its expression differential to AP180 was reported (Kim and Lee, 1999). This gene product promotes the polymerization of clathrin into clathrin cage and found to be a regulator in membrane trafficking between intracellular compartments in eukaryotic cells (Kim et al., 2000). In this study, we have purified the CALM protein from clathrin-coated vesicles of rat liver using the monoclonal antibody against the recombinant N-terminal region of the CALM. The coated proteins extracted from the coated vesicle fraction was further purified by multi-step procedures involving gel-filtration and ion-exchange chromatography and SDS-PAGE. The purified protein with an apparent molecular weight of 100 kD promoted the assembly of clathrin triskelia into clathrin cage. In this respect the CALM protein bears a functional resemblance to the AP180 that has been previously described.

Inhibition of hsc70-catalysed clathrin uncoating by HSJ1 proteins.

The uncoating of clathrin-coated vesicles can be mediated in vitro by the 'uncoating ATPase' that has been identified as the constitutive 70 kDa heat shock protein (hsp70), hsc70. It is now established that the activity of hsp70 proteins can be regulated by another family of molecular chaperones, the DnaJ family. In this study, we have investigated the effects of DnaJ-like proteins (the human neuron-specific proteins HSJ1a and HSJ1b) on clathrin uncoating. In order to measure the kinetics of clathrin release from coated vesicles, we have developed a quantitative, two-site ELISA for clathrin triskelions and demonstrated that stoichiometric amounts of HSJ1 proteins inhibit the initial burst of hsc70-mediated clathrin uncoating by over 40%. This inhibition is not a consequence of ADP binding by hsc70 or the aggregation of hsc70, but correlates with an increase in the hsc70 associated with the coated vesicle fraction, suggesting that the inhibition is a consequence of a non-productive stabilization of hsc70 with a component of the coated vesicle fraction. These results strongly suggest that HSJ1 proteins interfere with an endogenous DnaJ-like protein that is involved in uncoating. Recent evidence suggests that the brain-specific vesicle-associated protein auxilin could play such a role. Although we find no evidence for auxilin in our coated vesicle preparation, our results predict that an auxilin-like protein will be a general factor in clathrin uncoating.

Rat liver endocytic coated vesicles do not exhibit ATP-dependent acidification in vitro.

Coated vesicle fractions from a variety of tissues have been found to contain a vacuolar proton ATPase. Since these fractions contain both plasma membrane- and Golgi-derived coated vesicles, we sought to determine specifically whether endocytic coated vesicles from rat liver contain an active vacuolar proton ATPase. Endocytic vesicles (coated vesicles and endosomes) were selectively labeled with pH-sensitive endocytic tracers (fluorescein isothiocyanate-dextran or -asialoorsomucoid). Coated vesicles were then separated from endosomes by sucrose density gradient centrifugation. Although the endosomal fractions were found to exhibit significant ATP-dependent acidification activity, highly purified coated vesicles containing pH-sensitive endocytic tracers were unable to generate a pH gradient in response to ATP addition. The coated vesicles could be passively acidified, however, by creating potassium diffusion potentials, indicating that they were in fact capable of maintaining proton gradients. Moreover, significant ATP-dependent acidification activity was observed when the coated vesicle fractions were assayed using the nonselective externally added pH probe acridine orange. Thus, it appears that rat liver endocytic coated vesicles do not contain a functional proton pump. The active vacuolar proton ATPase found in these fractions instead reflected the presence of Golgi-derived coated vesicles or contaminating membranes.

Differential distribution of vesicular carriers during differentiation and synapse formation

Coated and noncoated vesicles participate in cellular protein transport. Both acetylcholine receptors (AChR) and acetylcholinesterase (AChE) are transported via coated vesicles, some of which accumulate beneath the neuromuscular synapse where AChRs cluster. To investigate the mechanisms by which these proteins are transported during postsynaptic remodeling, we purified coated vesicles from the bovine brain via column chromatography (Sephacryl S-1000) and raised monoclonal antibodies to epitopes of the vesicular membranes enriched in AChE. We assayed for AChE (coated vesicle enriched), hexosaminidase (lysosomal contaminants), NADH cytochrome C reductase (mitochondrial containing), and protein and demonstrated electron microscopically using negative staining that the vesicular fraction contained 95% pure coated vesicles. We then injected coated vesicle fractions and the fractions from which the coat was removed intraperitoneally into mice and obtained three monoclonal antibodies: C-33, C-172, and F-22. On immunoblots of purified vesicles and cultured skeletal muscle, mAb C-33 stained a 180 Kd band and mAb C-172 stained a 100 kd band. MAb F-22 stained 50 kd and 55 kd bands and was not characterized further. Immunofluorescent microscopy with C-33 and C-172 revealed punctate fluorescence whose distribution depends upon the stage of myotube development. Four days after plating, myotubes showed punctate fluorescence throughout the myotube, whereas those stained 8 days after plating showed a punctate perinuclear distribution. Myotubes innervated by ciliary neurons show punctate fluorescence limited to the nuclear periphery and most concentrated around nuclei which line up beneath neuronal processes. This differential vesicular distribution, observed during myotube differentiation and innervation, suggests that these vesicles participate in vesicular membrane traffic.

The prominent 28 kDa polypeptide in clathrin coated vesicle fractions from developing pea cotyledons is contaminating ferritin

SDS-PAGE of clathrin coated vesicle (CCV) fractions prepared from developing pea cotyledons are characterized by the presence of a 28 kDa polypeptide. Like clathrin light chains, this polypeptide is heat-stable and can bind calcium ions. However, the distribution of this polypeptide is not identical to that of clathrin in the rate zonal gradients used to purify CCV. Negatively stained preparations reveal small, 12 nm diameter hollow particles, in addition to CCV. As judged by the electron dense centre to these particles, we infer that CCV preparations from pea cotyledons are contaminated with phytoferritin. This has been confirmed by immuno-blotting with pea ferritin antibodies. The degree of phytoferritin contamination in CCV fractions is greater when RNase digestion of postmicrosomal pellets is performed at cold, rather than warm temperatures.

Identification of membrane‐bound carbonic anhydrase in white matter coated vesicles: The fate of carbonic anhydrase and other white matter coated vesicle proteins in triethyl tin‐induced leukoencephalopathy

We have extended our studies on the content of white matter derived coated vesicles (WMCVs) to show that they are enriched in membrane-bound carbonic anhydrase. Within the myelin complex membrane-bound carbonic anhydrase is concentrated in the periaxolemmal domain; however, this protein is enriched almost sevenfold in the bilayer of coated vesicles even relative to this myelin membrane region. These data suggest that some vesicles are derived from a site at which this enzyme is highly localized. The enrichment observed for membrane-bound carbonic anhydrase is unique since other periaxolemmal proteins such as CNPase and plasmolipin are only present in equal amounts in periaxolemmal-myelin fractions and WMCVs. Based on their known localization, the presence of CNPase coupled with the absence of MAG in WMCVs suggest that these vesicles are derived from the paranodal region. The identification in WMCVs of periaxolemmal-myelin proteins associated with ion and fluid movement, such as carbonic anhydrase, Na+,K+ ATPase, and the putative K+ channel protein plasmolipin, prompted us to examine the status of these vesicles in triethyl tin (TET)-induced myelin edema. Coated vesicles and other membrane fractions were isolated from whole brains of control and TET-treated rats. Whole brains were used so we could compare the effects of TET on WMCV proteins with the effect on proteins enriched in gray matter coated vesicles. The results indicated that TET had no detectable effect on compact or periaxolemmal-myelin, however, Western blot analysis showed that WMCV proteins, such as carbonic anhydrase, CNPase, and plasmolipin, were virtually absent or greatly diminished from the whole brain coated vesicle fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of GABAA/benzodiazepine receptors on clathrin-coated vesicles from rat brain.

To investigate the subcellular compartments that are involved in the endocytosis and intracellular trafficking of GABAA/benzodiazepine receptors, we have studied the distribution and properties of clonazepam-displaceable binding of [3H]flunitrazepam to membrane fractions from rat brain. The microsomal fraction was subjected to density centrifugation and gel filtration to isolate clathrin-coated vesicles. Homogeneity of the coated-vesicle fraction was demonstrated by using electron microscopy and by analysis of clathrin subunits and clathrin light-chain kinase. Vesicles exhibiting specific binding of [3H]flunitrazepam eluted from the sieving gel as a separate peak, which was coincident with that for coated vesicles. Scatchard analysis of equilibrium binding of [3H]flunitrazepam to coated vesicles yielded a KD value of 21 +/- 4.7 nM and a Bmax value of 184 +/- 28 fmol/mg. The KD value for coated vesicles was 12-19-fold that found with microsomal or crude synaptic membranes. This low-affinity benzodiazepine receptor was not identified on any other subcellular fraction and thus appears to be a novel characteristic of coated vesicles. The Bmax value for coated vesicles, expressed per milligram of protein, corresponded to 16 and 115% of that found for crude synaptic and microsomal membrane fractions, respectively. Because the trafficking of neurotransmitter receptors via clathrin-coated vesicles is most likely to occur through endocytosis, the data suggest that an endocytotic pathway may be involved in the removal of GABAA/benzodiazepine receptors from the neuronal surfaces of the rat brain. This mechanism could play a role in receptor sequestration and down-regulation that is produced by exposure to GABA and benzodiazepine agonists.

Lysosome-Associated Membrane Protein-1 (LAMP-1) Is the Melanocyte Vesicular Membrane Glycoprotein Band II

Coated vesicles play a critical role in the process of melanogenesis. Antisera raised against a coated vesicle fraction from mouse melanoma cells recognize two major glycoprotein antigens, band I (47-55 kd) and band II (90-120 kd). We demonstrate that band II is lysosome-associated membrane protein 1 (LAMP-1) by the following criteria: 1) the molecular weight and abundance of LAMP-1 varies among tissues but is always identical to that of band II; 2) band II and LAMP-1 co-migrate in sucrose gradient sedimentation studies; 3) immunodepletion of cell extracts with antivesicle serum removes all LAMP-1; and 4) intact organelles immunoisolated with antivesicle serum contain band II and LAMP-1. Our results further confirm the long-suspected relationship between melanosomes and the lysosomal lineage of organelles.

Storage Protein Polypeptides in Clathrin Coated Vesicle Fractions from Developing Pea Cotyledons are not Due to Endomembrane Contamination

We have isolated clathrin coated vesicles (CCV) from developing pea cotyledons and have purifiedthem extensively. On the basis of particle counting in negatively stained preparations these CCV fractions have less than 10% contamination. The activities of endo- and plasma membrane marker enzymes in CCV fractions have been determined and compared to those for plasma membrane and mixed ER/GApp fractions. According to these measurements contamination of the CCV fractions with smooth surfaced membranes is also less than 10%. Activities of the specific endoplasmic reticulum and Golgi apparatus markers NADPH-cytochrome c-reductase and glucan synthase I were not detected in CCV fractions. Through Western blotting with legumin and vicilin antibodies the relative amounts of prolegumin and provicilin polypeptides in a dilution series of endoplasmic reticulum/Golgi apparatus membranes were compared with that of the CCV fraction. From this it is clear that endomembrane contaminants are not sufficient to account for the presence of storage polypeptides in CCV fractions from pea cotyledons. Storage polypeptides are not detectable in CCV fractions isolated from slow frozen/slow thawed pea cotyledons, whereas previously isolated CCV are hardly affected by such a treatment. Storage polypeptides are also still present in CCV which have been subjected to detergent. These results are discussed in terms of receptor-ligand interaction.

In vitro, insulin receptor catalyses phosphorylation of clathrin heavy chain and a plasma membrane 180,000 molecular weight protein

Insulin receptor mutation studies that the receptor tyrosine kinase activity is necessary for receptor endocytosis, and several insulin receptor-containing tissues have a plasma membrane-associated protein ( M r ⋍ 180,000, p180 ) whose tyrosine phosphorylation is receptor catalysed. Since clathrin heavy chain ( M r ⋍ 180,000 in dodecyl sulphate gel electrophoresis) is a major component of coated vesicles, the latter functioning in receptor endocytosis, we investigated whether insulin receptors can catalyse clathrin phosphorylation and whether p180 is clathrin. Bovine brain triskelion or coated vesicles and 32P-ATP were added to prephosphorylated insulin receptor preparations (wheat ferm agglutinin-purified human placenta membrane proteins). Antiphosphotyrosine immunoprecipitated a phosphorylated 180,000 molecular weight protein. Insulin (10 −7M) increased the rate of phosphorylation. Monoclonal anti-clathrin antibody immunoprecipitated the phosphorylated 180,000 molecular weight protein, whereas monoclonal anti-insulin receptor antibodies (α-IR1, MA10) immunoprecipitated both insulin receptors and the phosphorylated 180,000 molecular weight protein. In the absence of added clathrin, anticlathrin immunoprecipitated no proteins, and α-IR1 imunoprecipitated only the insulin receptor. Density gradient (glycerol 7.5–30%, w/v) centrifugation separated human placenta microsomal membrane proteins into endosomal, plasma membrane, cytoplasmic and coated vesicle fractions. Antiphosphotyrosine immunoprecipitated phosphorylated-microsomal proteins that centrifugated into endosomal and plasma membrane fractions. Addition of glycerol gradient fractions to a prephosphorylated insulin receptor preparation, however, gave a tyrosine-phosphorylated 180,000 molecular weight protein when cytoplasmic and coated vesicle fractions were added. Taken together these results suggest: (1) that, in vitro, human placenta insulin receptors can phosphorylate bovine brain and human placenta clathrin heavy chain; (2) that both assembled and unassembled clathrin can be phosphorylated; and (3) that p180, the plasma membrane-associated insulin receptor substrate, is not clathrin heavy chain.

Bovine Brain Coated Vesicles Contain Guanine Nucleotide Regulatory Proteins

Binding of [3H]guanosine triphosphate (GTP) with a high affinity was found to be present in the coated vesicle fraction prepared from bovine cerebral cortex. The binding was saturable and displaced by 1 μΜ of GTP, guanosine diphosphate and guanosine 5’-(3-0-thio)triphosphate. Incubation of the vesicles with islet-activating protein and p’PJNAD resulted in ADP-ribosylation of a 39,000 - 41,000-dalton polypeptide. Antibodies to the α-subunit of stimulatory guanine nucleotide regulatory proteins (G-proteins) immunoblotted 52,000- and 45,000-dalton polypeptides. The results indicate that stimulatory and inhibitory G-proteins are contained in a fraction of the bovine brain coated vesicles.

Bovine brain coated vesicles contain guanine nucleotide regulatory proteins.

Binding of [3H]guanosine triphosphate (GTP) with a high affinity was found to be present in the coated vesicle fraction prepared from bovine cerebral cortex. The binding was saturable and displaced by 1 microM of GTP, guanosine diphosphate and guanosine 5'-(3-O-thio)triphosphate. Incubation of the vesicles with islet-activating protein and [32P]NAD resulted in ADP-ribosylation of a 39,000-41,000-dalton polypeptide. Antibodies to the alpha-subunit of stimulatory guanine nucleotide regulatory proteins (G-proteins) immunoblotted 52,000- and 45,000-dalton polypeptides. The results indicated that stimulatory and inhibitory G-proteins are contained in a fraction of the bovine brain coated vesicles.

Biochemical characterization of algal coated vesicles

Thin sections of tissue preparations from a green alga, Ulva lactuca (Ulvophyceae), and brown alga, Laminaria digitata (Pheophyceae) showed the presence of coated pits and coated vesicles in these 2 species. A discontinuous sucrose gradient after subcellular fractionation of the tissue homogenate resulted in an enriched coated vesicle fraction. Electron microscopy of negatively stained samples revealed the presence of coated vesicles of diameter ranging from 40–125 nm, together with large sheets of polygonal nets of clathrin. Electrophoresis of the CV purified fraction revealed various polypeptide components. Two of them, a 175 kDa and a 70 kDa, exhibited a positive response to bovine brain anticlathrin antibodies raised in goat or in rabbit. A third component of 30–40 kDa also gave a faint positive response. These 3 components corresponded to the clathrin heavy and light chains already described in higher plants. Clathrin was released from the CV algal preparations by treatment with 2M urea in Tris buffer, pH 8.5. Interestingly, in Ulva lactuca, the proportion of clathrin relative to the other proteins from the CV decreased with plant growth. Biochemical analysis of the purified CV revealed the presence of all the major phospholipids characterized in mammalian CV. The ratio of protein over lipid was also in the same range as that calculated for mammalian CV [1]. Carbohydrate analysis demonstrated a high proportion of N-acetylgalactosamine and N-acetylglycosamine in both algal CV whereas these sugars were not detectable in the crude homogenate. These results demonstrate the presence of clathrin and coated vesicles in 2 species of algae. The effect of the biochemical composition of the CV and the variation of the clathrin content in Ulva lactuca upon plant growth suggest the involvement of these organelles with the cellular transport of glycoproteins, glycolipids and polysaccharides.

Coated Vesicles from the Protozoan Parasite Trypanosoma brucei: Purification and Characterization

A procedure was developed to purify a coated vesicle fraction from the protozoan parasite Trypanosoma brucei. Electron microscopy revealed a difference between T. brucei coated vesicles and clathrin-coated vesicles from other eukaryotes: trypanosome vesicles were larger (100 to 150 nm in diameter) and contained an inner coat of electron-dense material in addition to the external coat. Evidence suggests that the internal coat is the parasite's variant surface glycoprotein (VSG) coat. The SDS-PAGE analysis shows the major protein of T. brucei coated vesicles has a molecular mass of 61 kD, similar to VSG; this protein was recognized in an immunoblot by anti-VSG serum. Trypanosome coated vesicles also contain a protein which comigrates with the major protein (clathrin) of coated vesicles purified from rat brains. However, this protein is a minor component and it is not serologically cross-reactive with mammalian clathrin. Immunoblot analysis demonstrated that the parasite vesicles contained host IgG, IgM, and serum albumin.

Purification and Characterization of Clathrin‐Coated Vesicles from Chlamydomonas

Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas reinhardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter +/- SD of 95 +/- 17 nm, n = 300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.

Legumin antibodies recognize polypeptides in coated vesicles isolated from developing pea cotyledons

A highly enriched coated vesicle fraction has been isolated from cotyledons of developing pea seeds. This, and coated vesicles isolated from bovine brain as well as from bean leaves were subjected to SDS-PAGE followed by Western blotting with legumin antibodies. A distinct cross reaction with two polypeptides at around 60 kDa was seen, but only with the coated vesicles isolated from peas. Since legumin is synthesized as a 60 kDa precursor, but occurs as 40 and 20 kDa polypeptides in the protein body, we interpret our results as giving support to the idea that reserve proteins, like lysosomal proteins, are transported via coated vesicles.

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.

Preparation of a homogeneous coated vesicle fraction from bean leaves

Using a procedure previously developed for suspension-cultured carrot cells, we have been able to isolate two different coated vesicle-containing fractions from green bean leaves (Vicia faba). The two fractions differ in their isopycnic densities in D2O-Ficoll as well as in their diameters. One of the fractions (the less dense of the two) is almost 100% pure as judged by negative staining. Because of this the polypeptide pattern obtained from SDS-PAGE is most clear and has enabled a clear recognition of clathrin light chains, in addition to the 190 kDa heavy chain coat component. Significantly the 100k Da and 50k Da polypeptides typical of brain coated vesicles are absent from bean leaf coated vesicles. Due to a) the high degree of vacuolation b) the presence of large amounts of ribulose bisphosphate carboxylase in the postmicrosomal supernatant, the yield of coated vesicles from bean leaves, as compared to nongreen plant cells, or to bovine brain tissue is extremely low (1 mg coated vesicles from 2.4 kg leaf tissue).

The isolation and enrichment of coated vesicles from suspension-cultured carrot cells

A method has been developed to isolate and purify coated vesicles from suspension cultured carrot (Daucus carota L.) cells. It incorporates features of centrifugation methods (sucrose step gradient; Ficoll/D2O gradient) previously employed in the isolation of coated vesicles from mammalian brain tissue. Most important is the treatment of the crude coated vesicle fraction (postmicrosomal supernatant) with ribonuclease to remove ribosomes which are a serious source of contamination in such fractions. The fraction finally obtained is contaminated to the extent of 30% of total observed particles in negatively stained preparations with naked vesicles whose diameter are smaller than those of the coated vesicles. These vesicles are interpreted as being coated vesicles which have been stripped of their coats. SDS-PAGE of coated vesicle fractions purified by this method reveal significant differences in the polypeptide patterns obtained from plant and animal systems.

Stereospecific opiate-binding sites occur in coated vesicles

We prepared clathrin-coated vesicles from bovine forebrain utilizing sucrose or deuterium oxide-Ficoll density gradient centrifugation followed by permeation chromatography. Homogeneity was monitored by electron microscopy (EM) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). EM revealed that the predominant (up to 98% of the total) organelles were coated vesicles and empty hexagonal baskets. Diameters of the coated vesicles ranged from 37 to 120 nm with a mean of 65.2 +/- 2.2. Upon SDS-PAGE of the coated vesicle fraction, the most prominent band appeared at 180,000 daltons. There were also three additional bands at 100,000, 50,000 and 35,000 daltons, giving the overall pattern characteristic of coated vesicles. Both 0.5 nM tritiated naltrexone and etorphine displayed specific binding to coated vesicles. Naltrexone binding in coated vesicles from gradient fractions was increased 2.5-fold over the original 100,000 X g pellet. An additional 4-fold enrichment in specific binding was observed after permeation chromatography which was concomitant with an increase in the volume density of coated vesicles in electron micrographs. Naltrexone binding was stereospecific and etorphine binding was inhibited by 100 mM NaCl (40%). Both naltrexone and etorphine binding were inhibited by 50 microM guanyl-5'-yl imidodiphosphate (40 to 50%). In summary, purified bovine brain-coated vesicles contained high affinity stereospecific opiate alkaloid-binding sites with characteristic opioid binding properties.