scholarly journals Immunofluorescent localization of 100K coated vesicle proteins.

1986 ◽  
Vol 102 (1) ◽  
pp. 48-54 ◽  
Author(s):  
M S Robinson ◽  
B M Pearse
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Limited Proteolysis ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Cell Protein ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Double Labeling ◽  
Vesicle Proteins

A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.

scholarly journals 100-kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies

1987 ◽  
Vol 104 (4) ◽  
pp. 887-895 ◽  
Author(s):  
MS Robinson
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Proteins ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Molecular Heterogeneity ◽  
Coated Pits ◽  
Double Labeling ◽  
Culture Cells ◽  
Punctate Pattern

Proteins with molecular weights of around 100,000 (designated 100K) are found in all coated vesicles. Five monoclonal antibodies have been raised against the major 100K proteins of bovine brain coated vesicles, which migrate on SDS gels as three closely spaced bands. One antibody stains the middle band (band B), two stain both upper and lower bands (bands A and C), and two stain the lower band (band C) only. Thus, the polypeptides in bands A and C are related (but not identical), a result confirmed by NH2-terminal sequencing. Other tissues were found to express proteins corresponding to, and co-migrating with, bands B and C but not band A. Only the two antibodies that recognize both A and C stained fixed and permeabilized tissue culture cells; they both showed a punctate pattern in the plane of the plasma membrane. Double labeling with anti-clathrin antibodies confirmed that the dots correspond to coated pits and vesicles. However, perinuclear staining seen with anti-clathrin, corresponding to Golgi-derived coated vesicles, was conspicuously absent with the two monoclonal antibodies. Affinity-purified polyclonal antisera against the 100K proteins, reported earlier, gave perinuclear as well as punctate staining; these included one antiserum which gave mainly perinuclear staining (Robinson, M. S., and B. M. F. Pearse, 1986, J. Cell Biol., 102:48-54). Thus, different 100K proteins appear to be found in different membrane compartments. Since the 100K proteins are thought to lie between clathrin and the membrane proteins of the vesicle, these results may help to explain how different membrane proteins can be sorted into coated vesicles in different parts of the cell.

scholarly journals The Role of Dynamin and Its Binding Partners in Coated Pit Invagination and Scission

2001 ◽  
Vol 152 (2) ◽  
pp. 309-324 ◽  
Author(s):  
Elaine Hill ◽  
Jeroen van der Kaay ◽  
C. Peter Downes ◽  
Elizabeth Smythe
Keyword(s):  
Plasma Membrane ◽  
Sh3 Domain ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coated Pits ◽  
Binding Partners ◽  
Late Stages

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain–containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.

scholarly journals Induction of mutant dynamin specifically blocks endocytic coated vesicle formation.

1994 ◽  
Vol 127 (4) ◽  
pp. 915-934 ◽  
Author(s):  
H Damke ◽  
T Baba ◽  
D E Warnock ◽  
S L Schmid
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Wild Type ◽  
Coated Pits ◽  
Bulk Fluid ◽  
Vesicle Budding ◽  
Gtp Binding ◽  
Hela Cell Lines

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline-inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.

scholarly journals AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

1998 ◽  
Vol 140 (5) ◽  
pp. 1055-1062 ◽  
Author(s):  
Alexandre Benmerah ◽  
Christophe Lamaze ◽  
Bernadette Bègue ◽  
Sandra L. Schmid ◽  
Alice Dautry-Varsat ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Coated Vesicle ◽  
Mutant Form ◽  
A431 Cells ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Terminal Domain

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.

scholarly journals Adaptor and Clathrin Exchange at the Plasma Membrane andtrans-Golgi Network

2003 ◽  
Vol 14 (2) ◽  
pp. 516-528 ◽  
Author(s):  
Xufeng Wu ◽  
Xiaohong Zhao ◽  
Rosa Puertollano ◽  
Juan S. Bonifacino ◽  
Evan Eisenberg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescence Recovery ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
Rapid Exchange ◽  
Dynamic Structures ◽  
Golgi Network ◽  
Network Exchange

We previously demonstrated, using fluorescence recovery after photobleaching, that clathrin in clathrin-coated pits at the plasma membrane exchanges with free clathrin in the cytosol, suggesting that clathrin-coated pits are dynamic structures. We now investigated whether clathrin at the trans-Golgi network as well as the clathrin adaptors AP2 and AP1 in clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of clathrin-coated vesicle is blocked without significantly affecting the structure of clathrin-coated pits, both clathrin and AP2 at the plasma membrane and clathrin and AP1 at thetrans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K+ depletion, conditions that markedly affect the structure of clathrin-coated pits, clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that clathrin-coated pits are dynamic structures with rapid exchange of both clathrin and adaptors and that adaptors are able to exchange independently of clathrin when clathrin exchange is blocked.

scholarly journals Identification of minor components of coated vesicles by use of permeation chromatography.

1981 ◽  
Vol 91 (2) ◽  
pp. 385-391 ◽  
Author(s):  
S R Pfeffer ◽  
R B Kelly
Keyword(s):  
Glass Bead ◽  
Intracellular Transport ◽  
Standard Procedure ◽  
Electrophoretic Analysis ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Major Protein ◽  
Minor Components ◽  
Uniform Diameter

Coated vesicles are thought to be vehicles for the intracellular transport of membranes. Clathrin is the major protein component of coated vesicles. Minor components of these organelles can be identified in highly purified preparations if they can be shown to copurify with clathrin. To show copurification we have made use of the relatively uniform diameter of coated vesicles (50-150 nm) to fractionate conventionally purified coated vesicles according to size in glass bead columns of 200-nm pore size. We have found that bovine brain coated vesicles prepared by the standard procedure of Pearse can be contaminated with large membrane fragments that are removed by permeation chromatography on such glass bead columns. Gel electrophoretic analysis of column fractions shows that only three major polypeptide chains, and a family of polypeptides with molecular weights close to 100,000 are always in constant ratio to clathrin, and are unique to fractions containing coated vesicles. Two other major polypeptides that appear to be components of coated vesicles are also present in other membrane fractions. We have also used permeation chromatography to monitor artifactual membrane trapping during vesicle isolation. Pure radiolabeled synaptic vesicle membranes were added to bovine brain tissue before homogenization. Considerable amounts of the added radioactivity could be recovered in the fractions conventionally pooled in the preparation of coated vesicles. After permeation chromatography, the radioactivity in the coated vesicle peak was reduced essentially to background.

scholarly journals Gangliosides and sialosylglycoproteins in coated vesicles from bovine brain

1985 ◽  
Vol 225 (3) ◽  
pp. 713-721 ◽  
Author(s):  
D Gravotta ◽  
H J F Maccioni
Keyword(s):  
Rat Brain ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Molar Ratio ◽  
Coated Vesicle ◽  
Synaptic Plasma Membranes ◽  
Morphological Criteria

The content of gangliosides and sialosylglycoproteins was investigated in a coated-vesicle-enriched fraction prepared from bovine brain by the method of Pearse [(1975) J. Mol. Biol. 97, 93-98] and further purified by g.p.c. (glass-permeation chromatography) [Pfeffer & Kelly (1981) J. Cell Biol. 91, 385-391]. From morphological criteria and from the analysis of the polypeptide pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the coated-vesicle fraction (CV-fraction) appeared more than 95% pure. The ganglioside-NeuAc (N-acetylneuraminate), glycoprotein-NeuAc, phospholipid and cholesterol contents of CV-fraction were compared with those of bovine brain synaptic plasma membranes (SPM). The cholesterol to phospholipid molar ratio was 0.47 +/- 0.07 in CV-fraction and 1.06 +/- 0.08 in SPM. The ganglioside-NeuAc and glycoprotein-NeuAc to phospholipid molar ratios were 0.047 and 0.020 respectively in CV-fraction and 0.039 and 0.016 respectively in SPM. The (Na+ + K+)-dependent ATPase activity sensitive to ouabain (in mumol of Pi/h per nmol of phospholipid) was 1.04 in CV-fraction and 0.63 in SPM; the ratio between this activity and the activity resistant to ouabain was 2 in CV-fraction and 1.4 in SPM. A t.l.c. analysis of the ganglioside fractions showed that most of the ganglioside species present in SPM were present in CV-fraction. In a rat brain coated-vesicle preparation not subjected to g.p.c., the activities [as sugar-radioactivity (c.p.m.) transferred/h per mumol of phospholipid] of the enzymes CMP-NeuAc:sialosyl-lactosylceramide (GM3) sialosyl-, UDP-Gal:N-acetylgalactosaminyl(sialosyl)lactosylceramide (GM2) galactosyl- and UDP-GalNAc:sialosyl-lactosylceramide (GM3) N-acetylgalactosaminyl-transferases, which were considered Golgi-apparatus markers, were about 19, 16 and 10% respectively of those determined in rat brain neuronal perikaryon-enriched fractions. Taken together, the results indicate that most of the major gangliosides are constituents of coated vesicles.

scholarly journals alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.

1979 ◽  
Vol 82 (3) ◽  
pp. 614-625 ◽  
Author(s):  
M C Willingham ◽  
F R Maxfield ◽  
I H Pastan
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Coated Vesicles ◽  
The Other ◽  
Con A ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Complexes ◽  
Transmission Electron ◽  
Epidermal Growth

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.

scholarly journals A novel class of clathrin-coated vesicles budding from endosomes.

1996 ◽  
Vol 132 (1) ◽  
pp. 21-33 ◽  
Author(s):  
W Stoorvogel ◽  
V Oorschot ◽  
H J Geuze
Keyword(s):  
Plasma Membrane ◽  
Microscopic Examination ◽  
Transferrin Receptor ◽  
Brefeldin A ◽  
Coated Vesicles ◽  
Integral Membrane Proteins ◽  
Transferrin Receptors ◽  
Receptor Recycling ◽  
Coated Pits ◽  
Novel Technique

Clathrin-coated vesicles transport selective integral membrane proteins from the plasma membrane to endosomes and from the TGN to endosomes. Recycling of proteins from endosomes to the plasma membrane occurs via unidentified vesicles. To study this pathway, we used a novel technique that allows for the immunoelectron microscopic examination of transferrin receptor-containing endosomes in nonsectioned cells. Endosomes were identified as separate discontinuous tubular-vesicular entities. Each endosome was decorated, mainly on the tubules, with many clathrin-coated buds. Endosome-associated clathrin-coated buds were discerned from plasma membrane-derived clathrin-coated vesicles by three criteria: size (60 nm and 100 nm, respectively), continuity with endosomes, and the lack of labeling for alpha-adaptin. They were also distinguished from TGN-derived clathrin-coated vesicles by their location at the periphery of the cell, size, and the lack of labeling for gamma-adaptin. In the presence of brefeldin A, a large continuous endosomal network was formed. Transferrin receptor recycling as well as the formation of clathrin-coated pits at endosomes was inhibited in the presence of brefeldin A. Together with the localization of transferrin receptors at endosome-associated buds, this indicates that a novel class of clathrin-coated vesicles serves an exit pathway from endosomes. The target organelles for endosome-derived clathrin-coated vesicles remain, however, to be identified.

scholarly journals Three-dimensional visualization of coated vesicle formation in fibroblasts.

1980 ◽  
Vol 84 (3) ◽  
pp. 560-583 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Receptor Mediated Endocytosis ◽  
Replication Method ◽  
Complete Contraction ◽  
Partial Contraction ◽  
Selective Mechanism

Fibroblasts apparently ingest low density lipoproteins (LDL) by a selective mechanism of receptor-mediated endocytosis involving the formation of coated vesicles from the plasma membrane. However, it is not known exactly how coated vesicles collect LDL receptors and pinch off from the plasma membrane. In this report, the quick-freeze, deep-etch, rotary-replication method has been applied to fibroblasts; it displays with unusual clarity the coats that appear under the plasma membrane at the start of receptor-mediated endocytosis. These coats appear to be polygonal networks of 7-nm strands or struts arranged into 30-nm polygons, most of which are hexagons but some of which are 5- and 7-sided rings. The proportion of pentagons in each network increases as the coated area of the plasma membrane puckers up from its planar configuration (where the network is mostly hexagons) to its most sharply curved condition as a pinched-off coated vesicle. Coats around the smallest vesicles (which are icosahedrons of hexagons and pentagons) appear only slightly different from "empty coats" purified from homogenized brain, which are less symmetrical baskets containing more pentagons than hexagons. A search for structural intermediates in this coat transformation allows a test of T. Kanaseki and K. Kadota's (1969. J. Cell Biol. 42:202--220.) original idea that an internal rearrangement in this basketwork from hexagons to pentagons could "power" coated vesicle formation. The most noteworthy variations in the typical hexagonal honeycomb are focal juxtapositions of 5- and 7-sided polygons at points of partial contraction and curvature in the basketwork. These appear to precede complete contraction into individual pentagons completely surrounded by hexagons, which is the pattern that characterizes the final spherical baskets around coated vesicles.

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