Common features of coated vesicles from dissimilar tissues: composition and structure

1978 ◽  
Vol 30 (1) ◽  
pp. 87-97 ◽  
Author(s):  
J.W. Woods ◽  
M.P. Woodward ◽  
T.F. Roth
Keyword(s):  
Membrane Vesicle ◽  
Coated Vesicles ◽  
Sodium Dodecylsulphate ◽  
Exterior Surface ◽  
Porcine Brain ◽  
Molecular Weights ◽  
Composition And Structure ◽  
Structural Subunit ◽  
Ordered Array ◽  
Coat Material

Coated vesicles were purified and characterized from porcine brain and chicken oocyte. Electrophoresis on sodium dodecylsulphate (SDS) gels showed that coated vesicles from either source have three major proteins in common with apparent molecular weights of 180000, 120000, and 55000 Daltons. Negatively stained specimens from both sources appear to consist of a highly ordered array of short interconnected rods or ridges of material on the exterior surface of a membrane vesicle. Coated vesicles purified from porcine brain and chicken oocyte have mean external diameters of 75.0 and 85.0 nm, respectively. In coated vesicles of the appropriate orientation, portions of the coat material appear to be organized into hexagons and pentagons. Based on the observed variability of size and apparent structure, it is postulated that the basic structural subunits of coated vesicles are the short rods or ridges of material observed on the exterior membrane vesicle surface. It is suggested that multiples of the subunits can be assembled into many arrangements to yield coated vesicles of different sizes and coat structure. It is also proposed that the structural subunit is a complex of 3 proteins of molecular weight of 180000, 120000 and 55000 Daltons.

Coated Vesicles in Absorptive Cells of Hydra

1967 ◽  
Vol 2 (4) ◽  
pp. 563-572
Author(s):  
D. B. SLAUTTERBACK
Keyword(s):  
Hexagonal Lattice ◽  
Coated Vesicles ◽  
Selective Absorption ◽  
Luminal Surface ◽  
Gastrovascular Cavity ◽  
Apical Cytoplasm ◽  
Absorptive Cells ◽  
Ordered Array

The apical cytoplasm of absorptive cells in hydra gastroderm contains numerous vesicles having a discoidal shape, an asymmetrical membrane and a peculiar coat firmly attached to the luminal surface of the membrane. The coat of these ‘discoidal coated vesicles’ consists of a highly ordered array of subunits made up of ‘pegs’ and globules. That these vesicles are involved in selective absorption and transport of materials from the gastrovascular cavity can be seen readily using ferritin as tracer. They are also involved in the uptake of particulate glycogen and the phagocytosis and possibly the subsequent digestion of complex food droplets. It may reasonably be supposed that the array of the coat subunits indicates a similar array of the molecular constituents of the membrane to which it is attached. In tangential sections of the membrane a compressed hexagonal lattice is seen which could fit the coat array.

scholarly journals Tubulin as a molecular component of coated vesicles.

1983 ◽  
Vol 97 (4) ◽  
pp. 1191-1199 ◽  
Author(s):  
W G Kelly ◽  
A Passaniti ◽  
J W Woods ◽  
J L Daiss ◽  
T F Roth
Keyword(s):  
Microscopic Analysis ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Antigenic Determinants ◽  
Molecular Component ◽  
Electron Microscopic ◽  
Molecular Weights ◽  
Controlled Pore Glass ◽  
Sds Page ◽  
Pore Glass

Two proteins of 53,000 and 56,000 mol wt have been found to be associated with coated vesicles (CV) purified from bovine brain and chicken liver. These proteins share molecular weights, isoelectric points, and antigenic determinants with alpha- and beta-tubulins purified from bovine brain. Based on SDS PAGE and electron microscopic analysis of controlled pore glass bead exclusion column fractions, both the tubulins and the major CV polypeptide clathrin were found to chromatograph as components of a single kinetic particle. In addition, tubulin and CV antigens assayed by a sensitive enzyme-linked-immunoadsorbent method eluted from the columns with constant stoichiometry. These data provide evidence that tubulin is a molecular component of coated vesicles.

Trafficking of apical proteins into clathrin-coated vesicles isolated from rat renal cortex

1994 ◽  
Vol 266 (4) ◽  
pp. F554-F562 ◽  
Author(s):  
T. G. Hammond ◽  
P. J. Verroust
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Renal Cortex ◽  
Coated Vesicles ◽  
Brush Border Enzymes ◽  
Molecular Weights ◽  
Endosomal Pathway ◽  
Fluorescent Dextran

The endosomal pathway of the rat renal cortex was labeled by intravenous infusion of fluorescent dextran small enough to cross the glomerular ultrafiltration barrier and be taken up by luminal endocytosis in the proximal tubule. Clathrin-coated vesicles (CCV) were isolated from the rat renal cortex utilizing discontinuous sucrose density gradients and negative lectin selection. More than 99 +/- 1% (n = 4) of the isolated vesicles contain entrapped fluorescein dextran when analyzed by small-particle flow cytometry techniques. Similarly, flow cytometry analysis demonstrates brisk H(+)-adenosinetriphosphatase activity in virtually all the vesicles. Western blot analysis of the vesicle proteins with a polyclonal anticlathrin antibody stains bands consistent with clathrin and adaptins. When the isolated vesicles are decoated by exposure to 0.5 M tris(hydroxymethyl)aminomethane, the proteins released match the molecular weights of the proteins identified on Western blot analysis. Flow cytometry demonstration of brush border enzymes in > 99% of the vesicles and Western blot identification of maltase suggests both that these vesicles are of apical origin and that apical enzymes traffic into endosomal elements. Additionally, two glycoproteins detectable in this fraction on Western blot analysis and flow cytometry immunocytochemistry are derived from intermicrovillar clefts traffic into the endosomal pathway. Hence, apical proteins traffic into a population of CCV isolated from the rat renal cortex.

scholarly journals Subunit composition and structure of subcomponent C1q of the first component of human complement

1976 ◽  
Vol 155 (1) ◽  
pp. 19-23 ◽  
Author(s):  
K B M Reid ◽  
R R Porter
Keyword(s):  
Ionic Strength ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Helical Structure ◽  
Guanidinium Chloride ◽  
Molecular Weights ◽  
Composition And Structure ◽  
Covalently Linked ◽  
Low Ionic Strength ◽  
Expected Position

1. Unreduced human subcomponent C1q was shown by electrophoresis on polyacrylamide gels run in the presence of sodium dodecyl sulphate to be composed of two types of non-covalently linked subunits of apparent mol.wts. 69 000 and 54 000. The ratio of the two subunits was markedly affected by the ionic strength of the applied sample. At a low ionic strength of applied sample, which gave the optimum value for the 54 000-apparent mol.wt. subunit, a ratio of 1.99:1.00 was obtained for the ratio of the 69 000-apparent mol.wt. subunit to the 5400-apparent-mol.wt. subunit. The amount of the 54 000-apparent-mol.wt. subunit detected in the expected position on the gel was found to be inversely proportional to increases in the ionic strength of the applled sample. 2. Human subcomponent C1q on reduction and alkylation, or oxidation, yields equimolar amounts of three chains designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. The results obtained by Yonemasu & Stroud [(1972) Immunochemistry 9, 545-554], which showed that the 69 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the A and B chains and that the 54 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the C chain, were confirmed. 3. Gel filtration on Sephadex G-200 in 6.0M-guanidinium chloride showed that both types of unreduced subunit were eluted together as a single symmetrical peak of apparent mol.wt. 49 000-50 000 when globular proteins were used as markers. The molecular weights of the oxidized or reduced A, B and C chains have been shown previously to be very similar all being in the range 23 000-24 000 [Reid et al. (1972) Biochem. J. 130, 749-763; Reid (1974) Biochem. J. 141, 189-203]. 4. It is proposed that subcomponent C1q (mol.wt. 410000) is composed of nine non-covalently linked subunits, i.e. six A-B dimers and three C-C dimers. 5. A structure for subcomponent C1q is proposed and is based on the assumption that the collagen-like regions of 78 residues in each of the A, B and C chains are combined to form a triple-helical structure of the same type as is found in collagens.

scholarly journals Clathrin assembly proteins: affinity purification and a model for coat assembly.

1987 ◽  
Vol 105 (5) ◽  
pp. 1989-1998 ◽  
Author(s):  
J H Keen
Keyword(s):  
Molecular Weight ◽  
Affinity Purification ◽  
Characteristic Size ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Mean Diameter ◽  
Assembly Proteins

Assembly protein (AP) preparations from bovine brain coated vesicles have been fractionated by clathrin-Sepharose affinity chromatography. Two distinct fractions that possess coat assembly activity were obtained and are termed AP-1 and AP-2. The AP-1, not retained on the resin, has principal components with molecular weights of 108,000, 100,000, 47,000, and 19,000. The AP-2, bound to the resin and eluted by Tris-HCl at a concentration that parallels the latter's effect on coat disassembly, corresponds to the active complex described previously (Zaremba, S., and J. H. Keen, 1983, J. Cell Biol., 97:1339-1347). Its composition is similar to that of the AP-1 in that it contains 100,000-, 50,000-, and 16,000-mol-wt polypeptides in equimolar amounts; minor amounts of 112,000- and 115,000-mol-wt polypeptides are also present. Both are distinct from a recently described assembly protein of larger subunit molecular weight that we term AP-3. These results indicate the existence of a family of assembly proteins within cells. On incubation with clathrin both AP-1 and AP-2 induce the formation of coat structures, those containing AP-1 slightly smaller (mean diameter = 72 nm) than those formed in the presence of AP-2 (mean diameter = 79 nm); both structures have been detected previously in coated vesicle preparations from brain. Coats formed in the presence of AP-2 consistently contain approximately one molecule each of the 100,000-, 50,000-, and 16,000-mol-wt polypeptides per clathrin trimer. By low angle laser light scattering the molecular weight of native AP-2 was determined to be approximately 343,000, indicating that it is a dimer of each of the three subunits, and implying that it is functionally bivalent in clathrin binding. A model for AP-mediated coat assembly is proposed in which a bivalent AP-2 molecule bridges the distal legs or terminal domains of two clathrin trimers that are destined to occupy adjacent vertices in the assembled coat. Binding of a second AP-2 molecule locks these two trimers in register for assembly and further addition of AP-2 to free trimer legs promotes completion of the clathrin lattice. Effects of AP binding on the angle and flexibility of the legs at the hub of the trimer (the "pucker") are suggested to account for the characteristic size distributions of coats formed under varied conditions and, more speculatively, to contribute to the transformation of flat clathrin lattices to curved coated vesicles that are thought to occur during endocytosis.

scholarly journals Mass distributions of coated vesicles isolated from liver and brain: analysis by scanning transmission electron microscopy.

1983 ◽  
Vol 97 (6) ◽  
pp. 1714-1723 ◽  
Author(s):  
A C Steven ◽  
J F Hainfeld ◽  
J S Wall ◽  
C J Steer
Keyword(s):  
Weighted Average ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Diameter Distribution ◽  
Mass Distributions ◽  
Molecular Weights ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Kolmogorov Smirnov

Populations of coated vesicles purified from bovine brain (BCV) and from rat liver (LCV) have been characterized with respect to the parameters of mass and diameter by analysis of scanning transmission electron micrographs of unstained specimens. Coated vesicles from both sources are heterogeneous, particularly in their masses. The respective distributions, compiled from mass measurements of many individual particles, are complex and markedly different. BCV range from 20 Mdaltons to approximately 100 Mdaltons with a weighted average of 35 Mdaltons: most BCV (80%) lie between 20 and 40 Mdaltons, including peaks at approximately 26 Mdaltons and at approximately 34 Mdaltons. In contrast, LCV masses tend to be substantially higher, ranging from 20 to 220 Mdaltons with a weighted average of 66 Mdaltons. There is a prominent subpopulation at approximately 35 Mdaltons, and 59% of all LCV belong to a broad peak between 50 and 120 Mdaltons. The Kolmogorov-Smirnov distribution-free test was used to affirm the statistical reproducibility of these isolates. BCV diameters vary from 50 to 90 nm, and those of LCV from 50 to 150 nm. Both protein compositions, determined by SDS PAGE, are dominated by clathrin and they are generally similar, except that corresponding secondary bands, notably the clathrin-associated light chains, appear to have lower molecular weights in the case of LCV. From consideration of the joint mass-diameter distribution, it is apparent that coated vesicles of a given diameter vary considerably in mass and that this variation is due primarily to widely differing amounts of material enclosed within the clathrin coat.

scholarly journals Plasma membrane coat and a coated vesicle-associated reticulum of membranes: their structure and possible interrelationship in Chara corallina.

1984 ◽  
Vol 98 (4) ◽  
pp. 1537-1545 ◽  
Author(s):  
T C Pesacreta ◽  
W J Lucas
Keyword(s):  
Plasma Membrane ◽  
Chara Corallina ◽  
Peripheral Region ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Structural Components ◽  
The Core ◽  
Membrane Bound ◽  
Zinc Iodide ◽  
Coat Material

Primary fixation with buffered glutaraldehyde plus 2.0 mM CaCl2 and 0.1% tannic acid results in the preservation of certain portions of the plasma membrane coat of Chara when seen with the electron microscope. Such a coat is not observable after fixation with glutaraldehyde alone. The coat appears to be present on all the above ground, vegetative cells of the male plant. Within complex invaginations of the plasma membrane, which are known as charasomes, the coat has two structural components, a central core that is either tubular or solid and a fibrous or granular peripheral region that surrounds the core. The coat material appears to be at least partially derived, via exocytosis, from the contents of single membrane-bound organelles known as glycosomes. Glycosomes seem to originate from within an assemblage of membranes and coated vesicles that can be described, in purely structural terms, as a partially coated reticulum. Such a reticulum is distinguishable from Golgi stacks because the reticulum (a) is not composed of stacked membranes, (b) is extensively involved with large, clearly detailed coated vesicles and coated invaginations, (c) is closely associated with glycosomes, and (d) is only slightly stained by the zinc-iodide-osmium tetraoxide reagent.

Controlling the aggregation of sodium dodecylsulphate in aqueous poly(ethylene glycol) solutions

RSC Advances ◽  
2015 ◽  
Vol 5 (91) ◽  
pp. 74744-74752 ◽  
Author(s):  
J. Dey ◽  
N. Sultana ◽  
S. Kumar ◽  
V. K. Aswal ◽  
S. Choudhury ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Self Assembly ◽  
The Self ◽  
Poly Ethylene Glycol ◽  
Sodium Dodecylsulphate ◽  
Molecular Weights ◽  
Poly Ethylene

The role of PEG's of different molecular weights on the self-assembly of SDS and the role of added NaCl has been addressed opening new scopes for further studies.

scholarly journals Identification of three coated vesicle components as alpha- and beta-tubulin linked to a phosphorylated 50,000-dalton polypeptide.

1983 ◽  
Vol 97 (1) ◽  
pp. 40-47 ◽  
Author(s):  
S R Pfeffer ◽  
D G Drubin ◽  
R B Kelly
Keyword(s):  
Intracellular Transport ◽  
Gel Filtration ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Tau Proteins ◽  
Coated Vesicle ◽  
Beta Tubulin ◽  
Two Dimensional Gel Electrophoresis ◽  
Molecular Weights ◽  
Membrane Compartments

Coated vesicles are involved in the intracellular transport of membrane proteins between a variety of membrane compartments. The coats of bovine brain coated vesicles contain at least six polypeptides in addition to an 180,000-dalton polypeptide called clathrin. In this report we show that the 54,000- and 56,000-dalton coated vesicle polypeptides are alpha- and beta-tubulin, determined by immunoblotting and two-dimensional gel electrophoresis. An affinity-purified tubulin antiserum can precipitate coated vesicles. The tubulin polypeptides are tightly associated with a 50,000-dalton coated vesicle polypeptide, which is phosphorylated. The phosphorylated 50,000-dalton polypeptide appears to be related to brain microtubule-associated tau proteins since it can be specifically immunoprecipitated by an affinity-purified antiserum directed against these proteins. In addition, gel filtration experiments indicate that at least a fraction of the 50,000-dalton polypeptide may associate with the 100,000-dalton coated vesicle polypeptide. Since brain is a tissue rich in tubulins, liver coated vesicles were analyzed for the presence of alpha- and beta-tubulin. Like brain coated vesicles, liver coated vesicles also contain an endogenous kinase activity, which phosphorylates polypeptides of the same molecular weights and isoelectric points as the brain coated vesicle 50,000-dalton, tau-like polypeptide, and alpha- and beta-tubulin. The phosphorylated 50,000-dalton polypeptide may link the membrane and contents of coated vesicles with components of the cytoskeleton.

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