Clathrin Baskets Research Articles

Dissociation of clathrin from coated vesicles by the uncoating ATPase.

The uncoating ATPase has been shown to dissociate clathrin from both clathrin-coated vesicles and synthetic clathrin baskets (Rothman, J. E., and Schmid, S. L. (1986) Cell 46, 5-9). In the present study, we investigated the mechanism of action of the uncoating ATPase using intact coated vesicles isolated from bovine brain. We observed an initial burst of uncoating followed by much slower steady-state uncoating. The initial burst of uncoating was essentially stoichiometric with each molecule of uncoating ATPase apparently binding to one leg of the clathrin triskelion. When the enzyme was preincubated with equimolar ADP, Pi, and ATP, rather than just ATP alone, both the initial burst and the slow steady-state uncoating were markedly inhibited, suggesting that the combination of ADP and Pi is a strong competitive inhibitor of ATP binding. However, kinetic studies suggested that ADP and Pi dissociates from the enzyme relatively rapidly unless clathrin is also bound to the enzyme. These results suggest that, after the uncoating ATPase rapidly removes a stoichiometric amount of clathrin while ATP is hydrolyzed at the active site, slow release of ADP and Pi from the resulting enzyme.clathrin.ADP.Pi complex limits the rate at which further uncoating occurs.

An intermediate polymer in the assembly of clathrin baskets.

Clathrin (8 S) is known to polymerize into two varieties of basket structures (150 S or 300 S) under the normal buffer conditions [100 mM 2-(N-morpholino)ethanesulfonic acid (Mes), pH 5.9-6.7] used for the isolation of coated vesicles. However, it is now observed that under very low salt conditions (2 mM Mes, pH 5.9), it forms a homogeneous species with a sedimentation coefficient of 27 S. Increasing the salt concentration to 50 mM Mes completely converts all the 27S species into 150S baskets. Sedimentation equilibrium data show that this 27S species has a molecular weight that is 6 times that of the clathrin protomer and is the result of highly cooperative reversible self-association of the 8S protomer. Light-scattering studies show that the stabilities of 27S species and baskets (150 S or 300 S) are comparable. Fluorescent labeling of sulfhydryl groups with N-(1-anilinonaphthalenyl)maleimide indicates that the conformation of clathrin in 27S species and baskets (150 S or 300 S) is similar. Trypsin digestion reveals that in the 27S species clathrin has a conformation differing from that in both the 8S species and baskets.

Freeze-etch visualizations of some elements in the stria vascularis.

A quick-freeze, deep-etch study was made to observe the three-dimensional aspect of both membranes and organelles in the stria vascularis of the guinea pig. Our replicas showed various membrane characteristics of the vesicles, e.g., a clathrin basket on the inner surface membrane, aggregation of membrane particles on the protoplasmic fracture face, and regularly arranged surface protuberances on the outer surface membrane. In the cell processes of marginal cells, there were not only bundles of microtubules but also another type of fibril. Many gap junctions were observed between marginal, intermediate, and basal cells. In some replicas, the inner surfaces of the gap junctions were covered with particle protrusions with some ordered arrangements. The functional roles of these elements in the stria vascularis are discussed.

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.

Ultrastructure of the Cytoskeleton Purified From Macrophages

The cytoskeleton of mammalian cells consists of families of protein fibers and networks which include microtubules (MT), microfilaments (MF), clathrin baskets (CB), intermediate filaments (IF), and the microtrabeculae lattice (MTL). The networks function in a coordinated, integrated manner to bring about movement/motility of intracellular organelle and membrane traffic. Although much is known about the structure and function of MT and MF, more knowledge is needed to elucidate the ultrastructure, assembly and function of intermediate filaments in non-keratinized mammalian cells.The objective of this study was to elucidate the ultrastructure of intermediate filaments isolated and purified from cultured mammalian cells.Macrophages were harvested from the peritoneal cavities of mice. Triton- insoluble fractions of IF were isolated from spread cultures using high salt and low ionic strength buffers. Purified IF were analyzed biochemically. Using poly acrylamide gel electrophoresis IF consists of 4-7 major bands in the molecular weight ranges of 14-90Kd3.

30] Dissociation and reassociation of clathrin

Publisher Summary This chapter describes the dissociation and reassociation of clathrin. The clathrin trimer is capable of assembling into the geodesic dome structure observed on coated vesicles by simply decreasing the pH of clathrin solutions from 7.5 to some value between 6 and 6.8, until chaotropic anions are absent and the ionic strength is not too high. The available procedures for clathrin purification depend on the relative enrichment of this protein by virtue of its tendency to form or remain assembled as macromolecular aggregates under conditions of slightly acidic pH. Cosedimentation of both coated vesicles and clathrin baskets in sucrose gradients allows a further enrichment from the starting homogenate. A second property utilized is clathrin's sensitivity to conditions of basic pH. When assemblies of clathrin are incubated in buffers of low ionic strengths and basic pH, they disassemble into trimers, which can be separated from the bulk of the vesicular membrane and other proteins by sedimentation and column chromatography. There are two principal approaches to clathrin purification described in the literature: one utilizes 2 M urea following low-ionic strength extraction of a crude coated vesicle fraction; the other utilizes a high concentration of Tris buffer and a more highly purified coated vesicle preparation.

Brain clathrin and clathrin-associated proteins.

The assembly of clathrin into baskets or cages in vitro may depend on formation of complex between clathrin and a polypeptide doublet migrating in the 30000-mol.wt. region. Clathrin with several associated proteins was isolated from coated-vesicle fractions of bovine cerebral cortex. Most associated proteins were separated by Sepharose 4B column chromatograhy. The eluted clathrin retained only the 30000-mol.wt. doublet and assembled into baskets at pH 6.5. Limited proteolysis of coated vesicles or clathrin assembled as baskets removed these clathrin-associated proteins (CAPs) without detectably altering clathrin. Enzyme-treated clathrin assembled into open-lattice structures but no longer formed baskets in vitro. Latex particles with bound enzyme cleaved the CAPs from coated vesicles and clathrin baskets, suggesting that the CAPs protrude from the exterior of the clathrin lattice.

Functional and structural studies on clathrin triskelions and baskets.

Recently it has been shown that the basic unit of clathrin coats of vesicles, is a trimer of clathrin organized together with probably three low-molecular-weight polypeptides in three-legged structures, called triskelions. An overview is provided of the clathrin work currently in progress at the Biological Laboratories at Harvard University. First the conditions for the binding of triskelions to the membrane of clathrin-depleted vesicles is described and then the regions of triskelions that are involved in membrane binding, basket formation, and binding of light chains are identified.

Clathrin-coated vesicles: Isolation, dissociation and factor-dependent reassociation of clathrin baskets

The nature of the protein coat on clathrin-coated vesicles and the interactions responsible for the structure and reformation of clathrin baskets have been investigated. Coated vesicles were isolated from bovine brain using a rapid, one-day modification of the method of Pearse (1975). The vesicles are composed predominantly of clathrin (175,000) with smaller amounts of 110,000 and 55,000 molecular weight polypeptides. Clathrin was released in a solubilized form from these vesicles by treatment with 0.5 M Tris(hydroxymethyl)methyl ammonium chloride (Tris-Cl) or other protonated amines at neutral pH. It was not released on treatment with thiols, thiol reagents, Triton X-100 or sodium chloride, leading us to suggest that specific amino-carboxylate salt linkages are necessary for maintenance of the basket structure. When viewed by electron microscopy, the solubilized proteins in Tris-Cl are present in the form of filamentous aggregates and no basket structures are observed. Gel filtration of the extract in Tris-Cl resolves a clathrin-containing fraction (I) from one consisting predominantly of the 110,000 molecular weight polypeptide (II). We were able to reconstitute basket structures from the unfractionated Tris-Cl extract by dialyzing it against the vesicle isolation buffer, a solution of moderate ionic strength (Γ/2 = 0.11). Neither of the resolved fractions (I or II) alone yielded baskets on dialysis, but reconstitution was successful when both I and II were combined and dialyzed. The activity in II, which appears to be a basket-assembly factor, was heat-labile and could not be replaced by bovine serum albumin or brain calcium-dependent modulator protein. If a solution of low ionic strength (Γ/2 = 0.01) containing calcium was used as the dialysate, the clathrin fraction (I) alone was capable of reforming baskets. Thus the clathrin coat is a labile structure that can be solubilized by nondenaturing treatments, and baskets can be reformed from the extracted material.