Molecular properties of the reassembled coat protein of coated vesicles

Nandi, P. K.; Pretorius, Harold T.; Lippoldt, Roland E.; Johnson, Michael L.; Edelhoch, Harold

doi:10.1021/bi00566a039

Cited by 51 publications

(48 citation statements)

References 18 publications

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“…Such preparations of clathrin trimers have been repolymerized in varying ionic conditions (8)(9)(10)(11)(12)(13) to give empty cages resembling the outer coats of coated vesicles . (We propose to use the word "cage" to denote empty shells assembled from purified clathrin, whereas "coat" denotes the complete structure surrounding a vesicle and probably containing additional components besides the clathrin.)…”

mentioning

confidence: 99%

Assembly and packing of clathrin into coats.

Crowther¹,

Pearse²

1981

The Journal of Cell Biology

198

181

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We present a model for the packing of clathrin molecules into the characteristic hexagons and pentagons covering coated pits and vesicles. The assembly unit is a symmetrical trimer with three extended legs. Polymerization of these units occurs in seconds under suitable conditions, giving empty polyhedral cages resembling the structures around coated vesicles. Images of small, negatively stained fragments of cages, assembled directly on electron microscope grids, reveal details of the structure, which correlate well with the predicted features of the model. There is one clathrin trimer at each polyhedral vertex, and each leg of the trimer extends along two neighboring polyhedral edges. Quasi-equivalent packing in pentagons and hexagons in polyhedra of different sizes requires a variable joint at the vertex of the molecule and a hinge in each leg. The construction of clathrin coats is remarkable for the extended fibrous contacts that each molecule makes with many others. Such contacts may confer mechanical strength combined with flexibility needed when a vesicle is pinched off from the membrane.

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confidence: 99%

Assembly and packing of clathrin into coats.

Crowther¹,

Pearse²

1981

The Journal of Cell Biology

198

181

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“…Clathrin (3), the principal constituent of the basket-like coat structure, is a multifunctional protein that must be capable of assembly into coats (4)(5)(6)(7)(8)(9) as well as binding to membranes. Considering clathrin's large size (180,000 Mr), it might be anticipated that some of these distinct functions would reside in separate domains of the polypeptide chain.…”

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confidence: 99%

A domain of clathrin that forms coats.

Schmid¹,

Matsumoto²,

Rothman³

1982

Proc. Natl. Acad. Sci. U.S.A.

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Triskelions, trimeric complexes of clathrin and associated light chains, are the assembly units of clathrin coats [Ungewickell, E. & Branton, D. (1981) Nature (London) 289, 420-422;Kirchhausen, T. & Harrison, S. C. (1981) Cell 23,[755][756][757][758][759][760][761]. We report here that triskelions whose outer arms have been removed by trypsin digestion retain the ability to be assembled into coats. These digested trimers contain a 110,000 molecular weight domain of clathrin and lack intact light chains.Coated vesicles transport selected sets of proteins between membrane-bound compartments (1, 2). Clathrin (3), the principal constituent of the basket-like coat structure, is a multifunctional protein that must be capable of assembly into coats (4-9) as well as binding to membranes. Considering clathrin's large size (180,000 Mr), it might be anticipated that some of these distinct functions would reside in separate domains of the polypeptide chain. We report here the proteolytic dissection from clathrin of a domain (of about 110,000 Mr) that has the capacity to be assembled into cages in the absence of intact clathrin-associated light chains (33,000 and 36,000 Mr), a property not possessed by native clathrin (7). MATERIALS AND METHODSPurification of Clathrin. Coat proteins were purified from calf brain as described by Kirchhausen and Harrison (7) with slight modifications. All procedures were carried out at 0-40C. Coated vesicles (approximately 25 mg) obtained from the second sucrose gradient [as described by Pearse (3)] were pelleted and resuspended in 15 ml of buffer I [0.75 M Tris/25 mM 4-morpholineethanesulfonic acid (Mes), pH 6.2/0.25 mM EGTA/ 0.12 mM MgCl2/0.02% NaN3] by using a Dounce homogenizer, incubated on ice for 30 min, and then centrifuged in a Beckman SW 50.1 rotor at 35,000 rpm for 90 min. The supernatant was precipitated with ammonium sulfate (50% of saturation). The precipitate was dissolved in 3 ml of buffer 11 (0.5 M Tris/50 mM Mes, pH 6.2/0.5 mM EGTA/0.25 mM MgCl2/ 0.02% NaN3) and chromatographed on a Bio-Gel A-1.5m column (3 x 80 cm) equilibrated with buffer II. The void volume (containing clathrin) was precipitated with ammonium sulfate (80% of saturation) and dissolved in 10 ml of buffer II. Remaining membrane contaminants were removed by a second centrifugation (SW 50.1 rotor, 35,000 rpm, for 1 hr). The resulting supernatant (referred to as "column-purified clathrin") was frozen in liquid nitrogen and stored at -70°C until use. Typically, 7-9 mg ofclathrin was purified from 1.5 kg ofbrain. As reported (7), these preparations consist almost entirely of clathrin (180,000 Mr) and light chains (33,000 and 36,000 Mr). The molar ratio of light chains (33,000 and 36,000 Mr species considered together) to clathrin was 0.78 ± 0.02 in four independent preparations.Reconstitution of Empty Cages and Mild Proteolysis. To form cages (7), column-purified clathrin (typically 0.5-1.0 mg/ ml) was dialyzed against buffer III (20 mM Mes, pH 6.2/1 mM EDTA/2 mM CaCl2) for 10-15 hr at 40C. For r...

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“…This clathrin assembles rapidly into baskets when the pH is lowered to 6.2 by adding 1 M MES (pH 6.0). Assembly can be monitored spectrophotometrically at 320 nm due to the increased light-scattering properties of assembled compared with disassembled clathrin (Nandi et al, 1980). Assembly-competent clathrin was preincubated with anti-clathrin IgG at molar concentrations of 3:1 (IgG:triskelion) which is equivalent to one IgG molecule per clathrin binding site.…”

Section: Resultsmentioning

confidence: 99%

“…Clathrin assembles into similar polyhedra under physiological conditions if specific additional polypeptide components of coated vesicles are present (Zaremba and Keen, 1983;Pearse and Robinson, 1984). Furthermore, when exposed to low pH (6.0-6.5), purified clathrin will self-assemble even in the absence of accessory proteins, indicating that clathrin-clathrin interactions alone are an important component of the assembly process (Woodward and Roth, 1978;Schook et al, 1979;Keen et al, 1979;Nandi et al, 1980). A molecule of depolymerized clathrin has a triskelion shape and is composed of three heavy chains (180 kd) and three light chains of two types (30-40 kd) (Ungewickell and Branton, 1981; Kirchhausen and Harrison, 1981;Crowther and Pearse, 1981).…”

Section: Introductionmentioning

confidence: 99%

Site-specific disruption of clathrin assembly produces novel structures.

Brodsky¹

1986

The EMBO Journal

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Clathrin assembly in vitro produces a highly ordered polyhedral structure (basket). This resembles clathrin assembled in situ on coated pits and vesicles which form during receptor-mediated endocytosis. Sites on clathrin involved in assembly were identified by assembling clathrin in the presence of anti-clathrin monoclonal antibodies. Three of the antibodies, as IgG, prevented the assembly of normal baskets, and their Fab fragments induced formation of two types of novel clathrin structures. Antibody effects on assembly and competitive binding data indicate these antibodies bind to two sites, critical for clathrin interactions, located in the same region of the clathrin heavy chain. Analysis of novel structures formed, suggested that nucleation but not further assembly was occurring, implying an ordered sequence of clathrin interactions during assembly.

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Molecular properties of the reassembled coat protein of coated vesicles

Cited by 51 publications

References 18 publications

Assembly and packing of clathrin into coats.

Assembly and packing of clathrin into coats.

A domain of clathrin that forms coats.

Site-specific disruption of clathrin assembly produces novel structures.

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