Energetics of clathrin basket assembly

A minimal thermodynamic model is used to study the in vitro equilibrium assembly of reconstituted clathrin baskets. The model contains parameters accounting for i) the combined bending and flexing rigidities of triskelion legs and hubs, ii) the intrinsic curvature of an isolated triskelion, and iii) the free energy changes associated with interactions between legs of neighboring triskelions. Analytical expressions for basket size distributions are derived, and published size distribution data (Zaremba S, Keen JH. J Cell Biol 1983;97: 1339–1347) are then used to provide estimates for net total basket assembly energies. Results suggest that energies involved in adding triskelions to partially formed clathrin lattices are small (of the order of kBT), in accord with the notion that lattice remodeling during basket formation occurs as a result of thermodynamic fluctuations. In addition, analysis of data showing the effects of assembly proteins (APs) on basket size indicates that the binding of APs increases the intrinsic curvature of an elemental triskelial subunit, the stabilizing energy of leg interactions, and the effective leg/hub rigidity. Values of effective triskelial rigidity determined in this investigation are similar to those estimated by previous analysis of shape fluctuations of isolated triskelia.     

Skeletal structure of clathrin triskelion in solution: experimental and theoretical approaches

The physicochemical properties of the clathrin triskelion were determined by dynamic and static light-scattering and sedimentation analyses in Tris and triethanolamine (TEA) buffers of about pH 8, in which the clathrin triskelion has been found to be in different conformational states by electron microscopy [Heuser, J., & Kirchhausen, T. (1985) J. Ultrastruct. Res. 92, 1-27]. Dynamic light-scattering measurements provided diffusion coefficients (D0(20,w)) of 1.22 x 10(-7) and 1.23 x 10(-7) cm2/s, and ultracentrifugal analysis gave sedimentation coefficients (S0(20,w)) of 8.39 and 8.32 S in Tris and TEA buffer, respectively. The average Stokes radius of the protein was determined to be 175 A from its diffusion and sedimentation coefficients and its molecular weight. Static light-scattering analysis provided molecular weights of 6.58 x 10(5) and 6.41 x 10(5) and radii of gyration of 311 and 301 A in the respective buffers. These results indicate that the clathrin triskelion has a similar conformation in the two buffers. For clarification of the skeletal structure of the clathrin triskelion in solution, the physicochemical parameters were calculated by using two models in which the clathrin arms are bent at various angles in a plane, on the basis of the Bloomfield approximation and a formula derived to estimate the radius of gyration of proteins consisting of various structural units. Values for the Stokes radius, diffusion and sedimentation coefficients, and radius of gyration in the ranges of 178-170 A, (1.20-1.26) x 10(-7) cm2/s, 8.26-8.66 S, and 316-266 A, respectively, were obtained with these models with the arms bent in the range of 0-60 degrees.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clathrin assembly involves a light chain-binding region

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.     

Visualization of the binding of Hsc70 ATPase to clathrin baskets: implications for an uncoating mechanism

Clathrin assembly into coated pits and vesicles is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. These interactions may be mimicked in vitro by the assembly and disassembly of clathrin "baskets." The chimera C58J is a minimal construct capable of supporting both reactions; it consists of the C58 moiety of AP180, which facilitates clathrin assembly, fused with the J domain of auxilin, which recruits Hsc70 to baskets. We studied the process of disassembly by using cryo-electron microscopy to identify the initial binding site of Hsc70 on clathrin-C58J baskets at pH 6, under which conditions disassembly does not proceed further. Hsc70 interactions involve two sites: (i) its major interaction is with the sides of spars of the clathrin lattice, close to the triskelion hubs and (ii) there is another interaction at a site at the N-terminal hooks of the clathrin heavy chains, presumably via the J domain of C58J. We propose that individual triskelions may be extricated from the clathrin lattice by the concerted action of up to six Hsc70 molecules, which intercalate between clathrin leg segments, prying them apart. Three Hsc70s remain bound to the dissociated triskelion, close to its trimerization hub.     

Deep-etch visualization of 27S clathrin: a tetrahedral tetramer

It has recently been reported that 8S clathrin trimers or "triskelions" form larger 27S oligomers upon dialysis into low ionic strength buffers (Prasad, K., R. E. Lippoldt, H. Edelhoch, and M. S. Lewis, 1986, Biochemistry, 25:5214-5219). Here, deep-etch electron microscopy of the 27S species reveals that they are closed tetrahedra composed of four clathrin triskelions. This was determined by two approaches. First, standard quick-freezing and freeze-etching of unfixed 27S species suspended in 2 mM 2-(N-morpholino)ethane sulfonic acid (MES) buffer, pH 5.9, yielded unambiguous images of tetrahedra that measured 33 nm on each edge. Second, the technique of freeze-drying molecules on mica (Heuser, J. E., 1983, J. Mol. Biol., 169:155-195) was modified to overcome the low affinity of mica in 2 mM MES, by pretreating the mica with polylysine. Thereafter, 27S species adsorbed avidly to it and collapsed into characteristic configurations containing four globular domains, each linked to the others by three approximately 33-nm struts. The globular domains look like vertices of deep-etched clathrin triskelions and the links, numbering 12 in all, look like four sets of triskelion legs. New light scattering and equilibrium centrifugation data confirm that 27S polymer is four times as massive as one clathrin triskelion. We conclude that in conditions that do not favor the formation of standard clathrin cages, low affinity interactions lead to closed, symmetrical assemblies of four triskelions, each of which assumes a unique puckered, straight-legged configuration to create the edges of a tetrahedron. Tetrahedra are similar in construction to the cubic octomers of clathrin recently found in ammonium sulfate solutions (Sorger, P. K., R. A. Crowther, J. T. Finch, and B. M. F. Pearse, 1986, J. Cell Biol., 103:1213-1219) but are still smaller, involving only half as many clathrin triskelions.     

A novel structural model for regulation of clathrin function.

The distinctive triskelion shape of clathrin allows assembly into polyhedral lattices during the process of clathrin-coated vesicle formation. We have used random and site-directed mutagenesis of the yeast clathrin heavy chain gene (CHC1) to characterize regions which determine Chc trimerization and binding to the clathrin light chain (Clc) subunit. Analysis of the mutants indicates that mutations in the trimerization domain at the triskelion vertex, as well as mutations in the adjacent leg domain, frequently influence Clc binding. Strikingly, one mutation in the trimerization domain enhances the association of Clc with Chc. Additional mutations in the trimerization domain, in combination with mutations in the adjacent leg domain, exhibit severe defects in Clc binding while maintaining near normal trimerization properties. The position of these trimerization domain mutations on one face of a putative alpha-helix defines a region on the trimer surface that interacts directly with Clc. These results suggest that Clc extends into the Chc trimerization domain from the adjacent leg, thereby bridging the two domains. On the basis of this conclusion, we propose a new model for the organization of the triskelion vertex which provides a structural basis for regulatory effects of Clc on clathrin function.     

Complete reconstitution of clathrin basket formation with recombinant protein fragments: adaptor control of clathrin self-assembly

Clathrin polymerization into a polyhedral basket, surrounding budding membrane vesicles, mediates protein sorting during endocytosis and organelle biogenesis. Adaptor proteins target clathrin assembly to specific membrane sites and sequester receptors into the clathrin coat. We have reconstituted complete clathrin basket formation from recombinantly expressed fragments of clathrin and adaptors. This reconstitution reveals a hierarchy of clathrin self-assembly interactions and demonstrates that adaptors control basket formation by alignment of the distal domains of the clathrin triskelion leg through their binding to the terminal domain.     

Clathrin self-assembly involves coordinated weak interactions favorable for cellular regulation

The clathrin triskelion self-assembles into a polyhedral coat surrounding membrane vesicles that sort receptor cargo to the endocytic pathway. A triskelion comprises three clathrin heavy chains joined at their C-termini, extending into proximal and distal leg segments ending in a globular N-terminal domain. In the clathrin coat, leg segments entwine into parallel and anti-parallel interactions. Here we define the contributions of segmental interactions to the clathrin assembly reaction and measure the strength of their interactions. Proximal and distal leg segments were found to lack sufficient affinity to form stable homo- or heterodimers under assembly conditions. However, chimeric constructs of proximal or distal leg segments, trimerized by replacement of the clathrin trimerization domain with that of the invariant chain protein, were able to self-assemble in reversible reactions. Thus clathrin assembly occurs because weak leg segment affinities are coordinated through trimerization, sharing a dependence on multiple weak interactions with other biopolymers. Such polymerization is sensitive to small environmental changes and is therefore compatible with cellular regulation of assembly, disassembly and curvature during formation of clathrin-coated vesicles.     

Extended circular dichroism measurements using synchrotron radiation show that the assembly of clathrin coats requires no change in secondary structure.

A number of models have been proposed for the assembly of clathrin triskelia into coats. However, little is known of the effects of assembly on triskelion structure. A more detailed knowledge of the way in which assembly affects triskelion structure would be valuable for assessing the relative merits of the proposed models. The development of a vacuum-ultraviolet circular dichroism (CD) instrument that uses synchrotron radiation as a light source has allowed us to extend the range of CD measurements to shorter wavelengths. This has greatly increased signal quality even for highly scattering samples. Also, we have improved CD data analysis to provide standard deviations for calculated secondary structure content. These developments have increased the precision of CD analysis beyond what has been thus far possible. Using these developments, we have determined the secondary structure content of all components of coat protein, under both assembly and dissociating conditions. The assembly of coats does not incur any change in secondary structure content, but a 10% loss of triskelion helical content accompanies assembly in the absence of AP-2. We conclude that coat assembly requires no detectable reorganization of triskelion structure. Our result indicates that AP-2 stabilizes helical structure in the triskelion, and we propose that this increases triskelion rigidity, restricting the range of coat sizes.     

Folding and trimerization of clathrin subunits at the triskelion hub.

The triskelion shape of the clathrin molecule enables it to form the polyhedral protein network that covers clathrin-coated pits and vesicles. Domains within the clathrin heavy chain that are responsible for maintaining triskelion shape and function were identified and localized. Sequences that mediate trimerization are distal to the carboxyl terminus and are adjacent to a domain that mediates both light chain binding and clathrin assembly. Structural modeling predicts that within this domain, the region of heavy chain-light chain interaction is a bundle of three or four alpha helices. These studies establish a low resolution model of clathrin subunit folding in the central portion (hub) of the triskelion, thus providing a basis for future mutagenesis experiments.     

Identification of a distinct 9S form of soluble clathrin in cultured cells and tissues

We have used a monoclonal antibody (CHC5.9) to identify clathrin (Mr 180,000; 'heavy chain') in coated vesicles, triskelion structures prepared in vitro and in high-speed supernatants (HSS) of cell homogenates from a variety of tissues and species (e.g., brain and liver from rat, cow and man; Xenopus ovaries). HSS proteins were subjected to sucrose density gradient centrifugation and gel filtration, and the fractions obtained were assayed for clathrin by enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE), followed by immunoblotting. The native soluble clathrin identified in such fractions was indistinguishable from triskelions produced in vitro from purified bovine brain clathrin by several criteria, e.g. by its sedimentation coefficient (9S) and elution profile on gel filtration using Sephacryl S 300. No other major forms of soluble clathrin were detected. The results indicate that cells contain a soluble pool of clathrin and that the predominant molecular form of this soluble clathrin has properties similar to those of the triskelion obtained by dissociation studies in vitro. We hypothesize that this distinct 9S form represents a major oligomeric subunit involved in assembly and disassembly of clathrin polyhedron coats in the living cell.     

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.     

The stability and transitions of tryptic-digested clathrin

The pH-dependence of dissociation of trypsin-digested baskets has been determined by light scattering and compared with that of undigested baskets. Essentially no difference was found between the two types of baskets. The molecular transitions of clathrin derived from digested baskets have been studied by fluorescence spectra and polarization measurements and compared with those of undigested baskets. The transitions in both forms of clathrin were very similar. It is clear, therefore, that removal of about 1/3 of the mass from the distal portions of the arms of the clathrin triskelion does not affect its structural transitions. The interactions between clathrin molecules in the basket structure and those within the molecule appear, therefore, to remain intact in the smaller clathrin chains remaining after tryptic digestion. The function of the distal portion of the clathrin chain still awaits elucidation.     

Clathrin coats at 21 A resolution: a cellular assembly designed to recycle multiple membrane receptors.

We present a map at 21 A resolution of clathrin assembled into cages with the endocytic adaptor complex, AP-2. The map was obtained by cryo-electron microscopy and single-particle reconstruction. It reveals details of the packing of entire clathrin molecules as they interact to form a cage with two nested polyhedral layers. The proximal domains of each triskelion leg depart from a cage vertex in a skewed orientation, forming a slightly twisted bundle with three other leg domains. Thus, each triskelion contributes to two connecting edges of the polyhedral cage. The clathrin heavy chains continue inwards under the vertices with local 3-fold symmetry, the terminal domains contributing to 'hook-like' features which form an intermediate network making possible contacts with the surface presented by the inner adaptor shell. A node of density projecting inwards from the vertex may correspond to the C-termini of clathrin heavy chains which form a protrusion on free triskelions at the vertex. The inter-subunit interactions visible in this map provide a structural basis for considering the assembly of clathrin coats on a membrane and show the contacts which will need to be disrupted during disassembly.     

THE CONFORMATION OF CLATHRIN LIGHT-CHAINS ON TRISKELIONS PROBED BY ANTIBODY INHIBITION

The conformation of clathrin light-chains along the proximal arm of the clathrin triskelion was studied by using rabbit anti-(light-chain peptides) to inhibit the binding of a mouse monoclonal antibody against an epitope in the amino-terminal region. Prior incubation of triskelions with rabbit antisera raised against the extreme carboxyl-terminal of the light-chains partially inhibited binding. The inhibition was largely removed when tested on light-chains that had been freed from triskelions. This suggests that when the light-chains bind the heavy-chain, they adopt a conformation in which the amino and carboxyl-terminal domains are not fully extended, but fold such that these two domains face each other.     

Enzymatic dissociation of clathrin cages in a two-stage process

Uncoating ATPase catalyzes the ATP-dependent dissociation of clathrin from coated vesicles and empty cages. Following an uncoating reaction, clathrin triskelions are released intact, in a stoichiometric complex with bound uncoating protein. This overall uncoating process was dissected into two partial reactions. In the first, ATP hydrolysis drives the transient displacement of a portion of a triskelion from a cage. Uncoating protein then captures the displaced triskelion, in the second stage, by binding to a newly exposed site on clathrin that had previously been buried in the cage lattice. Triskelion-uncoating protein complexes are released when all points of attachment of the triskelion to the cage have been severed. The uncoating protein interacts with a distinct site on clathrin for each of these reactions.     

Measuring the elasticity of clathrin-coated vesicles via atomic force microscopy

Using a new scheme based on atomic force microscopy (AFM), we investigate mechanical properties of clathrin-coated vesicles (CCVs). CCVs are multicomponent protein and lipid complexes of approximately 100 nm diameter that are implicated in many essential cell-trafficking processes. Our AFM imaging resolves clathrin lattice polygons and provides height deformation in quantitative response to AFM-substrate compression force. We model CCVs as multilayered elastic spherical shells and, from AFM measurements, estimate their bending rigidity to be 285 +/- 30 k(B)T, i.e., approximately 20 times that of either the outer clathrin cage or inner vesicle membrane. Further analysis reveals a flexible coupling between the clathrin coat and the membrane, a structural property whose modulation may affect vesicle biogenesis and cellular function.     

Clathrin triskelia show evidence of molecular flexibility

The clathrin triskelion, which is a three-legged pinwheel-shaped heteropolymer, is a major component in the protein coats of certain post-Golgi and endocytic vesicles. At low pH, or at physiological pH in the presence of assembly proteins, triskelia will self-assemble to form a closed clathrin cage, or “basket”. Recent static light scattering and dynamic light scattering studies of triskelia in solution showed that an individual triskelion has an intrinsic pucker similar to, but differing from, that inferred from a high resolution cryoEM structure of a triskelion in a clathrin basket. We extend the earlier solution studies by performing small-angle neutron scattering (SANS) experiments on isolated triskelia, allowing us to examine a higher q range than that probed by static light scattering. Results of the SANS measurements are consistent with the light scattering measurements, but show a shoulder in the scattering function at intermediate q values (0.016 Å⁻¹), just beyond the Guinier regime. This feature can be accounted for by Brownian dynamics simulations based on flexible bead-spring models of a triskelion, which generate time-averaged scattering functions. Calculated scattering profiles are in good agreement with the experimental SANS profiles when the persistence length of the assumed semiflexible triskelion is close to that previously estimated from the analysis of electron micrographs.     

New faces of the familiar clathrin lattice

The clathrin triskelion self-assembles into a lattice that coats transport vesicles participating in several key membrane traffic pathways. A new model of a clathrin lattice at approximately 8 angstrom resolution, generated by Fotin et al. (Nature 2004;432:573) confirmed the basic structural features of clathrin that were defined over many years of biochemical and structural analysis. In addition, new structural features of the clathrin trimerization domain were modelled for the first time, and the predictions correlated well with previous biochemical studies. A second model, placing auxilin within the lattice suggested a possible lattice contact targeted during lattice disassembly (Fotin et al. Nature 2004;432:649). This contact predicts interactions of the newly modelled trimerization domain with a newly defined extension of the clathrin triskelion, the ankle domain. These aspects of the new models were emphasized in the published reports describing them and in recent commentary (Brodsky, Nature 2004;432:568). Also emerging from the new models is a better picture of how the clathrin structure is distributed throughout the lattice, allowing the first predictions of interacting molecular interfaces contributing to contacts in the assembled lattice. The focus of this interchange is to emphasize these additional features revealed by the recently published models from Fotin and colleagues.     

The association of clathrin fragments with coated vesicle membranes

The association between clathrin triskelions and the clathrin-stripped membranes of coated vesicles has been investigated using a filter assay to separate bound from unbound clathrin. The filter assay is more sensitive and less cumbersome than a sedimentation assay used previously (1). While confirming the high affinity interaction between clathrin and the vesicle membrane, our results yield Scatchard plots that are curvilinear and consistent with a positively cooperative interaction between clathrin and the vesicle membranes. Controlled digestion with trypsin removes the distal portions of the triskelion legs leaving the proximal 31 nm portions that form the hub of the triskelions. These hubs are trimers of large 112,000- and 124,000-dalton fragments of clathrin heavy chains. They competitively inhibit the binding of 125I-labeled intact triskelions to stripped vesicles with a KI identical to the KD for the association of 125I-labeled intact triskelions to stripped vesicles. Furthermore, these large fragment trimers bind to stripped vesicles with approximately the same high affinity as do intact triskelions and also show evidence of a positively cooperative interaction. It is concluded that clathrin binds to coated vesicles by an interaction that is mediated by the proximal 112,000-dalton fragment of the clathrin heavy chains.