Two classes of binding sites for uncoating protein in clathrin triskelions

Clathrin released from coated vesicles or empty cages by the ATP-dependent action of uncoating protein exists as a complex with the uncoating protein. Despite its apparent consumption during a round of uncoating, we have found that uncoating protein functions as an enzyme in that it rapidly and spontaneously recycles from its product (triskelions) to its substrate (cages). The binding of uncoating protein to clathrin triskelions is a complex equilibrium that involves the interaction of uncoating protein with at least two distinct sites on the clathrin molecule. Limited proteolysis dissected clathrin into two domains, each of which contained distinct binding sites. Binding to one of these sites, located on the proximal leg of a triskelion, was dependent upon the presence of light chains and was unstable to gel filtration. Binding to the second kind of site, located on the distal portion of a triskelion leg, was stable to gel filtration and was independent of the presence of light chains.     

Brain ankyrin. Purification of a 72,000 Mr spectrin-binding domain

Polypeptides of Mr = 190,000-220,000 that cross-react with erythrocyte ankyrin were detected in immunoblots of membranes from pig lens, pig brain, and rat liver. The cross-reacting polypeptides from brain were cleaved by chymotrypsin to fragments of Mr = 95,000 and 72,000 which are the same size as fragments obtained with erythrocyte ankyrin. The brain 72,000 Mr fragment associated with erythrocyte spectrin, and the binding occurred at the same site as that of erythrocyte ankyrin 72,000 Mr fragment since (a) brain 72,000 Mr fragment was adsorbed to erythrocyte spectrin-agarose and (b) 125I-labeled erythrocyte spectrin bound to brain 72,000 Mr fragment following transfer of the fragment from a sodium dodecyl sulfate gel to nitrocellulose paper, and this binding was displaced by erythrocyte ankyrin 72,000 Mr fragment. Brain 72,000 Mr fragment was purified about 400-fold by selective extraction and by continuous chromatography on columns attached in series containing DEAE-cellulose followed by erythrocyte spectrin coupled to agarose, and finally hydroxylapatite. The brain 72,000 Mr fragment was not derived from contaminating erythrocytes since peptide maps of pig brain and pig erythrocyte 72,000 Mr fragments were distinct. The amount of brain 72,000 Mr fragment was estimated as 0.28% of membrane protein or 39 pmol/mg based on radioimmunoassay with 125I-labeled brain fragment and antibody against erythrocyte ankyrin. Brain spectrin tetramer was present in about the same number of copies (30 pmol/mg of membrane protein) based on densitometry of Coomassie blue-stained sodium dodecyl sulfate gels. The binding site on brain spectrin for both brain and erythrocyte ankyrin 72,000 Mr fragments was localized by electron microscopy to the midregion of spectrin tetramers about 90 nM from the near end and 110 nM from the far end. These studies demonstrate the presence in brain membranes of a protein closely related to erythrocyte ankyrin, and are consistent with a function of the brain ankyrin as a membrane attachment site for brain spectrin.     

Selective externalization of an ATP-binding protein structurally related to the clathrin-uncoating ATPase/heat shock protein in vesicles containing terminal transferrin receptors during reticulocyte maturation

Transferrin receptors are lost from reticulocytes in vesicles that are released during the final stage of erythrocyte maturation (Pan, B. T., and Johnstone, R. M. (1983) Cell 33, 967-977). Transferrin receptor-containing vesicles have a major protein component present in a 1:1 ratio with the receptor that migrates on sodium dodecyl sulfate gels as two polypeptides of Mr = 71,000 and 72,000. The Mr = 71,000/72,000 doublet is indistinguishable from the clathrin-uncoating ATPase/heat shock protein based on cross-reaction with affinity-purified antibody against the uncoating protein, by comparison of peptide maps of the Mr = 72,000 and 71,000 polypeptides and the uncoating protein, and by selective binding of these polypeptides to ATP-agarose. This finding suggests a possible activity of proteins related to the uncoating/heat shock protein family in the disposal of aged membrane proteins by a pathway independent of lysosomes.     

Biochemical and immunological studies on clathrin light chains and their binding sites on clathrin triskelions

Clathrin light chains from bovine brain tissue (LC alpha and LC beta) are monomeric proteins with an average mol. wt. of approximately 33,000, as determined by sedimentation equilibrium. Solution studies on purified light chains indicate a large Stokes radius (Re = 3.3 nm) and little defined secondary structure. Both light chains bind specifically and with high affinity (KA approximately 5 x 10(7)/M) to overlapping sites on clathrin heavy chains. These binding sites are contained within a 125,000 dalton heavy chain fragment that forms truncated triskelions with legs, 15 nm shorter than those of intact triskelions. As judged by immuno-electron microscopy, light chain-specific IgG molecules bind mostly to the center of triskelions, but there are also sites that are scattered some 16 nm along the proximal part of triskelion legs. From heterologous binding experiments using human placenta light chains and heavy chain fragments from bovine brain clathrin, it is concluded that the domains of light and heavy chains that are involved in the interaction are conserved across tissue and species boundaries.     

Purification of Clathrin Heavy and Light Chain fromDictyostelium discoideum

Clathrin, a protein important for endocytosis, is a hexamer composed of three heavy chains and three light chains. We report here the purification scheme used to isolate the clathrin protein from the simple eukaryote,Dictyostelium discoideum.Using a combination of differential centrifugation and column chromatography, we isolated ∼2 mg of clathrin triskelions from 150–200 g ofDictyosteliumcells. One additional step purified the 30-kDa clathrin light chain to homogeneity. Glycerol gradient centrifugation was used to determine anSvalue of 7.9 for purified clathrin. Rotary shadowed images ofDictyosteliumclathrin revealed trimeric molecules with extended legs measuring 48 ± 5 nm, similar in length to the legs of mammalian and yeast clathrin triskelions. The single clathrin light chain proved resistant to heat treatment, a property also similar to light chains from other species. The conservation of these physical properties inDictyosteliumclathrin demonstrates the potential of this model organism for the study of clathrin structure and function.     

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.     

Clathrin light chains are calcium-binding proteins

Clathrin light chains have been purified to near homogeneity. When analyzed by sodium dodecyl sulfate gel electrophoresis followed by silver stain for proteins, no bands corresponding to light chains were detected. As calmodulin and troponin C are known to behave in the same manner on silver staining, the possibility that clathrin light chains were Ca2+-binding proteins was investigated. Light chains fixed to nitrocellulose filters were found to bind 45Ca2+ in the presence of 5 mM Mg2+. The Ca2+-binding capacity of the light chains was further investigated, using gel filtration and equilibrium dialysis. The light chains were shown to bind, in the presence of 3 mM Mg2+, 1 mol of Ca2+ per mol of light chain with a Kd of 25-55 microM. Nitrocellulose binding and gel filtration studies showed that light chains present in triskelions are still capable of binding Ca2+, in this case with a calculated Kd of 45 microM.     

Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG

Clathrin depletion by ribonucleic acid interference (RNAi) impairs mitotic spindle stability and cytokinesis. Depletion of several clathrin-associated proteins affects centrosome integrity, suggesting a further cell cycle function for clathrin. In this paper, we report that RNAi depletion of CHC17 (clathrin heavy chain 17) clathrin, but not the CHC22 clathrin isoform, induced centrosome amplification and multipolar spindles. To stage clathrin function within the cell cycle, a cell line expressing SNAP-tagged clathrin light chains was generated. Acute clathrin inactivation by chemical dimerization of the SNAP-tag during S phase caused reduction of both clathrin and ch-TOG (colonic, hepatic tumor overexpressed gene) at metaphase centrosomes, which became fragmented. This was phenocopied by treatment with Aurora A kinase inhibitor, suggesting a centrosomal role for the Aurora A-dependent complex of clathrin, ch-TOG, and TACC3 (transforming acidic coiled-coil protein 3). Clathrin inactivation in S phase also reduced total cellular levels of ch-TOG by metaphase. Live-cell imaging showed dynamic clathrin recruitment during centrosome maturation. Therefore, we propose that clathrin promotes centrosome maturation by stabilizing the microtubule-binding protein ch-TOG, defining a novel role for the clathrin-ch-TOG-TACC3 complex.     

ATP catalyzes the sequestration of clathrin during enzymatic uncoating

ATP facilitates the sequestration of displaced triskelions by uncoating protein. In so doing, ATP is not hydrolyzed; nor does the concentration of ATP affect the equilibrium of this binding. However, the rates of both the binding of uncoating protein to clathrin and of their dissociation are greatly accelerated by ATP. These properties suggest that ATP acts catalytically to speed the capture of displaced triskelions by uncoating protein, as well as stoichiometrically in its hydrolysis to drive the displacement of triskelions from cages. The nucleotide specificity of this "catalytic" site for ATP on the uncoating protein is much less strict than that of the distinct "hydrolytic" site that drives the ATP-dependent displacement of triskelions from cages.     

An enzyme that removes clathrin coats: purification of an uncoating ATPase

Uncoating ATPase, an abundant 70,000-mol-wt polypeptide mediating the ATP-dependent dissociation of clathrin from coated vesicles and empty clathrin cages, has been purified to virtual homogeneity from calf brain cytosol. Uncoating protein is present in cells in amounts roughly stoichiometric with clathrin. This enzyme is isolated as a mixture of monomers and dimers, both forms being active. ATP can support protein-facilitated dissociation of clathrin at micromolar levels; all other ribotriphosphates as well as deoxy-ATP are inactive. The clathrin that is released from cages consists of trimers (triskelions) in a stoichiometric complex with uncoating ATPase. These complexes with clathrin have little tendency to self-associate at neutral pH, and at acidic pH they interfere with the assembly of free clathrin. The possible existence and function of these complexes as clathrin carriers in cells would explain why uncoating protein is made in quantities equivalent to clathrin.     

A motif in the clathrin heavy chain required for the Hsc70/auxilin uncoating reaction

The 70-kDa heat-shock cognate protein (Hsc70) chaperone is an ATP-dependent "disassembly enzyme" for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone auxilin together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is thought that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C terminus (residues 1631-1675) of the heavy chain at the foot of the tripod below the hub, containing the sequence motif QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP- and auxilin-dependent, Hsc70-catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70-facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone.     

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.     

Hsc70 chaperones clathrin and primes it to interact with vesicle membranes

When Hsc70 uncoats clathrin-coated vesicles in an auxilin- and ATP-dependent reaction, a single round of rapid uncoating occurs followed by very slow steady-state uncoating. We now show that this biphasic time course occurs because Hsc70 sequentially forms two types of complex with the dissociated clathrin triskelions. The first round of clathrin uncoating is driven by formation of a pre-steady-state assembly protein (AP)-clathrin-Hsc70-ADP complex. Then, following exchange of ADP with ATP, a steady-state AP-clathrin-Hsc70-ATP complex forms that ties up Hsc70, preventing further uncoating. This steady-state complex forms only during uncoating in the presence of APs; in the absence of APs, Hsc70 rapidly dissociates from the uncoated clathrin and continues to carry out uncoating. Whether it is complexed with ATP or ADP, the steady-state complex has very different properties from the pre-steady-state complex in that it cannot be immunoprecipitated by anti-clathrin antibodies and is readily dissociated by fast protein liquid chromatography. Remarkably, when the steady-state complex is incubated with uncoated vesicle membranes in ATP, the pre-steady-state complex reforms, suggesting that the clathrin triskelions in the steady-state complex rebind to the membranes and are again uncoated by Hsc70. We propose that Hsc70 not only uncoats clathrin but also chaperones it to prevent it from inappropriately polymerizing in the cell cytosol and primes it to reform clathrin-coated pits.     

Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics

Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat shock protein hsc70 control receptor-mediated pathways of intracellular transport. The clathrin light chains are major regulatory elements in these processes. These polypeptides consist of linear arrays of functional domains with distinctive sequence motifs. Comparison of unicellular and multicellular eukaryotes reveals differences in the numbers of clathrin light chains and in the functional domains they contain.     

Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats

A protein activity has been identified in extracts of coated vesicles that enables purified clathrin triskelions to reassemble in vitro into coat structures of uniform size. Coats formed in the presence of this preparation, regardless of the buffer system employed, are uniform in size with a mean diameter of 78 nm (+/- 5 nm SD) and a sedimentation coefficient (S20,w) of approximately 250S. Analysis of the reassembled coats on dodecyl sulfate acrylamide gels reveals that they have specifically incorporated three polypeptides from the preparation: those of Mr congruent to 52,000, 100,000, and 110,000. The 52,000-, 100,000-, and 110,000-mol-wt polypeptides are incorporated in molar ratios of 0.85, 1.11, and 0.26, respectively, per three clathrin monomers (equivalent to one triskelion). We therefore designate these as assembly polypeptides (AP). In contrast, coats formed from clathrin alone, under permissive buffer conditions, are larger (400S), more heterogeneous in size (101 nm +/- 15 nm SD), and are composed only of clathrin and its associated light chains. These biochemical and biophysical characteristics distinguish AP-reassembled coats from coats formed by triskelions alone. AP-reassembled coats can be isolated, dissociated, then reassembled in the absence of any other factors. This recycling indicates that all the information needed for reassembly is present in the coat-incorporated polypeptides themselves. Reassembly is stoichiometric and saturable with respect to both clathrin and AP concentration. In the presence of AP, significant coat reassembly occurs at clathrin concentrations as low as 0.06 mg/ml. AP-mediated reassembly proceeds at 4 degrees, 22 degrees, and 37 degrees C. Coat formation also proceeds efficiently at intracellular pH values (7.2- 7.5) in the presence of AP. In its absence, reassembly does not occur at all above pH 6.7. In summary, AP promotes clathrin reassembly into coat structures of uniform size and distinctive composition under physiologically relevant salt, temperature, and pH conditions. In addition, the close similarity in size between AP-reassembled coats in vitro and coated membranes in the Golgi region in vivo raises the possibility that AP in the cell may be associated with this subpopulation of coat structures.     

Phosphorylation Characteristics of Brain Clathrin-Coated Vesicle Endogenous Proteins

Clathrin-coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle-associated substrates: a 50,000-Mr polypeptide (pp50) and clathrin-associated protein2 (CAP2; the faster-migrating clathrin light chain). Various exogenous substrates, e.g., casein, phosvitin, histone II, and histone III, also are phosphorylated. The pp50 protein kinase activity of clathrin-coated vesicles is not modulated by Ca2+, calmodulin, phosphatidylserine, or cyclic AMP. On the other hand, phosphorylation of the other endogenous substrates requires certain activators, including histone, polylysine, polyarginine, or polyethylenimine. Phosphate incorporation into pp50 was sensitive to divalent cations that inhibit sulfhydryl-dependent enzymes in the following order of potency: Zn2+ greater than Hg2+ greater than Cd2+, Cu2+, and Pb2+. Phosphate incorporation into CAP2 with polylysine present was insensitive to divalent cations. The alkylating agents dithiodinitrobenzene, phenacyl bromide, and N-ethylmaleimide inhibited phosphate incorporation into pp50 up to 90% without affecting incorporation into the other substrates. Vanadium pentoxide inhibited phosphorylation of CAP2 but had a minimal effect on pp50. CAP2 kinase activity was separated from the coated vesicle membrane and from dis-assembled clathrin triskelions, coeluting with the assembly polypeptide complex on a Sepharose 4B column. It retained phosphorylation properties similar to those of intact vesicles. These data imply that clathrin-coated vesicle kinases are elements of the coat proteins and may be involved in the assembly/disassembly of clathrin triskelions or interactions of coated vesicles with other cellular components.     

Light-chain-independent binding of adaptors, AP180, and auxilin to clathrin

Binding of coated vesicle assembly proteins to clathrin causes it to assemble into regular coat structures. The assembly protein fraction of bovine brain coated vesicles comprises AP180, auxilin, and HA1 and HA2 adaptors. Clathrin heavy chains, separated from their light chains, polymerize with unimpaired efficiency when assembly proteins are added. The reassembled coats were purified by sucrose gradient centrifugation and examined for composition by SDS-PAGE and immunoblotting. We found that all four major coat proteins are incorporated in the presence and absence of light chains. Moreover, each of the purified coat proteins is able to associate directly with clathrin heavy chains in preassembled cages as efficiently as with intact clathrin. We conclude that light chains are not essential for the interaction of AP180, auxilin, and HA1 and HA2 with clathrin.     

Characterization of a Temperature-Sensitive Vertebrate Clathrin Heavy Chain Mutant as a Tool to Study Clathrin-Dependent Events In Vivo

Clathrin and clathrin-dependent events are evolutionary conserved although it is believed that there are differences in the requirement for clathrin in yeast and higher vertebrates. Clathrin is a long-lived protein and thus, with clathrin knockdowns only long-term consequences of clathrin depletion can be studied. Here, we characterize the first vertebrate temperature-sensitive clathrin heavy chain mutant as a tool to investigate responses to rapid clathrin inactivation in higher eukaryotes. Although we created this mutant using a clathrin cryo-electron microscopy model and a yeast temperature-sensitive mutant as a guide, the resulting temperature-sensitive clathrin showed an altered phenotype compared to the corresponding yeast temperature-sensitive clathrin. First, it seemed to form stable triskelions at the non-permissive temperature although endocytosis was impaired under these conditions. Secondly, as a likely consequence of the stable triskelions at the non-permissive temperature, clathrin also localized correctly to its target membranes. Thirdly, we did not observe missorting of the lysosomal enzyme beta-glucuronidase which could indicate that the temperature-sensitive clathrin is still operating at the non-permissive temperature at the Golgi or, that, like in yeast, more than one TGN trafficking pathway exists. Fourthly, in contrast to yeast, actin does not appear to actively compensate in general endocytosis. Thus, there seem to be differences between vertebrates and yeast which can be studied in further detail with this newly created tool.     

Characterization of the coated vesicle uncoating ATPase: tissue distribution, association with and activity on intact coated vesicles

We have analyzed the uncoating process of clathrin-coated vesicles (CV) performed by an ATPase (UA; apparent molecular mass 70 kDa) prepared from various mammalian tissues. Our data show that this enzyme removes the clathrin coat from isolated, intact coated vesicles, as seen by sedimentation analysis on gels and also by electron microscopy. The isolated UA does not discriminate between CV from homologous or heterologous tissues. This finding implies that the brain-specific insertion in clathrin light chains cannot be essential for the binding of brain UA to target vesicles. Polyclonal antibodies were raised against UA and were found to inhibit UA activity. Immunoblotting of purified CV and immunoblotting of CV in situ indicate that a subpopulation of CV contains bound UA. However, most of the uncoating enzyme is not associated with coated structures in mammalian tissue culture cells. Our data support the hypothesis that the 70 kDa uncoating ATPase is responsible for the in vivo uncoating of coated vesicles.     

Analysis of clathrin light chain-heavy chain interactions using truncated mutants of rat liver light chain LCB3

Clathrin light chains are extended molecules located along the proximal segment of each of the three heavy chain legs of a clathrin trimer. All mammalian light chains share a central segment with 10 repeated heptad motifs believed to mediate the interaction with clathrin heavy chains. In order to test this model in more detail, we have expressed intact rat liver clathrin light chain LCB3 in Escherichia coli and find that it binds tightly to calf clathrin heavy chains. Using a set of expressed truncated mutants of LCB3, we show that the presence of seven to eight heptads is indeed necessary for a successful interaction. More extensive deletions of the central segment completely abolish the ability to bind to heavy chains. Neither the amino- nor the carboxyl-terminal domain is essential for binding, but competition experiments show that the presence of the carboxyl-terminal domain does enhance the interaction with heavy chains.