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PubMed2003

The AP-2 complex is excluded from the dynamic population of plasma membrane-associated clathrin.

Numerous biologically relevant substrates are selectively internalized via clathrin-mediated endocytosis. At the plasma membrane the AP-2 complex plays a major role in clathrin coat formation, interacting with both cargo and clathrin. Utilizing simultaneous dual-channel total internal reflection fluorescence microscopy we have analyzed components of the AP-2 complex (alpha- and beta 2-adaptin) during clathrin-mediated endocytosis. Although in static images enhanced green fluorescent protein-tagged AP-2 markers significantly co-localized with clathrin and other components of clathrin-coated pits, AP-2 did not seem to be present in clathrin spots that appeared to undergo internalization or motility parallel to the plane of the plasma membrane. Two populations of clathrin at the plasma membrane seem to exist, the dynamic and the static, and AP-2 appears to be only found within the latter. These results suggest that colocalized clathrin/AP-2 puncta may represent loci for coated pit production and that previous models that assumed AP-2 was retained within clathrin coats during endocytosis may need to be re-evaluated.

Adaptor Protein Complex 2↗
PubMed2000

The interaction of beta-arrestin with the AP-2 adaptor is required for the clustering of beta 2-adrenergic receptor into clathrin-coated pits.

Beta-arrestins are cytosolic proteins that regulate the signaling and the internalization of G protein-coupled receptors (GPCRs). Although termination of receptor coupling requires beta-arrestin binding to agonist-activated receptors, GPCR endocytosis involves the coordinate interactions between receptor-beta-arrestin complexes and other endocytic proteins such as adaptor protein 2 (AP-2) and clathrin. Clathrin interacts with a conserved motif in the beta-arrestin C-terminal tail; however, the specific molecular determinants in beta-arrestin that bind AP-2 have not been identified. Moreover, the respective contributions of the interactions of beta-arrestin with AP-2 and clathrin toward the targeting of GPCRs to clathrin-coated vesicles have not been established. Here, we identify specific arginine residues (Arg(394) and Arg(396)) in the beta-arrestin 2 C terminus that mediate beta-arrestin binding to AP-2 and show, in vitro, that these domains in beta-arrestin 1 and 2 interact equally well with AP-2 independently of clathrin binding. We demonstrate in HEK 293 cells by fluorescence microscopy that beta(2)-adrenergic receptor-beta-arrestin complexes lacking the beta-arrestin-clathrin binding motif are still targeted to clathrin-coated pits. In marked contrast, receptor-beta-arrestin complexes lacking the beta-arrestin/AP-2 interactions are not effectively compartmentalized in punctated areas of the plasma membrane. These results reveal that the binding of a receptor-beta-arrestin complex to AP-2, not to clathrin, is necessary for the initial targeting of beta(2)-adrenergic receptor to clathrin-coated pits.

Adaptor Protein Complex alpha Subunits↗
PubMed2001

Multiple interactions of auxilin 1 with clathrin and the AP-2 adaptor complex.

The removal of the clathrin coat is essential for vesicle fusion with acceptor membranes. Disassembly of the coat involves hsc70, which is specifically recruited by members of the auxilin protein family to clathrin lattices. In vitro, this function of auxilin does not require the globular amino-terminal domain of the clathrin heavy chain, which is known to play a prominent role in the interaction of clathrin with adaptors and numerous endocytic accessory proteins. Here we report the unexpected finding that the neuron-specific form of auxilin (auxilin 1) can also associate with the clathrin amino-terminal domain. This interaction is mediated through tandemly arranged sites within the auxilin 1 carboxyl-terminal segment 547-910. The overlapping auxilin 1 fragments 547-714 and 619-738 bind the clathrin terminal domain with high affinity, whereas auxilin 1-(715-901) interacts only poorly with it. All three fragments also associate with the clathrin distal domain and the alpha-appendage domain of AP-2. Moreover, they support efficient assembly of clathrin triskelia into regular cages. A novel uncoating assay was developed to demonstrate that auxilin 1-(715-901) functions efficiently as a cofactor for hsc70 in the uncoating of clathrin-coated vesicles. The multiple protein-protein interactions of auxilin 1 suggest that its function in endocytic trafficking may be more complex than previously anticipated.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

Individual phosphoinositide 3-kinase C2alpha domain activities independently regulate clathrin function.

Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) is a member of the class II PI-3 kinases, defined by the presence of a second C2 domain at their C termini. The cellular functions of the class II enzymes are incompletely understood, though they have been implicated in receptor activation pathways initiated by epidermal growth factor, insulin, and chemokines. PI3K-C2alpha was recently found to be localized to clathrin-coated membranes in the trans-Golgi network and at endocytic sites on the plasma membrane. Further, a specific binding site was identified for clathrin on the N terminus of PI3K-C2alpha, whose occupancy resulted in lipid kinase activation. Expression of PI3K-C2alpha in cells dramatically affected clathrin distribution and function in cells, leading to accumulation of intracellular clathrin-coated structures, which are visualized here at the ultrastructural level, and inhibition of clathrin-mediated transport from both the plasma membrane and the trans-Golgi network. In this study we have demonstrated that the isolated clathrin binding domain of PI3K-C2alpha can drive clathrin lattice assembly and that both it and the lipid kinase activity of the protein can independently modulate clathrin distribution and function when expressed in cells. Together, these results suggest that PI3K-C2alpha employs both protein-protein interaction and localized production of 3-phosphoinositides to affect clathrin dynamics at sites of membrane budding and targeting.

Animals↗
PubMed1985

Assembled and unassembled pools of clathrin: a quantitative study using an enzyme immunoassay.

Using polyclonal antibodies raised against clathrin, we have developed an enzyme-linked immunoassay that can specifically measure the quantity of clathrin in crude cell extracts. We found that the quantity (weight percent of total protein) of clathrin was similar in cell types that exhibit large differences in their levels of endocytosis and exocytosis (lymphoid cells, 0.11%; liver cells, 0.07%, fibroblasts, 0.18%; myeloma cells, 0.16%). However, the quantity of clathrin was found to be significantly higher in brain cortex (0.75%). Cellular clathrin was separated by high-speed centrifugation into two fractions: an unassembled form present in high-speed supernatants and an assembled form (clathrin coats) present in the pellets. We show that the fraction of clathrin in the unassembled state varies considerably depending on the cell type studied (14% in brain cortex to 70% in lymphocytes). Our data support the view that the amount of clathrin (relative to total cell protein) in eucaryotic cells is not related to the extent of receptor-mediated endocytosis and intracellular membrane traffic. However, the fraction of assembled clathrin seems to be higher in endocytically and/or exocytically active cells.

Animals↗
PubMed1984

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.

Adenosine Triphosphatases↗
PubMed1993

Uncoating of clathrin-coated vesicles by uncoating ATPase from developing peas.

A cytosolic ATPase (an enzyme that dissociates clathrin from clathrin-coated vesicles in the presence of ATP) was isolated from developing pea (Pisum sativum L.) cotyledons using chromatography on ATP-agarose. After chromatography on phenyl Sepharose, the fraction with uncoating activity was enriched in a doublet of 70-kD peptides. Using chromatofocusing, it was possible to produce fractions enriched in the upper component of the doublet of 70-kD peptides; these fractions still retained ATP-dependent uncoating activity. In western blot analysis, antibodies against a member of the 70-kD family of heat-shock proteins interacted with the upper component of the doublet of the 70-kD peptides from the phenyl Sepharose-purified fractions. On the basis of these data, it appears that the uncoating ATPase may be a member of the 70-kD family of heat-shock proteins. The uncoating activity removed clathrin from both pea and bovine brain clathrin-coated vesicles. The uncoating ATPase from bovine brain also uncoated coated vesicles from peas. Pea clathrin-coated vesicles that were prepared by three different methods were uncoated to different extents by the plant uncoating ATPase. Different populations of clathrin-coated vesicles from the same preparation showed differential sensitivity to the uncoating ATPase. Limited proteolysis of the clathrin light chains in the protein coat abolished the susceptibility of the clathrin-coated vesicles to the uncoating ATPase. The properties of the uncoating ATPase isolated from developing pea cotyledons are similar to those of uncoating ATPases previously described from mammalian and yeast systems. It appears that despite dissimilarities in composition of the clathrin components of the vesicles from the respective sources, uncoating is achieved by a common mechanism.

Adenosine Triphosphatases↗
PubMed1982

Isolation and preliminary characterization of clathrin-associated proteins.

Clathrin-associated proteins were separated from clathrin under various clathrin-denaturing conditions, i.e. heating, freezing and isoelectric precipitation. The proteins retained biological activity; they were purified further by affinity chromatography on calmodulin-conjugated CNBr-Sepharose 4B and used for antibody purification. The affinity-purified anti-(clathrin-associated proteins) antibodies gave a fluorescent dotted pattern in cultured fibroblasts consistent with the known distribution of clathrin. Chemical cross-linking of pure clathrin-associated proteins indicated that these polypeptides exist as monomers in solution, each possessing Ca2+-dependent affinity for calmodulin to which they bind in a 1:1 molar ratio. Chymotryptic treatment of coated vesicles selectively cleaved the clathrin-associated proteins into a 15 000-18 000-Mr doublet polypeptide. These subfragments retained their Ca2+-dependent affinity for calmodulin. Our results support a regulatory role for clathrin-associated proteins in clathrin assemblies.

Calcium↗
PubMed1981

Purification and characterization of clathrin from bovine brain.

Clathrin has been purified to electrophoretic homogeneity by initial extraction of clathrin from purified coated vesicle fraction, followed by column chromatographies with gel filtration, DEAE-cellulose, and hydroxylapatite and finally by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Antibody specific to clathrin has also been obtained. Two forms of native clathrin, fast and slow components, have been prepared to about 95% purity by hydroxylapatite column chromatography. Both fast and slow components are believed to represent two different aggregates of clathrin subunit because they comigrate in agarose electrophoresis, pH 7.4, and also migrate as clathrin subunit on SDS-PAGE with a molecular weight of 175,000. Furthermore, both components cross-react with antibody against purified clathrin and compete for antibody binding site with labeled fast component. The fast component can also be converted to the slow component. In addition to clathrin, two proteins of about 38,000 and 35,000 M.W. that consistently copurified with native clathrin are probably also intrinsic to coated vesicle.

Animals↗
PubMed2006

Nerve growth factor-induced phosphorylation of amphiphysin-1 by casein kinase 2 regulates clathrin-amphiphysin interactions.

Amphiphysins interact directly with clathrin and have a function in clathrin-mediated synaptic vesicle recycling and clathrin-mediated endocytosis. The neuronal isoform amphiphysin-1 is a serine/threonine phosphoprotein that is dephosphorylated upon stimulation of synaptic vesicle endocytosis. Rephosphorylation was stimulated by nerve growth factor. We analysed the regulation of amphiphysin-clathrin interactions by phosphorylation. The N-terminal domain of clathrin bound to unphosphorylated amphiphysin-1, but not to the phosphorylated protein. A search for possible phosphorylation sites revealed two casein kinase 2 consensus motifs in close proximity to the clathrin binding sites in amphiphysin-1 and -2. We mutagenized these residues (T350 and T387) to glutamate, mimicking a constitutive phosphorylation. The double mutant showed a strong reduction in clathrin binding. The assumption that casein kinase 2 phosphorylates amphiphysin-1 at T350 and T387 was corroborated by experiments showing that: (i) casein kinase 2 phosphorylated these residues directly in vitro, (ii) when expressed in HeLa cells, the glutamate mutant showed reduced phosphorylation, and (iii) casein kinase 2 inhibitors blocked nerve growth factor-induced phosphorylation of endogenous amphiphysin-1 in PC12 cells. These observations are consistent with the hypothesis that, upon activation by nerve growth factor, casein kinase 2 phosphorylates amphiphysin-1 and thereby regulates the endocytosis of clathrin-coated vesicles via the interaction between clathrin and amphiphysin.

Animals↗
PubMed2006

Clathrin is important for normal actin dynamics and progression of Sla2p-containing patches during endocytosis in yeast.

Clathrin is a major vesicle coat protein involved in receptor-mediated endocytosis. In yeast and higher eukaryotes, clathrin is recruited to the plasma membrane during the early stage of endocytosis along with clathrin-associated adaptors. As coated pits undergo maturation, a burst of actin polymerization accompanies and helps drive vesicle internalization. Here, we investigate the dynamics of clathrin relative to the early endocytic patch protein Sla2p. We find that clathrin is recruited to the cortex prior to Sla2p. In the absence of clathrin, normal numbers of Sla2p patches form, but many do not internalize or are dramatically delayed in completion of endocytosis. Patches that do internalize receive Sla1p late, which is followed by Abp1, which appears near the end of Sla2p lifetime. In addition, clathrin mutants develop actin comet tails, suggesting an important function in actin patch organization/dynamics. Similar to its mammalian counterparts, the light chain (LC) subunit of yeast clathrin interacts directly with the coiled-coil domain of Sla2p. A mutant of Sla2p that no longer interacts with LC (sla2Delta376-573) results in delayed progression of endocytic patches and aberrant actin dynamics. These data demonstrate an important role for clathrin in organization and progression of early endocytic patches to the late stages of endocytosis.

Actins↗
PubMed2006

Novel binding sites on clathrin and adaptors regulate distinct aspects of coat assembly.

Clathrin-coated vesicles (CCVs) sort proteins at the plasma membrane, endosomes and trans Golgi network for multiple membrane traffic pathways. Clathrin recruitment to membranes and its self-assembly into a polyhedral coat depends on adaptor molecules, which interact with membrane-associated vesicle cargo. To determine how adaptors induce clathrin recruitment and assembly, we mapped novel interaction sites between these coat components. A site in the ankle domain of the clathrin triskelion leg was identified that binds a common site on the appendages of tetrameric [AP1 and AP2] and monomeric (GGA1) adaptors. Mutagenesis and modeling studies suggested that the clathrin-GGA1 appendage interface is nonlinear, unlike other peptide-appendage interactions, but overlaps with a sandwich domain binding site for accessory protein peptides, allowing for competitive regulation of coated vesicle formation. A novel clathrin box in the GGA1 hinge region was also identified and shown to mediate membrane recruitment of clathrin, while disruption of the clathrin-GGA1 appendage interaction did not affect recruitment. Thus, the distinct sites for clathrin-adaptor interactions perform distinct functions, revealing new aspects to regulation of CCV formation.

Adaptor Proteins, Vesicular Transport↗
PubMed1991

Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis.

The gene encoding clathrin heavy chain in Saccharomyces cerevisiae (CHC1) is not essential for growth in most laboratory strains tested. However, in certain genetic backgrounds, a deletion of CHC1 (chc1) results in cell death. Lethality in these chc1 strains is determined by a locus designated SCD1 (suppressor of clathrin deficiency) which is unlinked to CHC1 (S. K. Lemmon and E. W. Jones, Science 238:504-509, 1987). The lethal allele of SCD1 has no effect on cell growth when the wild-type version of CHC1 is present. This result led to the proposal that most yeast strains are viable in the absence of clathrin heavy chain because they possess the SCD1 suppressor. Discovery of another yeast strain that cannot grow without clathrin heavy chain has allowed us to perform a genetic test of the suppressor hypothesis. Genetic crosses show that clathrin-deficient lethality in the latter strain is conferred by a single genetic locus (termed CDL1, for clathrin-deficient lethality). By constructing strains in which CHC1 expression is regulated by the GAL10 promoter, we demonstrate that the lethal alleles of SCD1 and CDL1 are recessive. In both cases, very low expression of CHC1 can allow cells to escape from lethality. Genetic complementation and segregation analyses indicate that CDL1 and SCD1 are distinct genes. The lethal CDL1 allele does not cause a defect in the secretory pathway of either wild-type or clathrin heavy-chain-deficient yeast. A systematic screen to identify mutants unable to grow in the absence of clathrin heavy chain uncovered numerous genes similar to SCD1 and CDL1. These findings argue against the idea that viability of chc1 cells is due to genetic suppression, since this hypothesis would require the existence of a large number of unlinked genes, all of which are required for suppression. Instead, lethality appears to be a common, nonspecific occurrence when a second-site mutation arises in a strain whose cell growth is already severely compromised by the lack of clathrin heavy chain.

Chromosome Deletion↗
PubMed1998

The vitronectin receptor associates with clathrin-coated membrane domains via the cytoplasmic domain of its beta5 subunit.

Rat myotubes cultured in fetal calf serum adhere to vitronectin-coated substrates through two distinct structures, focal contacts and clathrin-coated membrane domains. We studied the integrins in myotubes to learn how they associate with these two domains. Double label immunofluorescence studies with antibodies specific for clathrin, vinculin and several forms of integrin showed that focal contacts and clathrin-coated membrane domains contain both vitronectin receptors (VnR, containing beta-3 and beta-5integrins) and fibronectin receptors (FnR, containing beta1-integrin). VnR but not FnR associates tightly with the substrate in both domains, as the VnR alone remains attached to the coverslip when the lipid bilayer and other membrane proteins are removed by detergent. Ultrastructural studies confirmed the localization of the beta5 subunit of the VnR at both domains. We used intracellular injection and affinity chromatography to test the possibility that clathrin at coated membrane domains associates with the cytoplasmic sequence of the beta5 subunit of the VnR. Injection of a synthetic peptide containing the NPXY motif from the cytoplasmic domain of the human beta5 subunit, SRARYEMASNPLYRKPIST, depleted clathrin from coated membrane domains without affecting clathrin in perinuclear structures or vinculin at focal contacts. Injection of the homologous beta1 peptide, MNAKWDTGENPIYKSAVITT, also containing an NPXY motif, had no significant effect on any of these structures. Affinity matrices containing the beta5 but not the beta1 peptide selectively retained clathrin from myotube extract, and bound clathrin could be selectively eluted by soluble forms of the beta5 but not the beta1 peptide. Thus, a sequence including the NPXY motif in the integrin beta5 subunit is involved in the specific anchoring of the VnR, but not the FnR, to clathrin-coated membrane.

Amino Acid Sequence↗
PubMed2004

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.

Adenosine Triphosphatases↗
PubMed2006

Trimerisation is important for the function of clathrin at the mitotic spindle.

Clathrin is a triskelion consisting of three heavy chains each with an associated light chain. During mitosis, clathrin contributes to kinetochore fibre stability. As the N-terminal domain at the foot of each leg can bind to the mitotic spindle, we proposed previously a ;bridge hypothesis' wherein clathrin acts as a brace between two or three microtubules within a kinetochore fibre to increase fibre stability. Here, we have tested this hypothesis by replacing endogenous clathrin heavy chain in human cells with a panel of clathrin constructs. Mutants designed to abolish trimerisation were unable to rescue the mitotic defects caused by depletion of endogenous clathrin. By contrast, stunted triskelia with contracted legs could partially rescue normal mitosis. These results indicate that the key structural features of clathrin that are necessary for its function in mitosis are a trimeric molecule with a spindle interaction domain at each end, supporting the bridge hypothesis for clathrin function in mitosis.

Cells, Cultured↗
PubMed2003

Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells.

To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the alpha-adaptin subunit of AP-2 in HeLa-cells using RNA interference. 48 h after transfection with clathrin heavy chain-specific short interfering RNA both, the heavy and light chains were depleted by more than 80%. Residual clathrin was mainly membrane-associated, and an increase in shallow pits was noted. The membrane-association of adaptors, clathrin assembly lymphoid myeloid leukemia protein (CALM), epsin, dynamin, and Eps15 was only moderately affected by the knockdown and all proteins still displayed a punctate staining distribution. Clathrin depletion inhibited the uptake of transferrin but not that of the epidermal growth factor. However, efficient sorting of the epidermal growth factor into hepatocyte growth factor-regulated tyrosine kinase substrate-positive endosomes was impaired. Depletion of alpha-adaptin abolished almost completely the plasma membrane association of clathrin. Binding of Eps15 to membranes was strongly and that of CALM moderately reduced. Whereas the uptake of transferrin was efficiently blocked in alpha-adaptin knockdown cells, the internalization and sorting of the epidermal growth factor was not significantly impaired. Since neither clathrin nor AP-2 is essential for the internalization of EGF, we conclude that it is taken up by an alternative mechanism.

Adaptor Protein Complex alpha Subunits↗
PubMed2001

Clathrin hub expression affects early endosome distribution with minimal impact on receptor sorting and recycling.

Clathrin-coated vesicles execute receptor-mediated endocytosis at the plasma membrane. However, a role for clathrin in later endocytic trafficking processes, such as receptor sorting and recycling or maintaining the organization of the endocytic pathway, has not been thoroughly characterized. The existence of clathrin-coated buds on endosomes suggests that clathrin might mediate later endocytic trafficking events. To investigate the function of clathrin-coated buds on endosomal membranes, endosome function and distribution were analyzed in a HeLa cell line that expresses the dominant-negative clathrin inhibitor Hub in an inducible manner. As expected, Hub expression reduced receptor-mediated endocytosis at the plasma membrane. Hub expression also induced a perinuclear aggregation of early endosome antigen 1-positive early endosomes, such that sorting and recycling endosomes were found tightly concentrated in the perinuclear region. Despite the dramatic redistribution of endosomes, Hub expression did not affect the overall kinetics of receptor sorting or recycling. These data show that clathrin function is necessary to maintain proper cellular distribution of early endosomes but does not play a prominent role in sorting and recycling events. Thus, clathrin's role on endosomal membranes is to influence organelle localization and is distinct from its role in trafficking pathways at the plasma membrane and trans-Golgi network.

Animals↗