Functional organization of clathrin in coats: combining electron cryomicroscopy and X-ray crystallography. (1/110)

The sorting of specific proteins into clathrin-coated pits and the mechanics of membrane invagination are determined by assembly of the clathrin lattice. Recent structures of a six-fold barrel clathrin coat at 21 A resolution by electron cryomicroscopy and of the clathrin terminal domain and linker at 2.6 A by X-ray crystallography together show how domains of clathrin interact and orient within the coat and reveal the strongly puckered shape and conformational variability of individual triskelions. The beta propeller of the terminal domain faces the membrane so that recognition segments from adaptor proteins can extend along its lateral grooves. Clathrin legs adapt to different coat environments in the barrel by flexing along a segment at the knee that is free of contacts with other molecules.  (+info)

ADP-ribosylation factor 6 and endocytosis at the apical surface of Madin-Darby canine kidney cells. (2/110)

We report that the small GTPase, ADP-ribosylation factor 6 (ARF6), is present only on the apical surface of polarized MDCK epithelial cells. Overexpression of a mutant of ARF6, ARF6-Q67L, which is predicted to be in the GTP-bound form, stimulates endocytosis exclusively at this surface. Surprisingly, overexpression of the mutant ARF6-T27N, which is predicted to be in the GDP-bound form, also stimulated apical endocytosis, though to a lesser extent. ARF6-stimulated endocytosis is inhibited by a dominant-negative form of dynamin, or a dominant-negative hub fragment of clathrin heavy chain, indicating that it is mediated by clathrin. Correspondingly, overexpression of either mutant of ARF6 leads to an increase in the number of clathrin-coated pits at the apical plasma membrane. When ARF6-Q67L is overexpressed in the presence of the dominant-negative dynamin, the ARF6-Q67L colocalizes with clathrin and with IgA bound to its receptor. We conclude that ARF6 is an important modulator of clathrin-mediated endocytosis at the apical surface of epithelial cells.  (+info)

Human monocyte activation by cleaved form of alpha-1-antitrypsin involvement of the phagocytic pathway. (3/110)

Production of alpha-1-antitrypsin (AAT) by human monocytes is an important factor in controlling tissue damage by proteases in the microenvironment of inflammation. Increases, of four- to eightfold, in numbers of macrophages and levels of AAT and its cleavage fragments have been found in various inflammatory loci. We have found that the C-terminal peptide (C-36) of AAT, produced by specific proteinase cleavage when added in its fibrillar form at concentrations >/=5 microM to monocytes in culture for 24 h, significantly increases low density lipoprotein (LDL) binding and uptake, up-regulates levels of LDL receptors and also induces proinflammatory cytokine (interleukin-1, interleukin-6 and tumour necrosis factor alpha) production and glutathione reductase activity. Because it is known that various cells selectively internalize surface receptors and their ligands through receptor-mediated endocytosis via clathrin-coated pits, we tested whether antibodies raised against the clathrin heavy chain would block the effects of the fibrillar form of C-36 on human monocytes in culture. Addition of excess anti-(clathrin HC) with 10 microM fibrillar C-36 diminished the stimulatory effects of the latter on LDL binding, uptake and LDL receptor levels. In contrast, however, in the presence of anti-(clathrin HC), the potentially cytotoxic effects of fibrils, such as induction of cytokines, free radicals and cytosolic activity of cathepsin D, were much greater than those observed when cells were treated with fibrils alone. These results suggest that endocytosis is the pathway by which C-36 fibrils upregulate LDL receptors, and may be the natural mechanism for fibril clearance. We infer that human monocytes clear C-36 fibrils by a clathrin-dependent pathway, presumably endocytotic, and that loss of this pathway amplifies the cytotoxic effects of the fibrils by increasing their availability to other specific or nonspecific sites through which they exert their cytotoxic effects.  (+info)

Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function. (4/110)

ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1Delta synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 null allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro-alpha-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2Delta cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.  (+info)

Spatially regulated recruitment of clathrin to the plasma membrane during capping and cell translocation. (5/110)

Clathrin-coated vesicles bud from selected cellular membranes to traffic-specific intracellular proteins. To study the dynamic properties of clathrin-coated membranes, we expressed clathrin heavy chain tagged with green fluorescent protein (GFP) in Dictyostelium cells. GFP-clathrin was functional and retained the native properties of clathrin: the chimeric protein formed classic clathrin lattices on cellular membranes and also rescued phenotypic defects of clathrin null cells. GFP-clathrin distributed into punctate loci found throughout the cytoplasm, on the plasma membrane, and concentrated to a perinuclear location. These clathrin-coated structures were remarkably motile and capable of rapid and bidirectional transport across the cell. We identified two local domains of the plasma membrane as sites for clathrin recruitment in motile cells. First, as cells translocated or changed shape and retracted their tails, clathrin was transiently concentrated on the membrane at the back of the cell tail. Second, as cells capped their cell surface receptors, clathrin was recruited locally to the membrane under the tight cap of cross-linked receptors. This suggests that local sites for clathrin polymerization on specific domains of the plasma membrane undergo rapid and dynamic regulation in motile cells.  (+info)

Clathrin heavy chain, light chain interactions. (6/110)

Purified pig brain clathrin can be reversibly dissociated and separated into heavy chain trimers and light chains in the presence of non-denaturing concentrations of the chaotrope thiocyanate. The isolated heavy chain trimers reassemble into regular polygonal cage structures in the absence of light chains. The light chain fraction can be further resolved into its two components L alpha and L beta which give different one-dimensional peptide maps. Radiolabelled light chains bind with high affinity (KD < 10(-10) M) to heavy chain trimers, to heavy chain cages and to a 110,000 mol. wt. tryptic fragment of the heavy chain. Both light chains compete with each other and with light chains from other sources for the same binding sites on heavy chains and c.d. spectroscopy shows that the two pig brain light chains possess very similar structures. We conclude that light chains from different sources, despite some heterogeneity, have a highly conserved, high affinity binding site on the heavy chain but are not essential for the formation of regular cage structures.  (+info)

Biochemical and immunological studies on clathrin light chains and their binding sites on clathrin triskelions. (7/110)

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.  (+info)

NGF signals through TrkA to increase clathrin at the plasma membrane and enhance clathrin-mediated membrane trafficking. (8/110)

Neurotrophin (NT) signals may be moved from axon terminals to neuron cell bodies via signaling endosomes-organelles in which NTs continue to be bound to their activated receptors. Suggesting that clathrin-coated membranes serve as one source of signaling endosomes, in earlier studies we showed that nerve growth factor (NGF) treatment increased clathrin at the plasma membrane and resulted in colocalization of clathrin with TrkA, the receptor tyrosine kinase for NGF. Strikingly, however, we also noted that most clathrin puncta at the surface of NGF-treated cells did not colocalize with TrkA, raising the possibility that NGF induces a general increase in clathrin-coated membrane formation. To explore this possibility further, we examined the distribution of clathrin in NGF- and BDNF-treated cells. NGF signaling in PC12 cells robustly redistributed the adaptor protein AP2 and the clathrin heavy chain (CHC) to surface membranes. Using confocal and epifluorescence microscopy, as well as biochemical assays, we showed the redistribution of clathrin to be attributable to the activation of TrkA. Significantly, NGF signaled through TrkA to induce an increase in clathrin-mediated membrane trafficking, as revealed in the increased endocytosis of transferrin. In that BDNF treatment increased AP2 and clathrin at the surface membranes of hippocampal neurons, these findings may represent a physiologically significant response to NTs. We conclude that NT signaling increases clathrin-coated membrane formation and clathrin-mediated membrane trafficking and speculate that this effect contributes to their trophic actions via the increased internalization of receptors and other proteins that are present in clathrin-coated membranes.  (+info)

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