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PubMed1990

Determination of the regions of the clathrin molecule inducing membrane fusion.

Clathrin induces fusion of liposome membranes containing phosphatidylserine at acidic pH [Maezawa, S., Yoshimura, T., Hong, K., Düzgüneş, N., & Papahadjopoulos, D. (1989) Biochemistry 28, 1422-1428]. The regions of the clathrin molecule inducing membrane fusion were determined by examining the fusion abilities of clathrin fragments obtained by limited proteolysis of clathrin cages with thermolysin. Membrane fusion was assessed by resonance energy transfer assay in terms of the dilution of fluorescent phospholipids in liposome membranes. Proteolysis of clathrin decreased the fusion rate and the amount of protein but did not affect the specific fusion rate (i.e., the fusion rate per unit of protein), indicating that clathrin fragments retain the ability to induce fusion. Of the two proteolytic fragments of the clathrin heavy chain, the terminal domain and the residual proximal part, which were separated by ultracentrifugation or gel chromatography, only the proximal part showed fusion activity. Light chains seemed to have no role in membrane fusion, since they are susceptible to proteolytic digestion. The terminal domain induced reversible liposome membrane aggregation, which was also induced by the residual proximal part of the heavy chain and the whole molecule of clathrin. These results suggest that the terminal domain and the proximal portion of clathrin have critical roles in the steps of close apposition and fusion of membranes, respectively.

Animals↗
PubMed2006

Activated Cdc42-associated kinase 1 is a component of EGF receptor signaling complex and regulates EGF receptor degradation.

Cdc42-associated tyrosine kinase 1 (ACK1) is a specific down-stream effector of Cdc42, a Rho family small G-protein. Previous studies have shown that ACK1 interacts with clathrin heavy chain and is involved in clathrin-coated vesicle endocytosis. Here we report that ACK1 interacted with epidermal growth factor receptor (EGFR) upon EGF stimulation via a region at carboxy terminus that is highly homologous to Gene-33/Mig-6/RALT. The interaction of ACK1 with EGFR was dependent on the kinase activity or tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 was colocalized with EGFR on EEA-1 positive vesicles upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNAi inhibited ligand-induced degradation of EGFR upon EGF stimulation, suggesting that ACK1 plays an important role in regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin-association (Uba) domain at the carboxy terminus, ACK1 binds to both poly- and mono-ubiquitin. Overexpression of the Uba domain-deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Taken together, our studies suggest that ACK1 senses signal of EGF and regulates ligand-induced degradation of EGFR.

Amino Acid Sequence↗
PubMed1990

Coated vesicles from thyroid cells carry iodinated thyroglobulin molecules. First indication for an internalization of the thyroid prohormone via a mechanism of receptor-mediated endocytosis.

We have tried to identify iodinated thyroglobulin molecules in purified thyroid-coated vesicles to determined whether the internalization of the thyroid prohormone could proceed via a mechanism of receptor-mediated endocytosis. Coated vesicles isolated from pig thyroids by differential centrifugation and centrifugation on 2H2O-sucrose cushion were characterized by transmission electron microscopy and analyses of the polypeptide composition by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and Western blot using anti-clathrin heavy chain and anti-thyroglobulin antibodies. Clathrin and thyroglobulin (Tg) appeared as the two major components of the purified thyroid coated vesicles (TCV). Purified TCV fraction was homogeneous when analyzed by isopycnic centrifugation on 30% Percoll gradient. TCV had an apparent buoyant density of 1.035 g/ml. The presence of Tg molecules inside TCV was ascertained by (a) immunogold labeling on cryosections of TCV pellet and (b) identification by gel electrophoresis and radio-immunoassay of a definite fraction of Tg (3-5% of total protein) in TCV treated by Triton X-100. The detergent-treated TCV also contained protein-bound iodine: 0.5-0.7 micrograms of iodine/mg protein. Pulse-chase experiments on in vitro reconstituted thyroid follicles have been used to further document the presence of iodinated Tg molecules in coated vesicles. TCV were isolated from reconstituted thyroid follicles previously labeled with [125I]iodide to radioiodinate Tg of the follicular lumen (the pre-endocytotic compartment) and incubated with or without thyrotropin or dibutyryl cyclic AMP to activate intraluminal 125I-Tg endocytosis. Autoradiographic analyses revealed the presence of 125I-Tg in purified TCV and Triton X-100-treated TCV. 125I-Tg present in TCV represented 1-2% of the total intracellular protein-bound radioactivity. Thyrotropin and dibutyryl cyclic AMP increased 2-3-fold the 125I-Tg content of TCV. Our results clearly show that iodinated Tg, the molecular form of the thyroid prohormone known to be internalized, is present into TCV. The data suggest that coated vesicles are involved in the uptake and transport of Tg from the follicular lumen to the lysosomal compartment and therefore, that the internalization of Tg could proceed, at least for a part, via a mechanism of receptor-mediated endocytosis.

Animals↗
PubMed2004

Efficient endosome-to-Golgi transport of Shiga toxin is dependent on dynamin and clathrin.

It has previously been shown that Shiga toxin, despite being bound to a glycolipid receptor, can be efficiently endocytosed from clathrin-coated pits. However, clathrin-independent endocytosis is also responsible for a proportion of the toxin uptake in some cells. After endocytosis the toxin can be transported in retrograde fashion to the Golgi apparatus and the endoplasmic reticulum, and then to the cytosol, where it exerts its toxic effect by inactivating ribosomes. In order to investigate the role of dynamin and clathrin in endosome-to-Golgi transport of Shiga toxin, we have used HeLa dyn(K44A) and BHK antisense clathrin heavy chain (CHC) cells that, in an inducible manner, express mutant dynamin or CHC antisense RNA, respectively. In these cell lines, one can study the role of dynamin and clathrin on endosome-to-Golgi transport because they, as shown here, still internalize Shiga toxin when dynamin- and clathrin-dependent endocytosis is blocked. Butyric acid has been shown to sensitize A431 cells to Shiga toxin by increasing the proportion of cell-associated toxin that is transported to the Golgi apparatus and the endoplasmic reticulum. Here, we find that, in HeLa and BHK cells also, butyric acid also increased toxin transport to the Golgi apparatus and sensitized the cells to Shiga toxin. We have therefore studied the role of dynamin and clathrin in both untreated and butyric-acid-treated cells by measuring the sulfation of a modified Shiga B fragment. Our results indicate that endosome-to-Golgi transport of Shiga toxin is dependent on functional dynamin in both untreated cells and in cells treated with butyric acid. Interestingly, the regulation of Shiga toxin transport in untreated and butyric-acid-treated cells differs when it comes to the role of clathrin, because only cells that are sensitized to Shiga toxin with butyric acid need functional clathrin for endosome-to-Golgi transport.

Amino Acid Substitution↗
PubMed1999

Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function.

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.

ADP-Ribosylation Factor 1↗
PubMed1995

Identification and characterization of membrane-bound calpains in clathrin-coated vesicles from bovine brain.

Calpains are intracellular cysteine proteases activated in a Ca(2+)-dependent manner. Previously, we found that the differentiation of K562 cells induced by 4 beta-phorbol 12-myristate 13-acetate treatment is accompanied by an increase in m-calpain levels and, at the same time, m-calpain becomes localized on the inside of plasma membranes, coated pits, and coated vesicles [Nakamura, M., Mori, M., Morishita, Y., Mori, S. & Kawashima, S. (1992) Exp. Cell Res. 200, 513-522]. We also reported that mu-calpain plays an essential role in morphological changes and membrane fusion of erythrocytes through the degradation of spectrin, a lining protein [Hayashi, M., Saito, Y. & Kawashima, S. (1992) Biochim. Biophys. Res. Commun. 182, 939-946]. Thus, calpains are implicated in endocytosis and/or exocytosis, processes stimulated by Ca2+ and involving intracellular membrane fusion. In this study, we report the biochemical characterization of calpains as components of purified coated vesicles from bovine brain. It was found by Western-blot analysis and chemical cross-linking of proteins that calpains are bound to the membranes of coated vesicles, and not to the coats. The binding of m-calpain to vesicles is Ca(2+)-dependent, while that of mu-calpain is less dependent on the presence of Ca2+. We also identified substrate proteins for calpains in coated vesicles. Upon activation of endogenous calpains, component proteins of coated vesicles such as the clathrin light chain, tubulins, and adaptins, but not the clathrin heavy chain, are highly sensitive to calpain digestion. In the case of exogenously added calpains, low concentrations degraded the same protein components. The degradation pattern differs slightly between added mu-calpain and m-calpain. These results strongly suggest that calpains are involved in the formation of coated vesicles and/or vesicle fusion to endosomes.

Animals↗
PubMed2006

Identification of genes that interact with Drosophila liquid facets.

We have performed mutagenesis screens of the Drosophila X chromosome and the autosomes for dominant enhancers of the rough eye resulting from overexpression of liquid facets. The liquid facets gene encodes the homolog of vertebrate endocytic Epsin, an endocytic adapter protein. In Drosophila, Liquid facets is a core component of the Notch signaling pathway required in the signaling cells for ligand endocytosis and signaling. Why ligand internalization by the signaling cells is essential for signaling is a mystery. The requirement for Liquid facets is a hint at the answer, and the genes identified in this screen provide further clues. Mutant alleles of clathrin heavy chain, Rala, split ends, and auxilin were identified as enhancers. We describe the mutant alleles and mutant phenotypes of Rala and aux. We discuss the relevance of all of these genetic interactions to the function of Liquid facets in Notch signaling.

Animals↗
PubMed2001

Role of clathrin in the regulated secretory pathway of pancreatic beta-cells.

The role of clathrin in the sorting of proinsulin to secretory granules, the formation of immature granules and their subsequent maturation is not known. To this end, primary rat pancreatic beta-cells were infected with a recombinant adenovirus co-expressing the Hub fragment, a dominant-negative peptide of the clathrin heavy chain and enhanced green fluorescent protein (EGFP as a marker of infected cells). A population of cells expressing the highest levels of EGFP (and thus Hub) was obtained using a fluorescence-activated cell sorter (FACS). Control cells were infected with an adenovirus expressing EGFP alone. By immunofluorescence, control cells showed intense staining for both clathrin light chain and proinsulin in a perinuclear region. In cells expressing high levels of Hub, the clathrin light-chain signal was faint and diffuse in keeping with its displacement from membranes. There was, however, no detectable effect of Hub expression on proinsulin staining or disposition within the cell. Proinsulin sorting and conversion, and the fate (release and/or degradation) of insulin and C-peptide, was studied by pulse-chase and quantitative reverse phase HPLC. In both Hub-expressing and control cells, >99% of all newly synthesized proinsulin was sorted to the regulated pathway and there was no effect of Hub on proinsulin conversion to insulin. In presence of Hub there was, however, a significant increase in the percentage of C-peptide truncated to des-(27-31)-C-peptide at early times of chase as well as more extensive degradation of C-peptide thereafter. It is concluded that clathrin is not implicated in the sorting or processing of proinsulin or in regulated exocytosis of secretory granules. These results confirm a role for clathrin in the removal of proteases from maturing granules, thus explaining the increased truncation and degradation of C-peptide in cells expressing Hub.

Adenoviridae↗
PubMed2005

Identification of nitrated proteins in the normal rat brain using a proteomics approach.

BACKGROUND: The nitration of tyrosine has been suggested to play a role in the pathogenesis of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). METHODS: In the present study, we identified four targets of protein nitration, T-complex polypeptide 1 alpha subunit (TCP-1), neurofilament L (NFL), glial fibrillary acidic protein (GFAP) and clathrin heavy chain (CHC), in the normal rat cortex using a proteomics approach. CONCLUSIONS: There have been no reports on these proteins being identified by proteomics as nitrated forms in the brain. For further study, we have to investigate alterations in these nitrated proteins during aging and in neurodegenerative disorders.

Amino Acid Sequence↗
PubMed2003

The synaptojanin-like protein Inp53/Sjl3 functions with clathrin in a yeast TGN-to-endosome pathway distinct from the GGA protein-dependent pathway.

Yeast TGN resident proteins that frequently cycle between the TGN and endosomes are much more slowly transported to the prevacuolar/late endosomal compartment (PVC) than other proteins. However, TGN protein transport to the PVC is accelerated in mutants lacking function of Inp53p. Inp53p contains a SacI polyphosphoinositide phosphatase domain, a 5-phosphatase domain, and a proline-rich domain. Here we show that all three domains are required to mediate "slow delivery" of TGN proteins into the PVC. Although deletion of the proline-rich domain did not affect general membrane association, it caused localization to become less specific. The proline-rich domain was shown to bind to two proteins, including clathrin heavy chain, Chc1p. Unlike chc1 mutants, inp53 mutants do not mislocalize TGN proteins to the cell surface, consistent with the idea that Chc1p and Inp53p act at a common vesicular trafficking step but that Chc1p is used at other steps also. Like mutations in the AP-1 adaptor complex, mutations in INP53 exhibit synthetic growth and transport defects when combined with mutations in the GGA proteins. Taken together with other recent studies, our results suggest that Inp53p and AP-1/clathrin act together in a TGN-to-early endosome pathway distinct from the direct TGN-to-PVC pathway mediated by GGA/clathrin.

ADP-Ribosylation Factors↗
PubMed1992

Possible involvement of clathrin in neuritogenesis induced by a protease inhibitor (benzyloxycarbonyl-Leu-Leu-Leu-aldehyde) in PC12 cells.

Our previous reports showed that benzyloxycarbonyl (Z)-Leu-Leu-Leu-al (ZLLLal) induces neurite outgrowth in PC12 cells, and that 33-, 35-, and 180-kDa proteins from PC12 cells elute specifically from a Leu-Leu-Leu-al (LLLal)-coupled affinity column. Several lines of evidence suggest that the 33-, 35-, and 180-kDa proteins are components of clathrin, well-known for its role in endocytosis. Separation of clathrin into its heavy and light chains showed that the clathrin heavy chains have the ability to bind to a LLLal affinity column directly. Furthermore, ZLLLal enhances the rate of polymerization of clathrin triskelion to the coat structure. ZLLL-COOH does not cause neurite outgrowth in PC12 cells, and has no effect on the rate of clathrin polymerization. On immunocytochemical analysis of PC12 cells with an anti-clathrin heavy chain antibody, enhanced staining of the clathrin heavy chain was observed concomitantly with neurite outgrowth initiated by ZLLLal, but not by NGF. This study provides new insights into both the role of the clathrin molecule and the regulatory mechanism of neurite outgrowth.

Amino Acid Sequence↗
PubMed2004

Clathrin-dependent targeting of receptors to the flagellar pocket of procyclic-form Trypanosoma brucei.

In trypanosomatids, endocytosis and exocytosis occur exclusively at the flagellar pocket, which represents about 0.43% of the pellicle membrane and is a deep invagination of the plasma membrane where the flagellum extends from the cell. Receptor molecules are selectively retained at the flagellar pocket. We studied the function of clathrin heavy chain (TbCLH) in the trafficking of the flagellar pocket receptors in Trypanosoma brucei by using the double-stranded RNA interference approach. It appears that TbCLH is essential for the survival of both the procyclic form and the bloodstream form of T. brucei, even though structures resembling large coated endocytic vesicles are absent in procyclic-form trypanosomes. Down-regulation of TbCLH by RNA interference (RNAi) for 24 h rapidly and drastically reduced the uptake of macromolecules via receptor-mediated endocytosis in procyclic-form trypanosomes. This result suggested the importance of TbCLH in receptor-mediated endocytosis of the procyclic-form trypanosome, in which the formation of large coated endocytic vesicles may not be required. Surprisingly, induction of TbCLH RNAi in the procyclic T. brucei for a period of 48 h prohibited the export of the flagellar pocket-associated transmembrane receptor CRAM from the endoplasmic reticulum to the flagellar pocket, while trafficking of the glycosylphosphatidylinositol-anchored procyclin coat was not significantly affected. After 72 h of induction of TbCLH RNAi, procyclics exhibited morphological changes to an apolar round shape without a distinct structure of the flagellar pocket and flagellum. Although trypanosomes, like other eukaryotes, use similar organelles and machinery for protein sorting and transport, our studies reveal a novel role for clathrin in the secretory pathway of trypanosomes. We speculate that the clathrin-dependent trafficking of proteins to the flagellar pocket may be essential for the biogenesis and maintenance of the flagellar pocket in trypanosomes.

Animals↗
PubMed1992

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.

Clathrin↗
PubMed1998

Genetic dissection of sperm individualization in Drosophila melanogaster.

The morphogenesis of spermatids generally takes place within a syncytium, in which all spermatid nuclei descended from a primary spermatocyte remain connected via an extensive network of cytoplasmic bridges. A late step in sperm maturation therefore requires the physical resolution of the syncytium, or cyst, into individual cells, a process sometimes referred to as sperm individualization. Despite the identification of specialized machinery involved in the individualization of Drosophila spermatids (Tokuyasu, K. T., Peacock, W. J. and Hardy, R. W. (1972) Z. Zellforsch 124, 479-506), and of many Drosophila genes mutable to male-sterile phenotypes, little is known of the mechanisms by which this extensive remodeling of the cyst is accomplished. Here, the identification of a major cytoskeletal component of the individualization complex as actin is confirmed with a simple fluorescence assay. Using rhodamine-phalloidin as a probe, the individualization complex is readily visualized forming around bundles of spermatid nuclei at one end of highly elongated cysts, then translocating along the length of the cysts. The structure of the individualization complex in a male-sterile clathrin heavy chain (Chc) mutant is observed to be reduced or disrupted relative to wild-type, consistent with the individualization-deficient phenotype of this mutant. Using the fluorescence assay, a sampling of male-sterile mutant phenotypes in which spermatogenesis proceeds to the assembly of highly elongated cysts distinguishes at least four different phenotypic classes: (1) mutations (nanking class) that block or significantly retard the assembly of the actin-based individualization complex around the nuclear bundle, (2) mutations (dud class) in which the individualization complex assembles in/around the nuclear bundle, but fails to translocate down the cyst, (3) mutations (mulet class) that allow the assembly of a morphologically normal individualization complex around the nuclear bundle, but result in a breakdown in the complex after it begins to translocate down the cyst, and (4) mutations (purity of essence class) that allow the assembly of a motile but morphologically altered or reduced individualization complex. Individualization also fails in a number of mutants with altered nuclear shape, consistent with the hypothesis that spermatid nuclei provide a physical scaffolding for the assembly of the individualization complex. Genetic analysis suggests that a substantial number of additional loci with phenotypes distinguishable with this assay remain to be identified. The large proportion of male-sterile mutations resulting in a late block to spermatogenesis, in which highly elongated cysts fail to be individualized, suggest a substantial susceptibility of this process to a broad range of cellular perturbations. The massive reorganization of cyst cytoplasm required at individualization is expected to be a correspondingly complex function requiring exquisite coordination of multiple cytoplasmic functions, and may account for the previously noted high frequency with which Drosophila genes are mutable to male-sterile phenotypes.

Actins↗
PubMed1995

Regulation of clathrin assembly and trimerization defined using recombinant triskelion hubs.

Clathrin polymerization into a polyhedral vesicle coat drives receptor sorting at cellular membranes during endocytosis and organelle biogenesis. To study clathrin self-assembly, we expressed the C-terminal third of the clathrin heavy chain in bacteria. The recombinant fragment trimerized, bound clathrin light chains, and morphologically resembled the hub domain of the triskelion-shaped clathrin molecule. Self-assembly of recombinant hubs demonstrated a regulatory role for clathrin light chains and for the distal portions of triskelion legs in clathrin coat formation. Deletion mutagenesis of the hub localized a domain mediating light chain binding and clathrin self-assembly and mapped a transferable trimerization domain. These studies define molecular interactions controlling clathrin self-assembly and establish a recombinant system for future analysis.

Animals↗
PubMed2005

Regulation of the clathrin-coated vesicle cycle by reversible phosphorylation.

Reversible phosphorylation has long been an attractive mechanism to control cycles of coat assembly and disassembly during clathrin-mediated endocytosis. Many of the coat proteins are phosphorylated in vivo and in vitro. Our work has focused on the role of phosphorylation of the mu2 subunit of AP-2 (adaptor protein 2), which appears to be necessary for efficient cargo recruitment. Studies to probe the regulation of mu2 phosphorylation demonstrated that clathrin is a specific activator of the mu2 kinase, and, in permeabilized cells, cargo sequestration, driven by exogenously added clathrin, results in elevated levels of m2 phosphorylation. Furthermore, phosphorylated mu2 is mainly associated with assembled clathrin in vivo and its steady-state level is strongly reduced in cells depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via modulation of phospho-mu2 levels. This is therefore a novel regulatory role for clathrin that is independent of its structural role and that provides elegant spatial control of AP-2 and cargo interactions, ensuring that AP-2 is only activated at the correct cellular location and in the correct functional context. Ongoing studies are exploring further the roles of reversible phosphorylation in the coated vesicle cycle.

Adaptor Protein Complex 2↗
PubMed2003

Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor micro2 kinase.

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo-AP2 interactions occur via the mu2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for mu2 phosphorylation is in cargo recruitment because mu2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of mu2 phosphorylation. We identify clathrin as a specific activator of the mu2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of mu2 phosphorylation. Furthermore, we show that AP2 containing phospho-mu2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-mu2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-mu2 levels.

Adaptor Protein Complex 2↗
PubMed2003

Clathrin-mediated endocytosis in AP-2-depleted cells.

We have used RNA interference to knock down the AP-2 mu2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2-depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2-depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

Adaptor Protein Complex 2↗