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Poliovirus receptor (hPVR/CD155) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily but its natural function remains unknown. Two membrane-bound isoforms, hPVRalpha and hPVRdelta, are known to date, and they differ only in the amino acid sequence of their cytoplasmic domains. To gain an insight into the possible function of the cytoplasmic domains, we examined the localization of introduced hPVRalpha and hPVRdelta in polarized epithelial cells deficient of native hPVRs. Basolateral sorting of hPVRalpha was observed in Madine-Darby canine kidney cells expressing mu1B, but not in LLC-PK1 porcine kidney cells deficient in mu1B. Distribution of hPVRdelta, however, occurred both on the apical and basolateral plasma membranes of these two cell lines. Basolateral sorting of hPVRalpha was also seen in LLC-PK1 cells that expressed an intact exogenous mu1B, but not in the cells that expressed a mutant mu1B lacking binding ability to tyrosine-containing signals. These results indicate that mu1B is involved in the distribution of hPVRalpha to the basolateral membrane. Comparative distribution analysis of hPVRalpha using a series of mutants with truncations and substitutions in the cytoplasmic tail demonstrated that determinant for the basolateral sorting resided in the tyrosine-containing motif of the cytoplasmic tail. Furthermore, yeast two hybrid analysis strongly suggested that the tyrosine motif directly interacted with mu1B protein. Thus, basolateral sorting of hPVRalpha appears to involve the interaction with mu1B through a tyrosine motif existing in the cytoplasmic domain., (Copyright 2001 Academic Press.)

Doublecortin is a microtubule-associated protein required for normal corticogenesis in the developing brain. We carried out a yeast two-hybrid screen to identify interacting proteins. One of the isolated clones encodes the mu1 subunit of the adaptor complex AP-1 involved in clathrin-dependent protein sorting. We found that Doublecortin also interacts in yeast with mu2 from the AP-2 complex. Mutagenesis and pull-down experiments showed that these interactions were mediated through a tyrosine-based sorting signal (YLPL) in the C-terminal part of Doublecortin. The functional relevance of these interactions was suggested by the coimmunoprecipitation of Doublecortin with AP-1 and AP-2 from mouse brain extracts. This interaction was further supported by RNA in situ hybridization and immunofluorescence studies. Taken together these data indicate that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in vivo and are consistent with a potential involvement of Doublecortin in protein sorting or vesicular trafficking., (Copyright 2001 Academic Press.)

The clathrin-coated pit is the major port of entry for many receptors and pathogens and is the paradigm for membrane-based sorting events in higher cells [1]. Recently, it has been possible to reconstitute in vitro the events leading to assembly, invagination, and budding off of clathrin-coated vesicles, allowing dissection of the machinery required for sequestration of receptors into these structures [2-6]. The AP2 adaptor complex is a key element of this machinery linking receptors to the coat lattice, and it has previously been reported that AP2 can be phosphorylated both in vitro and in vivo [7-10]. However, the physiological significance of this has never been established. Here, we show that phosphorylation of a single threonine residue (Thr156) of the mu2 subunit of the AP2 complex is essential for efficient endocytosis of transferrin both in an in vitro coated-pit budding assay and in living cells.

Tyrosine phosphorylation can upregulate NMDA receptor activity during pathological and physiological alterations of synaptic strength. Here we describe downregulation of recombinant NR1/2A receptors by tyrosine dephosphorylation that requires agonist binding, but is independent of ion flux. The tyrosine residues involved in this new form of NMDA receptor modulation likely form a 'ring' adjacent to the last transmembrane domain. The downregulation was due to a reduction in the number of functional channels, and was blocked by co-expressing a dominant-negative mu2-subunit of the clathrin-adaptor protein AP-2. Our results provide a mechanism by which synaptic NMDA receptors can be modulated in a use-dependent manner even when the postsynaptic membrane is not sufficiently depolarized to relieve channel block by magnesium ions.

The Drosophila dicistronic stoned locus encodes two distinctive presynaptic proteins, Stoned A (STNA) and Stoned B (STNB); STNA is a novel protein without homology to known synaptic proteins, and STNB contains a domain with homology to the endocytotic protein AP50. Both Stoned proteins colocalize precisely with endocytotic proteins including the AP2 complex and Dynamin in the "lattice network" characteristic of endocytotic domains in Drosophila presynaptic terminals. FM1-43 dye uptake studies in stoned mutants demonstrate a striking decrease in the size of the endo-exo-cycling synaptic vesicle pool and loss of spatial regulation of the vesicular recycling intermediates. Mutant synapses display a significant delay in vesicular membrane retrieval after depolarization and neurotransmitter release. These studies suggest that the Stoned proteins play a role in mediating synaptic vesicle endocytosis. We have documented previously a highly specific synaptic mislocalization and degradation of Synaptotagmin I in stoned mutants. Here we show that transgenic overexpression of Synaptotagmin I rescues stoned embryonic lethality and restores endocytotic recycling to normal levels. Furthermore, overexpression of Synaptotagmin I in otherwise wild-type animals results in increased synaptic dye uptake, indicating that Synaptotagmin I directly regulates the endo-exo-cycling synaptic vesicle pool size. In parallel with recent biochemical studies, this genetic analysis strongly suggests that Stoned proteins regulate the AP2-Synaptotagmin I interaction during synaptic vesicle endocytosis. We conclude that Stoned proteins control synaptic transmission strength by mediating the retrieval of Synaptotagmin I from the plasma membrane.

The interaction of adaptor protein (AP) complexes with signal structures in the cytoplasmic domains of membrane proteins is required for intracellular sorting. Tyrosine- or dileucine-based motifs have been reported to bind to medium chain subunits (mu) of AP-1, AP-2, or AP-3. In the present study, we have examined the interaction of the entire 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR46-CT) containing tyrosine- as well as dileucine-based motifs with mu2 and mu3A chains using the yeast two-hybrid system. Both mu2 and mu3A bind specifically to the MPR46-CT. In contrast, mu3A fails to bind to the cytoplasmic domain of the 300-kDa mannose 6-phosphate receptor. Mutational analysis of the MPR46-CT revealed that the tyrosine-based motif and distal sequences rich in acidic amino acid residues are sufficient for effective binding to mu2. However, the dileucine motif was found to be one part of a consecutive complex C-terminal structure comprising tyrosine and dileucine motifs as well as clusters of acidic residues necessary for efficient binding of mu3A. Alanine substitution of 2 or 4 acidic amino acid residues of this cluster reduces the binding to mu3A much more than to mu2. The data suggest that the MPR46 is capable of interacting with different AP complexes using multiple partially overlapping sorting signals, which might depend on posttranslational modifications or subcellular localization of the receptor.

Expression of the epithelial cell-specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD mu subunit, mu1A, being replaced by the closely related mu1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged mu1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.

Clathrin-adaptor complexes (APs) are vesicle coat components that participate in cargo selectivity and transport vesicle formation. Here we cloned and characterized apm1, apm3 and apm4 cDNAs encoding AP medium chains (mu) in D. discoideum. Amino acid comparison suggested that predicted proteins were homologous to known mu1, mu3 and mu4 subunits of mammalian APs as they shared 69, 51, and 26% identity with mouse mu1A, human mu3A and human mu4, respectively. In all chains, amino acid residues predicted to interact with tyrosine based sorting signals were conserved. Southern blot analysis indicated only one copy of each gene in D. discoideum genome. Expression of apm1 and apm3 mRNAs stayed relatively constant during vegetative growth and throughout development. In contrast, apm4 was poorly expressed in amoebae but became well detectable by RT-PCR upon cell differentiation. This regulated expression of coat proteins enlightens the importance of intracellular membrane transport vesicles during development in D. discoideum and strengthens this attractive model organism for studying the function of coat complexes in vivo.

Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, mu1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.

The synaptic vesicle protein synaptotagmin was proposed to act as a major docking site for the recruitment of clathrin coats implicated in endocytosis, including the recycling of synaptic vesicles. We show here that the C2B domain of synaptotagmin binds mu2- and alpha-adaptin, two of the four subunits of the endocytic adaptor complex AP-2. mu2 represents the major interacting subunit of AP-2 within this complex. Its binding to synaptotagmin is mediated by a site in subdomain B that is distinct from the binding site for tyrosine-based sorting motifs located in subdomain A. The presence of the C2B domain of synaptotagmin at the surface of liposomes enhances the recruitment of AP-2 and clathrin. Conversely, perturbation of the interaction between synaptotagmin and AP-2 by synprint, the cytoplasmic synaptotagmin-binding domain of N-type calcium channels, inhibits transferrin internalization in living cells. We conclude that a dual interaction of synaptotagmin with the clathrin adaptor AP-2 plays a key physiological role in the nucleation of endocytic clathrin-coated pits.

RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral-RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. Mu2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates mu2 from mu1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.

In the nematode Caenorhabditis elegans, there exist two micro1 medium chains of the AP-1 clathrin-associated protein complex. Mutations of unc-101, the gene that encodes one of the micro1 chains, cause pleiotropic effects (). In this report, we identified and analyzed the second mu1 chain gene, apm-1. Unlike the mammalian homologs, the two medium chains are expressed ubiquitously throughout development. RNA interference (RNAi) experiments with apm-1 showed that apm-1 and unc-101 were redundant in embryogenesis and in vulval development. Consistent with this, a hybrid protein containing APM-1, when overexpressed, rescued the phenotype of an unc-101 mutant. However, single disruptions of apm-1 or unc-101 have distinct phenotypes, indicating that the two medium chains may have distinct functions. RNAi of any one of the small or large chains of AP-1 complex (sigma1, beta1, or gamma) showed a phenotype identical to that caused by the simultaneous disruption of unc-101 and apm-1, but not that by single disruption of either gene. This suggests that the two medium chains may share large and small chains in the AP-1 complexes. Thus, apm-1 and unc-101 encode two highly related micro1 chains that share redundant and distinct functions within AP-1 clathrin-associated protein complexes of the same tissue.

HIV-1 Nef interacts with cellular adaptor protein (AP) complexes and their medium (mu) subunits. However, the role of the dileucine-based sorting motif within Nef in these interactions has been incompletely characterized. Here, yeast two-hybrid assays indicated that HIV-1 Nef interacted not only with the mu subunits of AP-1 and AP-2, but also with that of AP-3. The interactions with mu1 and mu3 were markedly stronger than the interaction with mu2. Leucine residues of the sorting motif were required for the interactions with mu3 and mu2 and contributed to the interaction with mu1. Confocal immunofluorescence microscopy indicated that Nef, AP-1, and AP-3 (but not AP-2) were concentrated in a juxtanuclear region near the cell center, potentially facilitating interaction between Nef and the mu1 and mu3 subunits. However, leucine residues of the sorting motif were not required for this subcellular localization of Nef. These data suggest that the dileucine motif, required for optimal viral replication, functions through interactions with a variety of AP complexes, including AP-3, potentially by recruiting adaptor complexes to subcellular locations specified by additional determinants in the Nef protein., (Copyright 2000 Academic Press.)

The heterotetrameric AP-1 complex is involved in the formation of clathrin-coated vesicles at the trans-Golgi network (TGN) and interacts with sorting signals in the cytoplasmic tails of cargo molecules. Targeted disruption of the mouse mu1A-adaptin gene causes embryonic lethality at day 13.5. In cells deficient in micro1A-adaptin the remaining AP-1 adaptins do not bind to the TGN. Polarized epithelial cells are the only cells of micro1A-adaptin-deficient embryos that show gamma-adaptin binding to membranes, indicating the formation of an epithelial specific AP-1B complex and demonstrating the absence of additional mu1A homologs. Mannose 6-phosphate receptors are cargo molecules that exit the TGN via AP-1-clathrin-coated vesicles. The steady-state distribution of the mannose 6-phosphate receptors MPR46 and MPR300 in mu1A-deficient cells is shifted to endosomes at the expense of the TGN. MPR46 fails to recycle back from the endosome to the TGN, indicating that AP-1 is required for retrograde endosome to TGN transport of the receptor.

Electron cryomicroscopy of the clathrin coat and X-ray crystallography of parts of the clathrin heavy chain combine to give a detailed picture of the clathrin molecule, assembled as a cage. Recently determined domain structures of other components of the endocytic machinery, particularly the mu2 subunit and the alpha-appendage domain of the AP2 adaptor complex, provide important information on the sequence of recognition events involved in the dynamic process of clathrin coat assembly.

Although polarized epithelial cells are well known to maintain distinct apical and basolateral plasma membrane domains, the mechanisms responsible for targeting membrane proteins to the apical or basolateral surfaces have remained elusive. We have identified a novel form of the AP-1 clathrin adaptor complex that contains as one of its subunits mu1B, an epithelial cell-specific homolog of the ubiquitously expressed mu1A. LLC-PK1 kidney epithelial cells do not express mu1B and missort many basolateral proteins to the apical surface. Stable expression of mu1B selectively restored basolateral targeting, improved the overall organization of LLC-PK1 monolayers, and had no effect on apical targeting. We conclude that basolateral sorting is mediated by an epithelial cell-specific version of the AP-1 complex containing mu1B.

The adaptor protein (AP) complexes AP-1, AP-2, and AP-3 mediate coated vesicle formation and sorting of integral membrane proteins in the endocytic and late exocytic pathways in mammalian cells. A search of the Drosophila melanogaster expressed sequence tag (EST) database identified orthologs of family members mammalian medium (mu) chain families mu1, mu2, and mu3, of the corresponding AP complexes, and delta-COP, the analogous component of the coatomer (COPI) complex. The Drosophila orthologs exhibit a high degree of sequence identity to mammalian medium chain and delta-COP proteins. Northern analysis demonstrated that medium chain and delta-COP mRNAs are expressed uniformly throughout fly development. Medium chain and delta-COP genes were cytologically mapped and the mu3 gene was found to localize to a region containing the pigmentation locus carmine (cm). Analysis of genomic DNA of the cm1 mutant allele indicated the presence of a large insertion in the coding region of the mu3 gene and Northern analysis revealed no detectable mu3 mRNA. Light microscopy of the cm1 mutant showed a reduction in primary, secondary, and tertiary pigment granules in the adult eye. These findings provide evidence of a role for mu3 in the sorting processes required for pigment granule biogenesis in Drosophila.

The Drosophila homologue of AP50, the medium chain of clathrin-associated protein complex AP-2, was identified and characterized from the Drosophila Expressed Sequence Tag database. The Drosophila AP50 is 86% identical to that of mouse and human, and 80% identical to the Caenorhabditis elegans homologue. It is a single-copy gene with two mini-introns in the coding region and it maps to position 94B1-B2 on polytene chromosomes. Two P1 clones, DS01102 and DS0104, were identified that contain the AP50 gene. Alternative 5' UTR splicing is involved in the regulation of AP50 expression. AP50 expression is highly enriched in the central nervous system and midgut caecum during embryo development, and its function is discussed. The two other Drosophila members of the medium-chain family of clathrin-associated protein complexes, AP47 and mu3, have also been identified and mapped to 85D20-D27 and 6E1-E4, respectively.

Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner.

The apical and basolateral plasma membrane domains of polarized epithelial cells contain distinct sets of integral membrane proteins. Biosynthetic targeting of proteins to the basolateral plasma membrane is mediated by cytosolic tail determinants, many of which resemble signals involved in the rapid endocytosis or lysosomal targeting. Since these signals are recognized by adaptor proteins, we hypothesized that there could be epithelial-specific adaptors involved in polarized sorting. Here, we report the identification of a novel member of the adaptor medium chain family, named mu1B, which is closely related to the previously described mu1A (79% amino acid sequence identity). Northern blotting and in situ hybridization analyses reveal the specific expression of mu1B mRNA in a subset of polarized epithelial and exocrine cells. Yeast two-hybrid analyses show that mu1B is capable of interacting with generic tyrosine-based sorting signals. These observations suggest that mu1B may be involved in protein sorting events specific to polarized cells.

Tyrosine based motifs conforming to the consensus YXXphi (where phi represents a bulky hydrophobic residue) have been shown to interact with the medium chain subunit of clathrin adaptor complexes. These medium chains are targets for phosphorylation by a kinase activity associated with clathrin coated vesicles. We have used the clathrin coated vesicle associated kinase activity to specifically phosphorylate a soluble recombinant fusion protein of mu2, the medium chain subunit of the plasma membrane associated adaptor protein complex AP-2. We have tested whether this phosphorylation has any effect on the interaction of mu2 with the tyrosine based motif containing protein, TGN38, that has previously been shown to interact with mu2. Phosphorylation of mu2 was shown to have no significant effect on the in vitro interaction of mu2 with the cytosolic domain of TGN38, indicating that reversible phosphorylation of mu2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs. In addition, although a casein kinase II-like activity has been shown to be associated with clathrin coated vesicles, we show that mu2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coated vesicles. Furthermore the kinase activity associated with clathrin coated vesicles was shown to be capable of phosphorylating dynamin 1. Phosphorylation of dynamin 1 has previously been shown to regulate its interaction with other proteins involved in clathrin mediated endocytosis.

The protein mu1B is a member of the medium chain family of the clathrin-associated adaptor complex and is expressed exclusively in epithelial cells. We determined the genomic structure of previously cloned murine genes for mu1B (Ap1m2) and its closely related homolog, mu1A (Ap1m1). Comparison of their genomic structures revealed that the positions of introns are identical between these two genes, except for the insertion of an additional intron in Ap1m1 (intron 4). By contrast, these structures are different from that of the more distantly related Ap2m1 gene encoding mu2. Taken together with the similarity of amino acid sequences among these genes, the data presented in this study suggest that Ap1m1/2 and Ap2m1 diverged long before the separation of Ap1m1 and Ap1m2, which most likely resulted from a relatively recent gene duplication. We also mapped AP1M2 to human chromosome 19p13.2 and Ap1m2 to the proximal region of mouse chromosome 9. The results are consistent with the fact that these regions are syntenic., (Copyright 1999 S. Karger AG, Basel)

Many cell surface proteins are marked for endocytosis by a cytoplasmic sequence motif, tyrosine-X-X-(hydrophobic residue), that is recognized by the mu2 subunit of AP2 adaptors. Crystal structures of the internalization signal binding domain of mu2 complexed with the internalization signal peptides of epidermal growth factor receptor and the trans-Golgi network protein TGN38 have been determined at 2.7 angstrom resolution. The signal peptides adopted an extended conformation rather than the expected tight turn. Specificity was conferred by hydrophobic pockets that bind the tyrosine and leucine in the peptide. In the crystal, the protein forms dimers that could increase the strength and specificity of binding to dimeric receptors.

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs found within the cytosolic domains of certain integral membrane proteins. Many of these tyrosine motifs conform to the consensus YXXPhi (where Phi represents a bulky hydrophobic residue). This YXXPhi motif has been shown to interact with the medium chain subunits of adaptor complexes that generally link relevant integral membrane protein cytosolic domains to the clathrin coat involved in vesicle formation. The motif YXXPhi is also very similar to motifs that are targets for phosphorylation by tyrosine kinases. Tyrosine kinase inhibitors known as tyrphostins are structural analogues of tyrosine, and so it is possible that tyrphostins could also inhibit interactions between medium chains and YXXPhi motifs. TGN38 is a type I integral membrane protein containing a tyrosine motif, YQRL, within the cytosolic domain. We have previously shown that this motif interacts directly with the medium chain subunit of the plasma membrane localized AP-2 adaptor complex (mu2). We have investigated a range of tyrphostins and demonstrated a specific inhibition of the interaction between mu2 and the TGN38 cytosolic domain by tyrphostin A23 through in vitro analysis and the yeast two-hybrid system. These data raise the exciting possibility that different membrane traffic events could be inhibited by specific tyrphostins.

Tyrosine-based sorting signals conforming to the motif YXXO (Y is tyrosine, X is any amino acid, and O is an amino acid with a bulky hydrophobic side chain (leucine, isoleucine, phenylalanine, methionine, valine)) interact with the medium (mu) subunits of clathrin adaptor (AP) complexes. We have analyzed the selectivity of interaction between YXXO signals and the mu1, mu2, and mu3 (A or B) subunits of the AP-1, AP-2, and AP-3 complexes, respectively, by screening a combinatorial XXXYXXO library using the yeast two-hybrid system. All the medium subunits were found to prefer proline at position Y+2, suggesting that YXXO signals are stabilized by a bend in the polypeptide backbone. Other than for this common preference, each medium subunit favored specific sets of residues at the X and O positions; these preferences were consistent with the proposed roles of the different adaptor complexes in rapid endocytosis and lysosomal targeting. A considerable specificity overlap was also revealed by these analyses, suggesting that additional factors, such as the context of the signals, must be important determinants of recognition.

The vacuolar type proton-translocating ATPase of clathrin-coated vesicles is composed of two large domains: an extramembranous catalytic sector and a transmembranous proton channel. In addition, two polypeptides of 50 and 57 kDa have been found to co-purify with the pump. These proteins, termed SFD (sub-fifty-eight-kDa dimer) activate ATPase activity of the enzyme and couple ATPase activity to proton flow (Xie, X.-S., Crider, B.P., Ma, Y.-M., and Stone, D. K. (1994) J. Biol. Chem. 269, 28509-25815). It has also been reported that the clathrin-coated vesicle proton pump contains AP50, a 50-kDa component of the AP-2 complex responsible for the assembly of clathrin-coated pits, and that AP50 is essential for function of the proton pump (Liu, Q., Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 31592-31597). We demonstrate through the use of anti-AP50 antibody, identical to that of the latter study, that hydroxylapatite chromatography removes AP50 from impure proton pump preparations and that purified proton pump, devoid of AP50, is fully functional. To determine the true molecular identity of SFD, both the 50- and 57-kDa polypeptides were directly sequenced. A polymerase chain reaction-based strategy was used to screen a bovine brain cDNA library, yielding independent full-length clones (SFD-4A and SFD-21); these were identical in their open reading frames and encoded a protein with a predicted mass of 54,187 Da. The SFD-21 clone was then used in a reverse transcription-polymerase chain reaction-based strategy to isolate a related, but distinct, transcript present in bovine brain mRNA. The nucleotide and predicted amino acid sequences of this isolate are identical to SFD-21 except that the isolate contains a 54-base pair insert in the open reading frame, resulting in a protein with a predicted mass of 55,933 Da. Both clones had 16% identity to VMA13 of Saccharomyces cerevisiae. No sequence homology between the SFD clones and AP50 was detectable. Anti-peptide antibodies were generated against an epitope common to the two proteins and to the unique 18-amino acid insert of the larger protein. The former reacted with both components of native SFD, whereas the latter reacted only with the 57-kDa component. We term the 57- and 50-kDa polypeptides SFDalpha and SFDbeta, respectively.

CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the mu2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The mu1 subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of mu2 and CTLA-4 were localized to residues, 161TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165YVKM, involved in CTLA-4 signaling. Mu2 interacted preferentially with CTLA-4 when residue 165Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 (165Y and 182Y) were phosphorylated by the T lymphocyte-associated tyrosine kinase, p56lck. Thus, phosphorylation of CTLA-4 residue 165Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.

The clathrin-associated adaptors AP-1 and AP-2 are heterotetrameric complexes involved in the recognition of sorting signals present within the cytosolic domain of integral membrane proteins. The medium chains of these complexes, mu1 and mu2, have been implicated in two types of interaction: assembly with the beta1 and beta2 chains of the corresponding complexes and recognition of tyrosine-based sorting signals. In this study, we report the results of a structure-function analysis of the mu1 and mu2 chains aimed at identifying regions of the molecules that are responsible for each of the two interactions. Analyses using the yeast two-hybrid system and proteolytic digestion experiments suggest that mu1 and mu2 have a bipartite structure, with the amino-terminal one-third (residues 1-145 of mu1 and mu2) being involved in assembly with the beta chains and the carboxyl-terminal two-thirds (residues 147-423 of mu1 and 164-435 of mu2) binding tyrosine-based sorting signals. These observations support a model in which the amino-terminal one-third of mu2 is embedded within the core of the AP-2 complex, while the carboxyl-terminal two-thirds of the protein are exposed to the medium, placing this region in a position to interact with tyrosine-based sorting signals.

CTLA-4 plays a critical role in regulating the immune response. It is mainly located in cytoplasmic vesicles and is expressed only transiently on the surface after T cell activation. In this study, we demonstrate that CTLA-4 is associated with AP50, the medium chain of the clathrin-associated coated pit adaptor protein complex AP2. In a yeast two-hybrid screen, three individual cDNA clones that encode mouse AP50 were isolated, all of which can interact specifically with the cytoplasmic domain of mouse CTLA-4, but not with the cytoplasmic domain of mouse CD28. We have shown that CTLA-4 can bind specifically to AP50 when CTLA-4 and AP50 are cotransfected into human 293T cells. A Y201 to F201 mutation in the YVKM intracellular localization motif of the CTLA-4 cytoplasmic domain significantly diminished its binding to AP50. We also found that AP50 bound to a CTLA-4 peptide containing unphosphorylated Y201 but not to a peptide containing phosphorylated Y201. Conversely, the p85 subunit of phosphatidylinositol 3-kinase and, to a lesser extent, protein tyrosine phosphatase SYP (SHP-2) and SHP (SHP-1) bind only to the CTLA-4 peptide containing phosphorylated Y201. Therefore, the phosphorylation status of Y201 in the CTLA-4 cytoplasmic domain determines the binding specificity of CTLA-4. These results suggest that AP50 and the coated pit adaptor complex AP2 may play an important role in regulating the intracellular trafficking and function of CTLA-4.

CTLA-4 is a lymphocyte cell surface receptor expressed by activated T cells that functions to down-regulate T cell responses induced by TCR and CD28 stimulation. Since CTLA-4 competes with CD28 for binding to the common ligands B7-1 and B7-2, the level of CTLA-4 surface expression is likely to play an important role in its ability to inhibit CD28-dependent T cell activation. The factors that regulate these levels are poorly understood. Recent studies have revealed that following T cell activation, the majority of CTLA-4 is localized intracellularly rather than on the cell surface, and surface CTLA-4 is rapidly reinternalized. In this study, we investigate the molecular mechanism underlying the rapid clearance of CTLA-4 from the cell surface. The data demonstrate that cell surface CTLA-4 is endocytosed into clathrin-coated vesicles even in the absence of ligand. The targeting of CTLA-4 to clathrin-coated vesicles is mediated by the clathrin-associated adaptor complex AP-2. The cytoplasmic domain of CTLA-4 was found to specifically bind to AP50, the medium chain subunit of AP-2 in both yeast two-hybrid and coimmunoprecipitation assays. The interaction requires the peptide sequence 199-GVYVKM-204 in the cytoplasmic tail of CTLA-4. Mutation of the CTLA-4 amino acid residue Y201 abrogates the interaction with AP50, resulting in the accumulation of CTLA-4 at the cell surface. Together these data suggest that the interaction of CTLA-4 with AP50 plays an important role in regulating the cell surface expression of CTLA-4.

TGN38 is a type I integral membrane protein that cycles between the trans-Golgi network and the plasma membrane. Internalization at the cell surface and targeting back to the trans-Golgi network is dependent on a hexapeptide motif, SDYQRL, in the cytosolic tail of the protein. It was recently demonstrated that this motif specifically interacts with the mu2 subunit of the AP2 adaptor complex. We have studied the interaction between the entire cytosolic domain of TGN38 and mu2 using the yeast two hybrid system, in vitro binding of recombinant fusion proteins and IAsys optical biosensor technology. A specific interaction has been demonstrated in each of the systems we have employed. We have shown an absolute requirement for Tyr-333 of TGN38 in binding to mu2. In addition we found that mutation of Ser-331 to alanine reduces the affinity of the interaction. By measuring tryptophan fluorescence at equilibrium, we have also determined the dissociation constant for the interaction between the entire cytosolic tail of TGN38 and mu2 as 58 nM. In contrast to previously published work, our data suggest that not only Tyr-333 but also its context is important in determining the specificity of binding of TGN38 to mu2.

Many plasma membrane proteins destined for endocytosis are concentrated into clathrin-coated pits through the recognition of a tyrosine-based motif in their cytosolic domains by an adaptor (AP-2) complex. The mu2 subunit of isolated AP-2 complexes binds specifically, but rather weakly, to proteins bearing the tyrosine-based signal. We now demonstrate, using peptides with a photoreactive probe, that this binding is strengthened significantly when the AP-2 complex is present in clathrin coats, indicating that there is cooperativity between receptor-AP-2 interactions and coat formation. Phosphoinositides with a phosphate at the D-3 position of the inositol ring, but not other isomers, also increase the affinity of the AP-2 complex for the tyrosine-based motif. AP-2 is the first protein known (in any context) to interact with phosphatidylinositol 3-phosphate. Our findings indicate that receptor recruitment can be coupled to clathrin coat assembly and suggest a mechanism for regulation of membrane traffic by lipid products of phosphoinositide 3-kinases.

CTLA-4 is a costimulation receptor that binds to the same ligands, CD80 and CD86, as CD28 with high affinity and is transiently expressed on the cell surface of activated T cells. CTLA-4 delivers an inhibitory signal through association of a phosphotyrosine-containing motif in the cytoplasmic domain with Syp tyrosine phosphatase. We now demonstrate that CTLA-4 interacts with the mu2 subunit of the plasma membrane-associated adaptor complex, AP-2, through the same motif involved in the interaction with Syp, except that the interaction with mu2 requires unphosphorylated tyrosine. The interaction with mu2 likely induces rapid internalization of CTLA-4 from the cell surface. Our results suggest that the phosphorylation state of a single tyrosine residue determines whether CTLA-4 delivers a negative signal or is internalized.

We report the cDNA cloning, primary structure and tissue distribution of two new proteins homologous to mu-adaptins, the medium chains of the clathrin coat adaptor complexes. Both predicted proteins share 60% amino acid sequence identity with each other and 27-31%, identity with mu1-adaptin (ap47) and mu2-adaptin (ap50). Lower similarity (23-25% identity) is found with two other mu-adaptin-related proteins, p47A/B, and there is similarity over the N-terminal 150 amino acids with the adaptin small chains and deltaCOP. The mRNAs of both molecules are expressed in all tissues analyzed, but with different profiles of relative abundance. mu-ARP1 is most abundant in brain, ovary and lung, whereas mu-ARP2 is prominently expressed in testis. These proteins suggest the existence of as yet uncharacterized types of clathrin- or non-clathrin-associated protein coats in cellular membrane traffic, of which they are probably prototype subunits, and provide molecular markers and probes for their characterization.

Many integral membrane proteins contain tyrosine-based signals within their cytoplasmic domains that mediate internalization from the cell surface and targeting to lysosomal compartments. Internalization depends on an interaction of the tyrosine-based signals with the clathrin-associated adaptor complex AP-2 at the plasma membrane, whereas lysosomal targeting involves interaction of the signals with an analogous complex, AP-1, at the trans-Golgi network. Recent studies have identified the medium chains mu2 of AP-2 and mu1 of AP-1 as the recognition molecules for tyrosine-based signals. We have now investigated the structural determinants for interaction of the signals with mu2 and mu1. The position of the signals was found to be an important determinant of interactions with mu2 and mu1; signals were most effective when present at the carboxyl terminus of a polypeptide sequence. Another important determinant of interactions was the identity of residues surrounding the critical tyrosine residue. Mutation of some residues affected interactions with mu2 and mu1 similarly, whereas other mutations had differential effects. These observations suggest that both the position and the exact sequence of tyrosine-based sorting signals are major determinants of selectivity in their interaction with clathrin-associated adaptor complexes.

The ubiquitous eps15 protein was initially described as a substrate of the EGF receptor kinase. Its functions are not yet delineated and this work provides evidence for its possible role in endocytosis. A novel anti-eps15 antibody, 6G4, coimmunoprecipitated proteins of molecular mass 102 kD. In human cells, these proteins were identified as the alpha- and beta-adaptins of the AP-2 complex on the basis of their NH2-terminal sequence and their immunoreactivity with anti-alpha- and anti-beta-adaptin antibodies but not with anti-gamma-adaptin antibody. In addition, the anti-eps15 antibody coimmunoprecipitated metabolically labeled polypeptides with molecular mass of 50 and 17 kD, comparable to those of the two other components of the AP-2 complex, mu2 and sigma 2. Constitutive association of eps15 with AP-2 was confirmed by two sets of experiments. First, eps15 was detected in immunoprecipitates of anti-alpha- and anti-beta-adaptin antibodies. Second, alpha- and beta- but not gamma-adaptins were precipitated by a glutathione-S-transferase eps15 fusion protein. The association of eps15 with AP-2 was ubiquitous and conserved between species, since it was observed in human lymphocytes and epithelial cells and in murine NIH3T3 fibroblasts. Our results are in keeping with a recent study showing homology between the NH2-terminal domains of eps15 and the product of the gene END3, involved in clathrin-mediated endocytosis of the pheromone alpha factor in Saccharomyces cerevisiae, and suggest a possible role for eps15 in clathrin-mediated endocytosis in mammals.

Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed "assembly proteins," or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2 alpha and AP2 beta, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2 beta (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with APs. Chromosomal in situ hybridization of a human genomic clone demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2 beta fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34.

There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN. The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19. To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains. We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting. Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19. These observations suggest that these other subunits may play an important role in targeting. In contrast, beta- and beta'-adaptins are clearly not involved in this event. Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal. We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system. Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma-adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19. These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane.

Mammalian clathrin-associated protein (AP) complexes, AP-1 (trans-Golgi network) and AP-2 (plasma membrane), are composed of two large subunits of 91-107 kDa, one medium chain (mu) of 47-50 kDa and one small chain (sigma) of 17-19 kDa. Two yeast genes, APM1 and APM2, have been identified that encode proteins related to AP mu chains. APM1, whose sequence was reported previously, codes for a protein of 54 kDa that has greatest similarity to the mammalian 47-kDa mu 1 chain of AP-1. APM2 encodes an AP medium chain-related protein of 605 amino acids (predicted molecular weight of 70 kDa) that is only 30-33% identical to the other family members. In yeast containing a normal clathrin heavy chain gene (CHC1), disruptions of the APM genes, singly or in combination, had no detectable phenotypic consequences. However, deletion of APM1 greatly enhanced the temperature-sensitive growth phenotype and the alpha-factor processing defect displayed by cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. In contrast, deletion of APM2 caused no synthetic phenotypes with clathrin mutants. Biochemical analysis indicated that Apm1p and Apm2p are components of distinct high molecular weight complexes. Apm1p, Apm2p, and clathrin cofractionated in a discrete vesicle population, and the association of Apm1p with the vesicles was disrupted in CHC1 deletion strains. These results suggest that Apm1p is a component of an AP-1-like complex that participates with clathrin in sorting at the trans-Golgi in yeast. We propose that Apm2p represents a new class of AP-medium chain-related proteins that may be involved in a nonclathrin-mediated vesicular transport process in eukaryotic cells.

We have previously shown that the 50-kDa subunit of the clathrin assembly complex AP-2 (AP50) stoichiometrically binds to and is immunoprecipitated with the vacuolar (H+)-ATPase (V-ATPase) from clathrin-coated vesicles (Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-9186). We now report that treatment of stripped coated vesicles with cystine results in a purified V-ATPase complex lacking the AP50 polypeptide. Removal of AP50 can be reversed upon treatment of the vesicles with dithiothreitol. Removal of AP50 reduces the ATPase activity of the purified V-ATPase by 90% relative to the enzyme containing AP50. This inhibition is not reversed upon treatment of the AP50-depleted enzyme with dithiothreitol in the absence of AP50. The reconstituted V-ATPase depleted of AP50 is devoid of ATP-dependent proton transport activity. We observe further that the peripheral V1 subunits are unable to reassemble onto the integral V0 domain in the absence of AP50. The addition of purified AP-2 containing the AP50 polypeptide restores the ability of the V1 subunits to assemble with the V0 sector to give a V-ATPase complex that is functional in ATP-dependent proton transport. These results indicate that the AP50 polypeptide is necessary for both activity and in vitro reassembly of the V-ATPase complex.

Our genetic analysis indicates that the unc-101 gene of Caenorhabditis elegans is required for many aspects of development and behavior, including negative regulation of vulval differentiation. We have cloned unc-101 and found that it encodes a homolog of the mammalian medium chains of clathrin-associated protein complexes located at the trans-Golgi and the plasma membrane, AP47 and AP50, respectively. Therefore, clathrin-mediated events might contribute to the negative regulation of vulval differentiation. Comparison of sequences, including a full-length sequence of a C. elegans AP50 homolog, reveals that UNC-101 is most closely related to AP47. Mouse AP47 and nematode UNC-101 proteins are functionally equivalent as assayed in transgenic nematodes. We have sequenced the mutant alleles of unc-101 identified in various genetic screens and shown that all but one are deletions or nonsense mutations, suggesting that these alleles severely reduce unc-101 function.

AP50 is a subunit of the assembly polypeptide (AP) subclass AP-2 from bovine brain coated vesicles. It can be phosphorylated in vivo and in vitro on a threonine residue by means of the AP50 kinase activity associated with AP. We have undertaken an analysis of the amino acid sequence around the AP50 phosphorylation site. After phosphorylation in vitro of AP50 followed by tryptic cleavage, only one radioactive peptide was isolated following Mono-Q ion-exchange f.p.l.c. and reverse-phase h.p.l.c. The amino acid sequence of this peptide: Glu146-Glu-Gln-Ser-Gln-Ile-Thr-Ser-Gln-Val-Thr*-Gly-Gly-Ile-Gly-Tr p-Arg162, displayed two threonine residues. Analysis of the yield and radioactivity of the product from automated Edman degradation indicated that only Thr-156 was phosphorylated, reflecting the presence of a single phosphorylation site in AP50. AP phosphorylated the corresponding synthetic peptide on the same threonyl residue. We demonstrated that AP50 was a phosphorylation substrate unable to autophosphorylate. The enzyme involved in the AP50 phosphorylation was shown to be associated with AP-1 and with a soluble protein complex co-purified with APs but resolved from the latter by hydroxyapatite-column exclusion chromatography. This AP50 kinase activity corresponded to a 280 kDa protein complex according to gel-filtration data.

We have previously noted a 50-kDa polypeptide (p50) co-purifying with preparations of the bovine brain clathrin-coated vesicle vacuolar (H+)-ATPase (V-ATPase) (Zhang, J., Myers, M., and Forgac, M. (1992) J. Biol. Chem. 267, 9773-9778). We show that p50 is also immunoprecipitated with the V-ATPase, further suggesting its specific association with the proton pump. To determine the identity of this 50-kDa polypeptide and the stoichiometry of its association with the V-ATPase, we performed N-terminal amino acid sequencing and quantitative amino acid analysis of the gel-purified protein. These results revealed the unknown polypeptide to be the 50-kDa subunit of the clathrin assembly protein AP-2 (AP50); we estimate the stoichiometry of association is one AP50 per V-ATPase complex. AP50 is an N-ethylmaleimide (NEM)-inhibitable autokinase and incubation of purified V-ATPase with [gamma-32P]ATP resulted in the NEM-sensitive phosphorylation of AP50 and the B subunit of the V-ATPase. The same phosphorylation pattern is seen if the labeling reaction is done with intact clathrin-coated vesicles and the V-ATPase subsequently immunoprecipitated from the solubilized vesicles. This represents the first report of phosphorylation of one of the V-ATPase subunits. The functional significance of this phosphorylation for regulation or targeting of the V-ATPase in vivo remains to be determined.

We have cloned and sequenced mouse brain AP47, the medium chain of the trans-Golgi network clathrin-associated protein complex AP-1. The predicted protein sequence of AP47 is closely related to rat and calf brain AP50, the corresponding medium chain of the plasma-membrane clathrin-associated protein complex AP-2. We have also identified in the yeast genome an open reading frame encoding a protein of previously unknown function. Referred to here as YAP54, its predicted protein sequence displays a striking homology to AP47. We therefore propose that Yap54 is the medium chain subunit of a putative AP-1 complex in yeast. From the analyses of the optimized sequence alignments of AP47, AP50 and Yap54p, we suggest a model for the domain organization of the medium chains.

AP-2 is the class of clathrin-associated protein complex found in coated vesicles derived from the plasma membrane of eukaryotic cells. We demonstrate here, using a chemical method, that an AP-2 complex is an asymmetric structure consisting of one large alpha chain, one large beta chain, one medium AP50 chain, and one small AP17 chain. The complex has been shown to contain a core and two appendages. The AP core includes the small AP17 and the medium AP50 chains together with the amino-terminal domains of the large alpha and beta chains. One appendage corresponds to the carboxy-terminal domain of the beta chain. We find that as in the case of the beta chains, the carboxy-terminal portion of the alpha chains is an independently folded domain corresponding to the second appendage. We use limited tryptic proteolysis of clathrin/AP-2 coats to show the release of the appendages from the interior of the coats and the retention of the AP core by the remaining clathrin lattice. In addition, we find that the AP core stabilizes the coat and prevents its depolymerization. These results are consistent with the proposal that the AP core contains the binding site(s) for clathrin, while the alpha- and beta-chain appendages interact with membrane components of coated pits and coated vesicles.