Epsin Binds to Clathrin by Associating Directly with the Clathrin-terminal Domain
2000; Elsevier BV; Volume: 275; Issue: 9 Linguagem: Inglês
10.1074/jbc.275.9.6479
ISSN1083-351X
AutoresMatthew T. Drake, Maureen A. Downs, Linton M. Traub,
Tópico(s)Biochemical and Structural Characterization
ResumoEpsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by associating directly with the globular appendage of the α subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence 257LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor β subunits, amphiphysin, and β-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the β subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence 480LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing clathrin-coated bud is an important aspect of epsin function. Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by associating directly with the globular appendage of the α subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence 257LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor β subunits, amphiphysin, and β-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the β subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence 480LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing clathrin-coated bud is an important aspect of epsin function. monoclonal antibody glutathione S-transferase heavy chain light chain polyacrylamide gel electrophoresis clathrin heavy chain-terminal domain trans-Golgi network 4-morpholineethanesulfonic acid phosphate-buffered saline Endocytosis occurs primarily at specific regions of the plasma membrane coated on the cytoplasmic surface with the AP-2 adaptor complex and clathrin. The ordered polymerization of clathrin into a polyhedral coat is thought to mechanically introduce curvature into the underlying membrane and thereby drive the formation of clathrin-coated vesicles. Because AP-2 and clathrin are the major protein components on clathrin-coated vesicles that bud from the cell surface, much work over the past decade has centered on carefully dissecting the specific roles that these proteins play in ordered coat assembly and the protein sorting process. It has become clear, however, that multiple factors in addition to clathrin and AP-2 are critical in regulating and coordinating endocytic events. Some additional molecules that have been demonstrated to affect endocytosis include dynamin (1.Schmid S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (356) Google Scholar, 2.Kelly R.B. Nat. Cell Biol. 1999; 1: E8-E9Crossref PubMed Scopus (19) Google Scholar), amphiphysin (3.Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (404) Google Scholar, 4.Wigge P. Vallis Y. McMahon H.T. Curr. 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Chem. 1997; 272: 30984-30992Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 20.Slepnev V.I. Ochoa G.C. Butler M.H. Grabs D. Camilli P.D. Science. 1998; 281: 821-824Crossref PubMed Scopus (275) Google Scholar). Likewise, eps15 interacts with AP-2 (21.Benmerah A. Gagnon J. Begue B. Megarbane B. Dautry-Varsat A. Cerf-Bensussan N. J. Cell Biol. 1995; 131: 1831-1838Crossref PubMed Scopus (151) Google Scholar), the 170-kDa long splice isoform of synaptojanin 1 (22.Haffner C. Takei K. Chen H. Ringstad N. Hudson A. Butler M.H. Salcini A.E. Di Fiore P.P. De Camilli P. FEBS Lett. 1997; 419: 175-180Crossref PubMed Scopus (132) Google Scholar), epsin (9.Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (272) Google Scholar), and intersectin/Ese (10.Yamabhai M. Hoffman N.G. Hardison N.L. McPherson P.S. Castagnoli L. Cesareni G. Kay B.K. J. Biol. Chem. 1998; 273: 31401-31407Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 11.Sengar A.S. Wang W. Bishay J. Cohen S. Egan S.E. EMBO J. 1999; 18: 1159-1171Crossref PubMed Scopus (182) Google Scholar). AP180, another clathrin coat component, binds to both AP-2 and clathrin. Thus, an emerging consensus from these more recent studies is that highly coordinated, multivalent protein-protein interactions are likely to contribute to the dynamics of AP-2-dependent clathrin coat formation that occurs during rapid endocytosis at the plasma membrane. Epsin was recently identified in screens for proteins that interact with the eps15 homology (EH) domain of eps15 (9.Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (272) Google Scholar, 23.Salcini A.E. Confalonieri S. Doria M. Santolini E. Tassi E. Minenkova O. Cesareni G. Pelicci P.G. Di Fiore P.P. Genes Dev. 1997; 11: 2239-2249Crossref PubMed Scopus (287) Google Scholar, 24.McPherson P.S. de Heuvel E. Phillie J. Wang W. Sengar A. Egan S. Biochem. Biophys. Res. Commun. 1998; 244: 701-705Crossref PubMed Scopus (21) Google Scholar). The carboxyl-terminal portion of epsin 1, the so-called NPF domain, contains three repeats of the tripeptide motif asparagine-proline-phenylalanine (NPF) (9.Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (272) Google Scholar). These repeats facilitate binding to the eps15 homology (EH) domains of not only eps15 but also to the eps15 homology (EH) domains of the recently identified intersectin/Ese protein family (10.Yamabhai M. Hoffman N.G. Hardison N.L. McPherson P.S. Castagnoli L. Cesareni G. Kay B.K. J. Biol. Chem. 1998; 273: 31401-31407Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 11.Sengar A.S. Wang W. Bishay J. Cohen S. Egan S.E. EMBO J. 1999; 18: 1159-1171Crossref PubMed Scopus (182) Google Scholar) and POB1 (13.Nakashima S. Morinaka K. Koyama S. Ikeda M. Kishida M. Okawa K. Iwamatsu A. Kishida S. Kikuchi A. EMBO J. 1999; 18: 3629-3642Crossref PubMed Scopus (187) Google Scholar). The central region of the epsin 1 molecule binds to the α subunit of AP-2, in an apparently phosphorylation-dependent manner (9.Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (272) Google Scholar). In fact, epsin was first discovered as a novel binding partner of the globular appendage domain of the AP-2 α chain (25.Wang L.-H. Sudhof T.C. Anderson R.G.W. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). This report describes the ability of epsin 1 to also interact directly with clathrin and identifies linear sequences within the epsin 1 (and epsin 2) primary structure that are responsible for this association. One binding site is adjacent to but distinct from the AP-2-binding site located within the central portion of the protein. A second site is upstream of the first NPF repeat within the carboxyl-terminal NPF domain. Thus, like AP-2, clathrin, amphiphysin, eps15, and β-arrestin, epsin is also capable of forming a multivalent protein network that likely influences the endocytic process. In addition, the potential relevance of related clathrin-binding sequences present in several additional proteins that participate in clathrin-coat assembly is also discussed. The anti-α subunit mAb1 100/2 and the anti-β1/β2 subunit mAb 100/1 were generously provided by Ernst Ungewickell. Polyclonal anti-μ2 subunit antiserum was a gift from Juan Bonifacino. The affinity-purified anti-AP-1 γ subunit antibody, AE/1, has been described elsewhere (26.Traub L.M. Kornfeld S. Ungewickell E. J. Biol. Chem. 1995; 270: 4933-4942Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The anti-AP-2 α subunit mAb AP.6, the clathrin heavy chain mAb X22, and mAb TD.1, which recognizes the clathrin-terminal domain, were kindly provided by Frances Brodsky, and the antibody specific for the clathrin light chain neuronal-specific insert, mAb Cl57.3, was from Reinhard Jahn. Polyclonal anti-epsin antibodies from rabbits injected with residues 229–575 of rat epsin 1 fused to glutathione S-transferase (GST) were affinity purified from serum on either purified epsin DPW domain immobilized on nitrocellulose or on histidine-tagged epsin 1 coupled to CNBr-activated Sepharose 6MB (Amersham Pharmacia Biotech). Polyclonal anti-peptide (RVDASDEDRISEVRKVLC) antibodies against synaptojanin were also raised in rabbits and affinity purified on the immunizing peptide immobilized on CNBr-activated Sepharose 4B. The pan-arrestin mAb F4C1 was kindly provided by Larry Donoso, whereas the polyclonal anti-β-arrestin serum was a gift from R. Lefkowitz. Monoclonal antibodies specific for amphiphysin, AP180, and the γ subunit of AP-1 (mAb 88) were purchased from Transduction Laboratories. A cDNA encoding residues 229–407 of rat epsin 1 cloned into pGEX-4T-1 (GST-DPW) was kindly provided by Pietro de Camilli. A construct containing residues 1–579 of the bovine clathrin heavy chain cloned into pGEX-2T, was kindly provided by Jim Keen. Mutagenesis of epsin 1 was performed using the QuikChange system (Stratagene) with the GST-DPW plasmid as the template. The sense primers used were 5′-GGCAAGGAGGAGTCATCTGCCGCGGCTCTTGCTGACGTCTTC-3′ for the GST-DPW (257LMD → AAA) substitution, 5′-GAGTCATCTCTTATGGATGCTGCCGCGGCCTTCACAACCCCAGCC-3′ for the GST-DPW (260LADV → AAAA) substitution and 5′-GGAGGAGTCATCTCTTTTGGATCTTATGGACGCCTTAACAACCCCAGCCC-3′ for the GST-DPW (1 → 2) substitution. The GST-DPW(2 → STOP), (4 → STOP), (6 → STOP), and (8 → STOP) constructs were all generated by converting the relevant TGG tryptophan codon in the GST-DPW plasmid to a TAA stop codon using the QuikChange system. The GST-ETLLDLDF, GST-SSLMDLADV, and GST-AALVDLDS constructs were each prepared by annealing sense and antisense oligonucleotides and ligating the double-stranded product into EcoRI/XhoI-digested pGEX-4T-1. The pGEX plasmid encoding the final eight amino acids (RGYTLIDL) of Saccharomyces cerevisiae Ent1p fused to GST was kindly provided by Beverly Wendland (27.Wendland B. Steece K.E. Emr S.D. EMBO J. 1999; 18: 4383-4393Crossref PubMed Scopus (206) Google Scholar). All of the constructs and mutations were confirmed by dideoxynucleotide sequencing. GST and the various GST fusion proteins were produced in Escherichia coli BL21 cells. The standard induction protocol entailed shifting log-phase cultures (A 600 = ∼0.6) from 37 °C to room temperature and adding isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 100 μm. After 3–5 h at room temperature with constant shaking, the bacteria were recovered by centrifugation at 8,000 rpm (JA-14 rotor) at 4 °C for 15 min and stored at −80 °C until used. GST fusion proteins were collected on glutathione Sepharose 4B after lysis of the bacteria in B-PER reagent (Pierce) and removal of insoluble material by centrifugation at 14,000 rpm (JA-20 rotor) at 4 °C for 15 min. Where necessary, proteins were cleaved with thrombin (Amersham Pharmacia Biotech) from GST while still immobilized on glutathione Sepharose. The conditions for the thrombin digestion were as recommended by the manufacturer, followed by addition of the irreversible thrombin inhibitor d-Phe-Pro-Arg chloromethyl ketone (Calbiochem) to a final concentration of 25 μm. To prepare the TD-Sepharose, thrombin-cleaved TD was coupled onto CNBr-activated Sepharose 4B to a density of ∼5 mg/ml using standard procedures. Rat brain cytosol was prepared from either fresh or frozen rat brains (PelFreez) exactly as described previously (28.Traub L.M. Bannykh S.I. Rodel J.E. Aridor M. Balch W.E. Kornfeld S. J. Cell Biol. 1996; 135: 1801-1804Crossref PubMed Scopus (103) Google Scholar). Before use, the cytosol was adjusted to 25 mm Hepes-KOH, pH 7.2, 125 mm potassium acetate, 2.5 mm magnesium acetate, 5 mm EGTA, and 1 mm dithiothreitol and centrifuged at 245,000 × g max (70,000 rpm, TLA-100.3 rotor) at 4 °C for 20 min to remove insoluble material. Clathrin-coated vesicles were prepared from fresh rat brain by standard procedures (29.Campbell C. Squicciarini J. Shia M. Pilch P.F. Fine R.E. Biochemistry. 1984; 23: 4420-4426Crossref PubMed Scopus (96) Google Scholar). For the preparation of purified clathrin, a crude coat extract was prepared from clathrin-coated vesicles using 1m Tris-HCl, pH 7.0 followed by gel filtration in 500 mm Tris-HCl, pH 7.0 (30.Ahle S. Ungewickell E. EMBO J. 1986; 5: 3143-3149Crossref PubMed Scopus (144) Google Scholar). Clathrin-containing fractions were pooled, the clathrin trimers were concentrated by addition of ammonium sulfate to 50% saturation, and then cages were assembled by dialysis against 100 mm MES-NaOH, pH 6.8, 2.5 mm MgCl2, 1 mm EGTA, 3 mm CaCl2 at 4 °C. The association of AP-2, clathrin, and other endocytic accessory proteins with the various GST fusion proteins was assayed in 25 mm Hepes-KOH, pH 7.2, 125 mmpotassium acetate, 2.5 mm magnesium acetate, 5 mm EGTA, and 1 mm dithiothreitol (assay buffer) in a final volume of 600 μl. Routinely, GST and the GST fusion proteins were first each immobilized on 50 μl of packed glutathione Sepharose to concentrations of ∼2–6 mg/ml. The immobilized proteins were then washed and resuspended to 100 μl in assay buffer. Rat brain cytosol was added, and the tubes were then incubated at 4 °C for 60 min with continuous gentle mixing. The Sepharose beads were then recovered by centrifugation at 10,000 ×g max for 1 min, and 30-μl aliquots of each supernatant were removed and adjusted to 100 μl with SDS-PAGE sample buffer. After washing the Sepharose pellets four times each with ∼1.5 ml of ice-cold PBS by centrifugation, the supernatants were aspirated, and each pellet was resuspended to a volume of 150 μl in SDS-PAGE sample buffer. Unless otherwise indicated, 10-μl aliquots, equivalent to 1200 of each supernatant and 115 of each pellet, were loaded on the gels. For the clathrin-cage binding experiments, preassembled cages were mixed with GST-DPW fusion proteins that had been centrifuged at 245,000 × g max for 20 min to remove insoluble material. The binding assays were performed in 100 mm MES-NaOH, pH 6.8, 2.5 mm MgCl2, 1 mm EGTA, 3 mm CaCl2 in a final volume of 100 μl. After 20 min at room temperature, the cages were recovered by centrifugation at 245,000 ×g max for 10 min, and then equal aliquots of the supernatant and pellet fractions were analyzed by SDS-PAGE. Samples were resolved on 10% polyacrylamide gels prepared with an altered acrylamide:bis-acrylamide (30:0.4) ratio stock solution. The decreased cross-linking generally improves resolution but also affects the relative mobility of several proteins, most noticeably AP180 and epsin 1. After SDS-PAGE, proteins were either stained with Coomassie Blue or transferred to nitrocellulose in ice-cold 15.6 mm Tris, 120 mm glycine. Blots were blocked overnight in 5% skim milk in 10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Tween 20, and then portions were incubated with primary antibodies as indicated in the individual figure legends. After incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG, immunoreactive bands were visualized with enhanced chemiluminescence (Amersham Pharmacia Biotech). Normal rat kidney cells were grown on 12-mm round glass coverslips in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 mml-glutamine. Cells were fixed either in methanol at −20 °C for 5 min or in 3.7% formaldehyde in PBS at room temperature for 15 min, washed with PBS, and then incubated in 10% normal goat serum, 0.2% saponin in PBS at room temperature for 30 min. The cells were then incubated with a mixture of affinity-purified anti-epsin 1 antibodies and mAb X22, mAb AP.6, or mAb 88 diluted in 10% normal goat serum, 0.05% saponin in PBS. After ∼60 min at room temperature, the cells were washed three times with PBS and then incubated with a mixture of goat anti-mouse Alexa 488 and sheep anti-rabbit Alexa 596 (Molecular Probes) conjugates diluted 1:250 in 10% normal goat serum, 0.05% saponin in PBS. After ∼30 min at room temperature, the cells were washed with PBS and mounted on Cytoseal. The central portion of rat epsin 1 (residues 249–401) contains eight repeats of the tripeptide motif aspartic acid-proline-tryptophan (DPW) (9.Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (272) Google Scholar). This region, termed the DPW domain, interacts with the AP-2 adaptor complex by binding directly to the carboxyl-terminal appendage of the α subunit (9.Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (272) Google Scholar, 31.Owen D.J. Vallis Y. Noble M.E. Hunter J.B. Dafforn T.R. Evans P.R. McMahon H.T. Cell. 1999; 97: 805-815Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 32.Traub L.M. Downs M.A. Westrich J.L. Fremont D.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8907-8912Crossref PubMed Scopus (145) Google Scholar). After a GST-DPW fusion protein is incubated with rat brain cytosol, a group of ∼100-kDa polypeptides associate with the sedimented glutathione-Sepharose (Fig.1 A, lane d). Immunoblotting with antibodies to the adaptor α and β subunits confirm the identity of these bands as the large subunits of AP-2 (Fig.1 B, lane d compared with lanes b andf). An anti-μ2 subunit antibody verifies the presence of this AP-2 subunit as well. The blots probed with the anti-adaptor β subunit antibody reveal that both the β1 chain of the AP-1 complex and the β2 chain of AP-2 are found with the GST-DPW fusion-protein pellet (Fig. 1 B, lane d). In rat brain sections, some epsin 1 staining has been observed in the perinuclear region and, when full-length epsin 1 was overexpressed in Chinese hamster ovary cells, the protein was also found in clathrin-coated structures at thetrans-Golgi network (TGN) (9.Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (272) Google Scholar). Because epsin binds to the carboxyl-terminal appendage of the α subunit of AP-2, targeting to the TGN could be via an analogous recognition site on the appendage domain of the γ subunit of the AP-1 adaptor complex. We therefore checked for the presence of AP-1 complexes bound to the epsin construct using an antibody against the γ subunit of AP-1 and, surprisingly, find that only limited amounts of the Golgi-adaptor associate with the GST-DPW (Fig. 1 C, lane d). The steady-state distribution of epsin 1 in normal rat kidney cells also indicates that only minor amounts of epsin 1 are associated with the clathrin-coated structures forming at the TGN (Fig. 2). In cells double-labeled for epsin 1 and AP-2, the staining patterns are almost identical (panels A and B). Cells labeled for both clathrin and epsin 1 (panels C and D) show that although almost all the peripheral clathrin-positive structures also contain epsin, the compact perinuclear clathrin population on the TGN is largely devoid of co-localizing epsin. This is evident despite the diffuse perinuclear epsin signal that corresponds to the soluble form of the protein. Staining of the AP-1 adaptor complex, concentrated at the TGN, is again clearly distinct from the staining pattern of epsin 1 (panels E and F). Together, these experiments suggest that AP-1 and AP-2 do not associate with the DPW domain of epsin 1 with similar affinities and instead argue that the bulk of the β1 subunit bound to the GST-DPW is most likely explained by the promiscuity of β1 subunit incorporation into brain AP-2 complexes noted previously by us (28.Traub L.M. Bannykh S.I. Rodel J.E. Aridor M. Balch W.E. Kornfeld S. J. Cell Biol. 1996; 135: 1801-1804Crossref PubMed Scopus (103) Google Scholar) and others (33.Page L. Robinson M. J. Cell Biol. 1995; 131: 619-630Crossref PubMed Scopus (129) Google Scholar, 34.Sorkin A. McKinsey T. Shih W. Kirchhausen T. Carpenter G. J. Biol. Chem. 1995; 270: 619-625Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The relative lack of epsin recruitment onto the TGN agrees well with the absence of eps15R on the clathrin coats assembling at the TGN (35.Coda L. Salcini A.E. Confalonieri S. Pelicci G. Sorkina T. Sorkin A. Pelicci P.G. Di Fiore P.P. J. Biol. Chem. 1998; 273: 3003-3012Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar).Figure 2Steady-state distribution of epsin, AP-2, and clathrin in normal rat kidney cells. Methanol-fixed (A–D) or formaldehyde-fixed (E and F) normal rat kidney cells were double labeled with affinity-purified antibodies against epsin (B, D, and F) and either mAb AP.6, against the α subunit of AP-2 (A) or mAb X22 directed against the clathrin HC (C) or mAb 88, against the γ subunit of AP-1 (E). Arrowheadspoint to representative examples of co-localization of epsin with AP-2- and clathrin-containing structures. Notice that in addition to the epsin found concentrated at clathrin bud sites on the cell surface, a pool of soluble epsin is also visible in the central region of each cell (B, D, and F). Careful inspection indicates that this staining pattern is, in general, quite distinct from that of the clathrin/AP-1 assembled at the TGN, however.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to the subunits of the AP-2 heterotetramer that associate with the immobilized GST-DPW fusion protein, a prominent ∼180-kDa protein is also found in the pellet (Fig. 1 A, lane d). The binding of both AP-2 and the ∼180-kDa protein is dependent on both the epsin portion of the fusion protein and cytosol as neither is found on the control GST Sepharose (lane b) or on GST-DPW Sepharose incubated without cytosol (lane f). To determine the identity of the large polypeptide, immunoblots were probed with antibodies against the clathrin heavy chain, AP180, and synaptojanin. Strong reactivity with the anti-clathrin antibody (Fig. 1 B, lane d) indicates that this protein is likely the clathrin heavy chain. Unlike clathrin, neither AP180 nor synaptojanin are found in the pellet (lane d), although both proteins are seen in the cytosol fraction (Fig. 1 C, lanes a and c), as expected. The simultaneous appearance of clathrin light chains in the pellet (Fig. 1 B, lane d) confirms the association of clathrin with the DPW domain of epsin 1. Incubating brain cytosol with increasing amounts of the GST-DPW fusion shows that the association of clathrin with epsin is dose-dependent, as is the binding of AP-2 (Fig. 3, lanes c–h). It is important to note, however, that although clathrin binding is still detectable when 25 μg of the GST-DPW fusion is immobilized (lane e), the association of clathrin trimers with the GST-DPW appears significantly more sensitive to dilution than the binding of soluble AP-2 (lanes d–h). The sequence 257LMDLADVF precedes the array of eight DPW repeats in the central portion of the rat epsin 1 molecule. A related sequence, 283LLDLMDAL is found in rat epsin 2 (TableI). These short regions of alternating aliphatic hydrophobic and acidic residues are similar to the clathrin-binding sequences found in the β1, β2, and β3 subunits of the adaptor complexes (36.Shih W. Galluser A. Kirchhausen T. J. Biol. Chem. 1995; 270: 31083-31090Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 37.Dell'Angelica E.C. Klumperman J. Stoorvogel W. Bonifacino J.S. Science. 1998; 280: 431-434Crossref PubMed Scopus (311) Google Scholar), in amphiphysin I and II (38.Ramjaun A.R. McPherson P.S. J. Neurochem. 1998; 70: 2369-2376Crossref PubMed Scopus (135) Google Scholar) and in β-arrestin 1 and β-arrestin 2 (39.Krupnick J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar) (Table I). To test whether this region of epsin 1 facilitates the association with clathrin, we separately mutated the 257LMD or 260LADV sequences to alanines in the context of the GST-DPW fusion protein. Either substitution causes a dramatic reduction in clathrin binding (Fig. 4 A, lanes dand e) compared with the GST-DPW (lane c). By contrast, the association of AP-2 with the epsin mutants (lanes d and e) is indistinguishable from native fusion protein (lane c). This rules out the possibility that clathrin simply associates with epsin only indirectly by interacting solely with the bound AP-2 adaptor. It also argues against gro
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