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Address correspondence to Harvey T. McMahon, MRC Laboratory of Molecular Biology, Hills Rd., Cambridge CB2 2QH, UK. Tel.: 44-1223-402311. Fax: 44-1223-402310. E-mail: hmm{at}mrc-lmb.cam.ac.uk
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Abstract |
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Key Words: epsin; GGA; TGN; endocytosis; ENTH
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Introduction |
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To recruit cargo, a number of adaptor proteins are needed. These can be classified into four subunit polymeric adaptor protein complexes (APs;* AP1, AP2, AP3, and AP4; Hirst and Robinson, 1998; Boehm and Bonifacino, 2002) and monomeric adaptors, e.g., Golgi-localized, gamma-earcontaining ADP-ribosylation factorbinding proteins (GGAs; Dell'Angelica et al., 2000; Hirst et al., 2000). Each polymeric AP contains a specific µ-subunit that recognizes variations of a sequence motif (Yxx) on the cytoplasmic tails of cargo molecules (Kirchhausen et al., 1997; Owen and Evans, 1998). Each AP also contains two large homologous subunits (
+ ß1 in the AP1 complex,
+ ß2 in the AP2 complex, and
+ ß3 in the AP3 complex) whose appendage domains bind to core and accessory proteins necessary for clathrin-coated vesicle (CCV) formation. The
-appendage has been shown to bind proteins that contain DxF motifs, normally in multiple copies (Owen et al., 1999, 2000; Brett et al., 2002), and as part of this paper, we investigate the motif for binding the
-appendage. AP2 adaptor complexes are found predominantly at the plasma membrane, whereas AP1 complexes are found concentrated at the TGN. The monomeric GGAs have recently been shown to bind cargo via a VHS domain (Doray et al., 2002; Misra et al., 2002; Shiba et al., 2002), and they also have a domain with very strong homology to the
-adaptin appendage.
The AP of 180 kD (AP180) is a brain-enriched clathrin/adaptor-binding protein that promotes clathrin assembly on membranes (Ford et al., 2001). It is not a conventional adaptor protein in that it does not link cargo into the clathrin cage. The protein is essential for CCV formation in Drosophila (Zhang et al., 1998) and in Caenorhabditis elegans (Nonet et al., 1999), and overexpression of the COOH-terminal domain blocks clathrin-coated budding events (Ford et al., 2001). The AP180 NH2-terminal homology (ANTH) domain of AP180 and its ubiquitous homologue, CALM, directs binding to PtdIns(4,5)P2 in the membrane.
By domain analysis, the epsin family of proteins are close relatives of the AP180 family. They both have lipid-binding domains at the NH2 terminus and clathrin/adaptor-binding domains centrally located. However, they are distinct in that the AP180 NH2-terminal homology (ANTH) domain binds lipids on its surface, coordinating the headgroup via a KxxKxH motif, whereas the epsin-NH2-terminal homology (ENTH) domain binds lipids in a pocket using residues spread over the first three helices. On binding, the first helix of the epsin ENTH domain folds around the lipid headgroup and exposes a hydrophobic surface on the outer surface of this new helix. We have proposed that on binding to membranes, this new helix buries itself between the lipid headgroups, pushing the lipids apart and thus promoting membrane curvature in the clathrin-coated bud (Ford et al., 2002). As this will need to be a coordinated event, the COOH terminus of epsin1 binds to both clathrin and the AP2 complex, thus inducing the curvature in newly forming coated pits.
Epsins were originally identified as Eps15-interacting proteins, but the family is now better described as being ENTH domain (phosphatidylinositol phosphate [PtdInsP] binding) proteins with clathrin/adaptor binding sequences. Epsins 1 and 2 are brain-enriched (Rosenthal et al., 1999), whereas epsin3 is expressed in wounded epithelia (Spradling et al., 2001). Previously, we identified another epsin in the database (Ford et al., 2002) that is more distantly related to epsins 13 in that it does not have any Eps15-binding motifs (NPFs), and so is not a classical "epsin," and thus, we named it epsinR (for epsin-related protein). The ENTH domain of epsinR is conserved, although the lipid specificity is predicted to be different (Ford et al., 2002). The clathrin/adaptor binding domain is also conserved, although the motifs present are distinctly different (Fig. 1 D). EpsinR does not have the ubiquitin interacting motifs present in epsins 13 that bind to ubiquitin and are essential for the monoubiquitination of the protein (Oldham et al., 2002; Polo et al., 2002). EpsinR homologues are also present in other species, including Drosophila and C. elegans (Fig. 1 C), and have no NPF motifs, but have multiple DxF motifs in place of the multiple DPWs in the epsin1 homologues. In this paper, we investigate the function of mammalian epsinR in vesicle budding events.
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Results |
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Epsin1 ENTH domain binds to PtdIns(4,5)P2 in liposome-binding assays and by isothermal titration calorimetry (ITC) with a low micromolar affinity (Itoh et al., 2001; Ford et al., 2002). In liposome-binding experiments and in overlay assays, epsinR showed a very weak preference for liposomes containing PtdIns(4)P while also binding to PtdIns(5)P (Fig. 2). This was similar to the specificity of the PH domain of oxysterol binding protein (OSBP; Levine and Munro, 2002) that is targeted in vivo to Golgi membranes (also for epsinR; see Fig. 6 and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200208023/DC1). Mutants of epsinR showed no binding in the overlay assay when tested using equivalent protein concentrations and incubation times (Fig. 2). In vivo, PtdIns(4)P is thought to be more TGN-enriched, and PtdIns 4-kinase activity is found on the Golgi (Godi et al., 1999), whereas PtdIns(5)P has not been localized in cells. The weak binding to lipids in vitro may well mean that multimerization and/or the presence of other proteins may play a role in membrane recruitment (see Discussion). We have limited evidence for self-association of epsinR from pulldown experiments (see Fig. 4 D and unpublished data), and it is therefore possible that endogenous epsinR may have a higher avidity for membranes than observed for the monomeric ENTH domain.
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Based on the truncation of N1, we saw a slight reduction in AP1 binding (as assessed by blotting for -adaptin) with a stop mutation after N379 and a major reduction in binding with a stop mutation after G345. We noted the presence of two DFG sequences sequentially deleted by these truncations. Point mutants of the sequences D349R and D371R both reduced AP1 binding. The presence of two binding sites is consistent with the affinity measurements for the N3 construct with the
-appendage domain (Fig. 5). Mutagenesis of both D349R and D371R in N3 both greatly weakened the high affinity interaction, implying that this may be due to secondary folding. However, secondary structure analysis of the N3 construct by CD spectroscopy showed that it is largely unstructured, though there is a mild change in the presence of
-appendage (Fig. S2).
To investigate AP interactions further, we made peptides for direct interaction studies (see Fig. 1 D for peptide locations). The P1 peptide contains the first DLF motif. It did not bind to the -, ß-, or
-appendages (Fig. 5), and mutagenesis of D325 and D328 had no effect on AP1 binding (Fig. 4 B). Peptides P2, P3, and P4 are overlapping; P2 and P3 contain the second DLF motif and P3 and P4 contain the first DFG sequence. All three peptides bound weakly to the
-appendage, thus excluding the possibility that the DFG alone is responsible for the interaction. P3 and P4 both interacted with the ß-appendage but only P3 with the
-appendage domain. The P5 peptide containing the second DFG sequence bound
-appendage with the highest affinity (48 µM) although still much weaker than the full N3 construct. P5 also showed a weak interaction with the ß- but not the
-appendage domain. Looking at the two peptides that bound with highest affinity to the
-appendage domain (P3 and P5), we concluded that Fxx[F/W] was a potential binding sequence. Using the P3 and P5 peptide consensus, we searched for homologous sequences in the protein database and found
-synergin. Next, we made a peptide of one of the homologous sequences from
-synergin, and found that it bound with an affinity of 13 µM. This was the highest affinity for any of our peptides. We now recompared the similarity between our two highest affinity peptides,
-synergin-P1 and epsinR-P5, and came up with the consensus DFxD[F/W]. The highest scoring protein in the database is now
-synergin, with three copies of this motif.
-synergin has already been characterized as a
-adaptin binding partner (Page et al., 1999) and thus, with this work, we identify a binding site within the protein for the
-appendage. Rabaptin5 (Nogi et al., 2002) and Eps15 (Kent et al., 2002) are also proposed to interact directly with the
-appendage domain. Both these proteins contain homologous sequences to the above consensus. Looking through the protein database for other potential
-adaptin appendage binding partners, we get a high score for an uncharacterized EF-hand protein (NP_060127) with three copies of this motif. This protein also contains potential clathrin-binding motifs and multiple DDF motifs, and thus, this is a future candidate to join the ranks as a
-appendage interacting protein.
Clathrin assembly
EpsinR is enriched in CCVs (Fig. 3 B). Clathrin binding to the truncations and mutants of N3 imply that there may be multiple clathrin-interacting motifs, including the one centered on D422. If this were the case, then it might be expected that epsinR will promote clathrin cage assembly by linking clathrin triskelia together. We and others have previously shown that epsin1 binding to membranes can promote clathrin recruitment and polymerization, and that this is due to the presence of multiple clathrin-binding motifs (Ford et al., 2002; Kalthoff et al., 2002a; and unpublished data). By analogy, epsinR might be expected to assemble clathrin. We tested this in comparison to AP180, which strongly exhibits this activity at neutral pH and with the ß-appendage + hinge domain, which exerts this effect more strongly at a lower pH (Owen et al., 2000; Ford et al., 2001). Under these conditions, the N3 domain does not promote clathrin cage assembly, and even antagonizes the limited assembly by ß-appendage + hinge domain at neutral pH. This does not exclude an assembly activity of epsinR, as the N3 construct does not include all the clathrin-binding activity (Fig. 4 A), but the N1 construct gave similar results. We conclude that clathrin assembly is not a primary function of this domain.
Subcellular localization of EpsinR
Epsin1 is brain-enriched, whereas epsinR is ubiquitous in its tissue distribution (Fig. 3), and homologues are found in both C. elegans and Drosophila. Thus, we used epsinR antibodies to detect endogenous protein in COS cells. It showed a perinuclear concentration with additional puncta distributed throughout the cytoplasm (Fig. 6 Ai). These puncta were completely distinct from those observed with overexpressed epsin1 (Fig. 6 Aii). Overexpressed Myc-epsinR showed the same expression pattern, although with a greater perinuclear concentration (Fig. 6 Aiii). Overexpressed myc-epsinR also overlapped with staining for other endogenous proteins (Fig. 6 B); AP1, perinuclear clathrin, GGA, M6P receptors and TGN46 (but not the medial Golgi marker GM130 and the lysosomal marker Lamp1; see http://www.jcb.org/cgi/content/full/jcb.200208023/DC1). Strong overexpression of WT epsinR led to enlarged perinuclear compartments (Fig. 6 Aiv). We have previously shown that overexpression of epsin1 leads to an inhibition of AP2/clathrin-coated vesicle formation from the plasma membrane (see Fig. 3 d in Ford et al., 2002), likewise, these enlarged epsinR positive compartments may be due to a budding deficiency (Fig. 7 B). This phenotype became stronger over time, thus, in these studies, we have been careful not to prolong the overexpression.
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Mutation of the L6 residue on helix zero of epsin1 has a profound effect on lipid curvature (Ford et al., 2002). The equivalent residue on epsinR is L10, and this was mutated to a hydrophilic residue (L10E) and the protein was overexpressed (Fig. 6 D). The TGN enrichment was severely reduced. Thus, like epsin1, the hydrophobicity of helix zero is important for the protein's normal distribution. The phenotype was distinguished by large puncta around the nucleus and in the cytoplasm, which cannot clearly be classified as a known compartment. AP1 was found in these epsinR puncta as a very diffuse stain, but we did not detect GGAs. We also did not find EEA1, Lamp1, GM130, or M6P receptors in the puncta (links to original images and extra data can be found at http://www.jcb.org/cgi/content/full/jcb.200208023/DC1). These puncta colocalized with clathrin and a minor component of TGN46, but most of the TGN46 was still in its perinuclear compartment. Furthermore, the intensities of epsinR and clathrin/TGN46 puncta were equivalent, and there was a disruption of the perinuclear clathrin organization (Fig. 6 D). Given the partial redistribution of TGN46 with WT epsinR, D34G + R67L, and the L10E mutants, we speculate that trafficking of this transmembrane protein is affected by epsinR. Further epsinR mutants are found at http://www.jcb.org/cgi/content/full/jcb.200208023/DC1.
EpsinR is involved in vesicle budding between the TGN and endosomes
Given the enrichment of endogenous epsinR in Golgi fractions and the distribution by immunofluorescence, we would predict that at least one trafficking step in which epsinR is involved is AP1/clathrin-mediated budding from the TGN. Numerous proteins are incorporated into AP1/clathrin-coated vesicles from the TGN, including the cation-independent M6P receptor and the lysosomal hydrolase cathepsin D. In mice deficient in the µ1A subunit of AP1, the trafficking of both the cation-independent M6P receptor and cathepsin D is perturbed (Meyer et al., 2000). As epsinR has a high affinity for AP1, we tested whether overexpression of epsinR could perturb cathepsin D trafficking. Cathepsin D is synthesized as an inactive 53-kD glycosylated protein (pro-cathepsin) in the Golgi and trafficked to lysosomes, via late endosomes (direct pathway) or via the cell surface (indirect pathway/secretion and reuptake). Pro-cathepsin D is de-glycosylated and further cleaved in lysosomes to generate the active 30-kD mature form. In mice deficient in the µ1A subunit of AP1, there is an increased secretion of glycosylated cathepsin D into the medium due to a disruption of its trafficking to the lysosome. We found that overexpression of epsinR caused a >10-fold increase (n = 4) in the quantity of cathepsin D precursors secreted into the medium versus untransfected control cells (Fig. 7 A). Overexpression of the -appendage domain also perturbed normal trafficking, though not to the same extent (Fig. 7 A), whereas epsin1 and the ENTH domain of epsinR had no effect (unpublished data). Thus, WT epsinR overexpression disrupts this AP1-dependent process in line with our observations of the enlarged perinuclear compartments with WT protein overexpression. The cation-independent M6P receptor also traffics from the TGN via CCVs (Meresse and Hoflack, 1993; Meyer et al., 2000), and is enriched in purified liver CCV fractions (Fig. 3 B). We were not able to detect a bulk change in the distribution by immunofluorescence, but there was a reduction in receptor incorporation in purified CCVs from COS cells transfected with epsinR (Fig. 7 B). This reinforces the argument that epsinR functions in CCV budding from the TGN.
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Discussion |
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The clathrin/adaptor-binding domain of epsinR contains multiple clathrin-binding sites and multiple DxF motifs that are predicted to bind to adaptor appendage domains. It is now generally assumed that these regions in the various endocytic proteins are unfolded and the motifs act like hooks on a fishing line to maximize the potential for ligand binding (Kalthoff et al., 2002a). The simultaneous binding of multiple ß- and/or -appendages to N3 supports this fishing line model. By CD spectroscopy, the N3 construct is largely unstructured and we did not detect any major change on binding of
-appendage (Fig. S2). However, our results imply that limited folding gives rise to the high affinity interactions. First, separate point mutants of the two
-appendage binding sites in N3 (D349R and D371R) both result in a reduction of the high affinity interaction. Second, the peptide binding data do not reproduce the high affinities achieved with the N3 construct.
In this paper, we have identified for the first time a -appendage binding motif, [D/E]FxD[F/W], present in epsinR,
-synergin, and an uncharacterized EF-hand protein, NP_060127, that will be the subject of future investigations. This motif should bind to a basic patch on the surface of the
-appendage (Kent et al., 2002; Nogi et al., 2002), and thus, the introduction of a negative charge into this patch (L762E) disrupted the interaction (Fig. 4 C). The Drosophila epsinR homologue has two copies of EFxDF, whereas the Drosophila epsin1 homologue has none. The C. elegans epsinR homologue conserves the GFxDF as present in epsinR-P3.
To enable the study of epsinR function, we modeled its ENTH domain on the structure of epsin1 ENTH bound to Ins(1,4,5)P3. Mutations reduced the binding to lipids and to purified Golgi membranes (Fig. 2 B and Fig. S1), and showed the ENTH domain primarily determines the location of the protein in cells, just as for epsin1 (Fig. 6). We found that the lipid binding of epsinR was not strong enough to enable us to do more biochemistry on the protein to assay for clathrin recruitment and liposome tubulation. However, this weaker affinity points to the importance of other factors in the localization of the protein, perhaps multimerization and/or the presence of other proteins. Arf could well play a role in recruitment, as it does in the localization of the AP1 complex, OSPB and PtdIns 4-kinase to the Golgi (Godi et al., 1999; Levine and Munro, 2002). Consistent with a role for oligomerization, we noted that the N3 construct of epsinR binds to full-length epsinR (Fig. 4 D). This is not due to N3 self-association (Fig. S3; ultracentrifugation data).
Another key feature of the epsin1 ENTH domain is the amphipathic nature of helix zero with an exposed ridge of hydrophobic residues. This is preserved in epsinR and is likely extended at the NH2 terminus for another turn of the helix (Fig. 1 B and yellow shading in Fig. 1 C). We would propose that epsinR, like epsin1, will fold this helix on binding to PtdInsP and insert the amphipathic helix between the lipid head groups, aiding membrane curvature during clathrin cage formation.
In this paper, we also propose that epsinR is functionally equivalent to epsin1, but in CCV budding from the TGN/endosomes rather than from the plasma membrane. We observe a trafficking defect in cathepsin D like that found in the µ1A-deficient cell line (Meyer et al., 2000). A budding defect from the TGN is supported by the observation of enlarged compartments on overexpression of epsinR (and especially with the lipid-binding mutants, for example L10E) and the disruption of M6P receptor incorporation into CCVs on overexpression of epsinR. Our data strongly suggest that these budding defects involve clathrin/AP1 trafficking pathways.
Having submitted this paper, other manuscripts have been published or are in press where epsinR has been found using a proteomics approach on purified CCVs (Wasiak et al., 2002; Hirst et al., 2003) and by homology searches for ENTH domain proteins (Kalthoff et al., 2002b). The authors give various names to the same protein "enthoprotin," epsinR, and "clint," respectively, and find a similar localization in cells and clathrin/adaptor interactions. Also, a yeast epsinrelated protein has been shown to be required for Golgiendosome traffic (Duncan et al., 2003).
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Materials and methods |
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Transfections and antibodies
COS-7 cells were transfected using GeneJuiceTM (Novagen) according to the manufacturer's protocol. 28 h after transfection, cells were transferred to serum-free medium and incubated with biotinylated transferrin for 15 min before fixation with 4% PFA. EpsinR-transfected cells were detected using either a polyclonal anti-Myc antibody (green in merged images; Cell Signaling), or an mAb (9E10; Sigma-Aldrich). AP1 distribution was followed using a -adaptin mAb (1003, red in merged images), and biotinylated transferrin was detected with labeled streptavidin (blue in merged images). Other antibodies used were as follows: polyclonal Epsin1 DPW domain (Ra14), monoclonal clathrin (X22, a gift of B. Pearse, MRC-LMB), monoclonal EEA1 (Transduction Laboratories), polyclonal cation-independent M6P receptor (1001, a gift of P. Luzio, Cambridge Institute for Medical Research, Cambridge, UK), and polyclonal GGA1 and 2 antibodies (a gift of J. Hirst and M. Robinson, Cambridge Institute for Medical Research). Cells were mounted and imaged using a confocal system (Radiance; Bio-Rad Laboratories). The Ra43 antibody raised against an NH2-terminal epsinR construct crossreacts with a band of 180 kD (likely AP180, which has a homologous NH2-terminal domain). By immunofluorescence, it does not recognize overexpressed epsin1, its closest homologue (Fig. 6 A), and the distribution of the overexpressed epsinR is the same as the endogenous protein, even in cells where there is an enlarged perinuclear compartment. The antibody works better on purified fractions than in total homogenates. We also raised another antibody against epsinR N3 that showed the same staining pattern. Biosynthetic labeling of cathepsin D was as described previously (Davidson, 1995), except that ProMix at 5 µl/ml of medium (Amersham Biosciences) was used for labeling.
Phospholipid binding
Protein binding to phospholipids was investigated in a protein lipid overlay assay using PIP StripsTM (Echelon Biosciences Incorporated) according to the manufacturer's protocol. In brief, the membrane was blocked in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20 [vol/vol] + 3% BSA [wt/vol]) for 1 h at RT. Protein was added at 0.5 µg/ml in TBST + BSA and incubated for 1 h at RT. The membrane was washed three times for 10 min in TBST + BSA. Proteins were detected by incubation with a 1/1,000 dilution of our polyclonal rabbit antibody Ra30 against the epsin1 ENTH domain, with a 1/1,000 dilution of our polyclonal rabbit antibody Ra43 for wild-type and mutant epsinR ENTH and with a 1/1,000 dilution of a monoclonal mouse antiGFP antibody (CLONTECH Laboratories, Inc.) for detection of His-GFP-OSBP in TBST + BSA, followed by incubation with antirabbit or antimouse HRP conjugate (Bio-Rad Laboratories) in a 1/10,000 dilution and by ECL (Amersham Biosciences). Lipid specificity was tested with a liposome pelleting assay as described on http://www.jcb.org/cgi/content/full/jcb.200208023/DC1. Proteins were detected with Coomassie stain.
Isothermal titration calorimetry
Binding of epsinR constructs and peptides to -, ß-, and
-adaptin appendage domains were investigated by ITC (Wiseman et al., 1989) using a VP-ITC (MicroCal, Inc.). All experiments were performed in 100 mM Hepes, pH 7.4, 50 mM NaCl, and 2 mM DTT at 10°C. The peptides or proteins were injected from a syringe in 4050 steps up to a 34-fold molar excess. The cell contained 1.4 ml protein solution, and typically, the ligand was added in steps of 48 µl every 3.5 min. Concentrations were chosen so that
-, ß-, and
-adaptin appendage domains were at least fivefold higher than the estimated dissociation constant, if possible. The epsinR protein or peptide ligands were again at least 10-fold more concentrated than the ear domains. The heat of dilution of the ligand was subtracted from the data before fitting. Titration curves were fitted to the data using the ORIGIN program (MicroCal, Inc.) yielding the stoichiometry N, the binary equilibrium constant KA (= KD-1) and the enthalpy of binding. The entropy of binding
S° was calculated from the relationship
G° = -RT lnKA and the Gibbs-Helmholtz equation.
Clathrin assembly and sedimentation assays
Purified clathrin was dialyzed into 0.1 M MES, pH 6.5, 1 mM EGTA, 0.5 mM MgCl2, and 1 mM DTT, and centrifuged at 100,000 g for 25 min to remove aggregates. Clathrin was incubated with proteins or an equivalent volume of buffer at 4°C for 10 min and spun at 100,000 g in a rotor (model TLA100; Beckman) for 25 min. Final protein concentrations were as follows: clathrin, 0.3 µM; epsinR N3, 50 µM; -appendage + hinge domain, 40 µM; AP180, 0.3 µM.
Online supplemental material
Fig. S1 shows the enrichment of epsinR in Golgi membrane fractions along with the cation-independent M6P receptor. It also shows that the epsinR lipid-binding mutant, D34G + R67L, no longer associates with Golgi membrane fractions. Fig. S2 shows that the N3 construct of epsinR is largely unstructured according to CD spectroscopy. Fig. S3 shows that epsinR is a monomer at all concentrations tested by analytical ultracentrifugation. Table SI supplies the detailed thermodynamic parameters measured by isothermal calorimetry for binding of the N3 construct of epsinR to various ligands. Original immunofluorescence images are presented as web pages that allow the reader to see individual channels and larger fields of cells. Additional antibody stains and mutants are also presented. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200208023/DC1.
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Footnotes |
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The online version of this article contains supplemental material.
* Abbreviations used in this paper: AP, adaptor protein complex; AP180, AP of 180 kD; CCV, clathrin-coated vesicle; EEA1, early endosome antigen 1; ENTH, epsin-NH2-terminal homology; GGA, Golgi-localized, gamma-earcontaining ADP-ribosylation factorbinding protein; ITC, isothermal titration calorimetry; OSBP, oxysterol binding protein; PtdInsP, phosphatidylinositol phosphate.
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Acknowledgments |
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I.G. Mills was supported by an MRC Postdoctoral Fellowship, G.J.K. Praefcke was supported by a Marie Curie Fellowship of the EU (MCFI-2000-01529) and B.J. Peter was supported by an EMBO Long Term Fellowship.
Submitted: 5 August 2002
Revised: 11 December 2002
Accepted: 12 December 2002
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