Previously, we have proposed that epsin1 has a fundamental role in generating the curvature of the clathrin-coated pit (Ford et al., 2002
). Epsin1 binds to clathrin and AP2 adaptors and to plasma membrane PtdIns(4,5)P2
. By sequence homology, epsin2 and 3 are also likely to act in clathrin-mediated endocytosis from the plasma membrane. In this paper, we show that epsinR is involved in clathrin-mediated budding from internal compartments. Consistent with this function, it binds to clathrin and AP1 adaptors and to PtdIns(4)P, and its distribution in cells did not overlap with that of epsin1. In fact, most of the clathrin puncta in transfected COS cells are accounted for by a combination of epsin1 and epsinR puncta (see http://www.jcb.org/cgi/content/full/jcb.200208023/DC1
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 β- and/or γ-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 γ-appendage (Fig. S2). However, our results imply that limited folding gives rise to the high affinity interactions. First, separate point mutants of the two γ-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 γ-appendage binding motif, [D/E]FxD[F/W], present in epsinR, γ-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 γ-appendage (Kent et al., 2002
; Nogi et al., 2002
), and thus, the introduction of a negative charge into this patch (L762E) disrupted the interaction ( 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 ( B and Fig. S1), and showed the ENTH domain primarily determines the location of the protein in cells, just as for epsin1 (). 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 ( 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 ( B and yellow shading in 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 epsin–related protein has been shown to be required for Golgi–endosome traffic (Duncan et al., 2003