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Epsin: Inducing membrane curvature
The International Journal of Biochemistry & Cell Biology 39 (2007) 1765–1770
Molecules in focus
Epsin: Inducing membrane curvature Caroline A.J. Horvath a,1 , Davy Vanden Broeck b,∗,1 , Ga¨elle A.V. Boulet a , Johannes Bogers a , Marc J.S. De Wolf b a
UA-Laboratory of Cell Biology and Histology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium b UA-Laboratory of Human Biochemistry, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium Received 16 November 2006; received in revised form 8 December 2006; accepted 8 December 2006 Available online 17 January 2007
Abstract Epsin was originally discovered by virtue of its binding to another accessory protein, Eps15. Members of the epsin family play an important role as accessory proteins in clathrin-mediated endocytosis. Epsin isoforms have been described that differ in intracellular site of action and/or in tissue distribution, although all epsins essentially contribute to membrane deformation. Besides inducing membrane curvature, epsin also plays a key function as adaptor protein, coupling various components of the clathrin-assisted uptake and fulfils an important role in selecting and recognizing cargo. Furthermore, epsin possesses the ability to block vesicle formation during mitosis. To perform all these functions, epsin, apart from interacting with PtdIns(4,5)P2 via its ENTH domain, also engages in several protein interactions with different components of the clathrin-mediated endocytic system. Recently, RNA interference has successfully been exploited to generate a cell line constitutively silencing epsin expression, which can be used to study internalization of multiple ligands. © 2007 Elsevier Ltd. All rights reserved. Keywords: Epsin; ENTH; Membrane curvature; Clathrin-endocytosis
1. Introduction Epsin (epsin1) is one of the best characterized members of a family of accessory proteins involved in coated pit formation. The protein is inextricably bound up with the clathrin-mediated internalization pathway, where its primary function is to add bending stress to the lipid bilayer, hereby inducing membrane curvature, followed by vesicle formation (Hurley & Wendland, 2002). Epsin was initially discovered in 1998 by virtue of its binding to another auxiliary protein factor, Eps15, and ∗ Corresponding author. Tel.: +32 3 265 33 25; fax: +32 3 265 33 26. E-mail address: [email protected] (D. Vanden Broeck). 1 These authors contributed equally to this work.
1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2006.12.004
hence was named the Eps15 Interacting protein or epsin for short (Chen et al., 1998). Subsequently, epsin and its isoforms (epsin2a, epsin2b, epsin3) were shown to be expressed in all vertebrates, were they are found in many tissues and cells, particularly in actively secreting cells. Recently, another epsin-like protein was identified, variously named epsinR (for epsin-related protein) (Mills et al., 2003), enthoprotin (Wasiak et al., 2002) and Clint (Kalthoff, Groos, Kohl, Mahrhold, & Ungewickell, 2002). Evolutionary, epsin is a well conserved protein with many homologues in lower species. A Xenopus epsin was identified as a mitotic phosphoprotein in oocytes (MP90), while the epsin orthologue, liquid facets (LqF), is present in Drosophila. Two characterized epsin homologues, Ent1 and Ent2, are found in Saccharomyces cerevisiae (De Camilli et al., 2002).
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2. Structure The members of the epsin family contain several conserved domains, as shown in Fig. 1(A and B) for
interaction with a variety of binding partners. A critically important feature of epsin, at the primary structure level, is an evolutionary conserved domain of ∼150 amino acids located at the NH2 -terminus and therefore
Fig. 1. Structure of different isoforms of the epsin superfamily. Panel (A) Schematic representation of epsins 1–3 and epsinR proteins. These proteins share a similar modular organization, with a NH2 -terminal ENTH domain. Immediately carboxyl terminal to this domain several ubiquitin-interacting motifs (EEElqLqlAlamSkE) are situated, responsible for interaction with ubiquitinated proteins. The central region of epsin is characterized by the presence of multiple DPW motifs, essential for AP2 binding. These motifs are flanked by clathrin boxes (LLDLD/LIELE) involved in clathrin binding. The COOH-terminal part of these proteins comprises NPF repeats, required for interaction with EH domain bearing proteins, such as Eps15, POB1 and intersectin. In epsinR, additional AP/GGA binding motifs are present, with a role in AP1 recruitment. Moreover, epsinR contains a methonine-rich domain with a not yet clearly defined function. Remarkably, no UIM and NPF repeats are observed in epsinR. Panel (B) Sequence alignment of the ENTH domain of epsins 1–3 and epsinR. The NH2 -terminal region of epsin proteins is known to be highly conserved, concerning its constitutive role in inducing membrane curvature. Amino acid residues, equal in all human epsin isoforms, including epsinR, are depicted in green, with a similarity of ∼40%. In addition, epsins 1–3, termed classical epsins, were found to share ∼70% homology (shaded in grey), clearly discriminating epsinR as a more distantly related ENTH-bearing protein. Panel (C) Ribbon diagram of the ENTH domain of human epsin, bound ˚ The ENTH domain displays an all ␣-helical structure and is composed of 7 to PtdIns(4,5)P2 , as dissolved by X-ray diffraction (resolution: 1.70 A). ␣-helices (Swiss-Model repository, UniProt AC: Q9Y6I3).
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known as the Epsin N-Terminal Homology (ENTH) domain (Chen et al., 1998; Wendland, 2002). Adjacent to the ENTH domain several ubiquitin-interacting motifs (UIMs) are situated, responsible for ubiquitin recognition. The central part of epsin is characterized by the presence of multiple DPW (Asp-Pro-Trp) motifs, flanked by clathrin boxes. This region functions as binding site for various components of the endocytic machinery, including AP2 adaptor proteins and clathrin (Legendre-Guillemin, Wasiak, Hussain, Angers, & McPherson, 2004; Wendland, 2002). The COOH-terminal region of the protein comprises NPF (Asn-Pro-Phe) repeats required for binding EH domain bearing proteins, such as Eps15, POB1 and intersectin (De Camilli et al., 2002; Salcini, Chen, Iannolo, De Camilli, & Di Fiore, 1999). The ENTH domain has been proposed to represent a separately folded protein module displaying a super helix of seven ␣-helices (Fig. 1C) with a supplementary phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2 )induced ␣0 helix misaligned with the super helical axis (Chen et al., 1998). The most highly conserved amino acids fall roughly into two classes: (i) internal residues, implicated in packing and therefore necessary for structural integrity; (ii) solvent accessible residues most likely involved in protein-protein interactions (De Camilli et al., 2002). The module most related to the ENTH domain is the ANTH (AP180 N-Terminal Homology) domain, found at the NH2 -termini of AP180, CALM and HIP (Ford et al., 2002). A common feature among many E/ANTH domain-bearing proteins is that their COOH-termini contain peptide motifs, including clathrin and clathrin adaptor protein-binding elements, indicative for a functional role in clathrin-mediated budding (De Camilli et al., 2002). EpsinR is more distantly related to epsins 1–3 having no Eps15 binding motifs (NPF repeats) and therefore not a classical epsin (Fig. 1A and B). On the other hand, this protein possesses the conserved ENTH domain, but the lipid specificity is predicted to be different (Ford et al., 2002). Furthermore, epsinR does not have UIMs, while the clathrin/adaptor binding domains are conserved, although the motifs are different (Mills et al., 2003). Recent insights in sorting at the trans-Golgi network revealed a possible link between ubiquitin signals and epsinR. It has been shown that epsinR interacts with GGA proteins (GGA: Golgi-localized, Gammaear-containing, Arf-binding proteins), a class of proteins interacting with ubiquitin (Pelham, 2004), thus forming a potential connection between ubiquitinated cargo and epsinR.
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3. Expression and activation Depending on the cell type, different isoforms of epsin are expressed. Epsin1 is abundantly expressed throughout most cell types, but is found to be enriched in brain (Rosenthal et al., 1999). Similar expression and localization patterns are described for epsin2a, epsin2b and epsinR (Kalthoff et al., 2002; Rosenthal et al., 1999; Wasiak et al., 2002). In contrast, epsin3 is exclusively associated with keratinocytes of wounded epithelia (Spradling, McDaniel, Lohi, & Pilcher, 2001). The gene encoding epsin1, EPN1 (GenBank accession no. NM013333), comprises 11 exons and encodes a protein composed of 551 amino acids. This gene is located on chromosome 19q13 and to date little is known about its regulation or expression pattern. However, based on the observation that epsin is present in all cell types investigated and regarding its function in a constitutively operating system, a widespread and persistent expression without specific spatiotemporal regulation can be inferred. Subcellular localization studies of epsin point to a general cytoplasmic distribution with a pronounced accumulation in puncta on the plasma membrane. Epsin colocalizes with AP2, clathrin, Eps15, and dynamin, suggesting that the puncta may represent endocytically incompetent coated pits (Ford et al., 2002). By blocking the nucleocytosolic export pathway, epsin has also been proven to occur inside the nucleus, indicating that epsin may also play a role in a signalling pathway, linking the endocytic machinery to the regulation of nuclear function (Hyman, Chen, Di Fiore, De Camilli, & Brunger, 2000). To assure blocking of clathrin-mediated endocytosis during cell mitosis, epsin is subjected to serinethreonine phosphorylation, which can be reversed by a stimulus-induced dephosphorylation. Dephosphorylation promotes the interaction of epsin with AP2, hereby facilitating a burst of clathrin-mediated uptake. On the other hand, mitotic phosphorylation of epsin by Cdc2 (Cdk1) results in a reduced interaction of the protein with AP2 and as such presumably contributes to the arrest of clathrin-mediated endocytosis during mitosis (Chen, Slepnev, Di Fiore, & De Camilli, 1999). 4. Biological function Epsin and its isoforms participate in clathrinmediated endocytosis in mammalian cells. This biological function depends on their localization at endocytic sites and the presence of the ENTH domain and other motifs in the unstructured COOH-terminal part.
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Fig. 2. Schematic overview of epsin function in membrane bending. (A) Certain microdomains of the plasma membrane are enriched in PtdIns(4,5)P2 , hotspots for clathrin-mediated endocytosis. Binding of PtdIns(4,5)P2 to the ENTH domain of epsin is the first step in bending the membrane. Upon binding, unstructured residues at the NH2 -terminus form an amphipathic ␣ helix, called ␣0, misaligned with the super helical axis of the ENTH domain. (B) The ␣0 helix inserts in the inner leaflet of the plasma membrane, deforming the lipid structure, hereby relieving the bending stress. (C) Insertion of the ␣0 helix induces membrane curvature, providing a mechanism which facilitates this energy demanding process.
This region is involved in interactions with clathrin and other components of the endocytic machinery (Ritter & McPherson, 2006). Epsin plays an important role in driving membrane deformation (Fig. 2). The creation of membrane curvature has been considered the major function of the ENTH domain (Ford et al., 2002; Ritter & McPherson, 2006). This domain has a compact ␣helical structure and binds PtdIns(4,5)P2 , which is found to be enriched in certain patches of the plasma membrane. Binding of PtdIns(4,5)P2 to the ENTH domain induces the formation of the NH2 -terminal amphipathic ␣0-helix that inserts into the inner leaflet of the lipid
bilayer inducing membrane curvature, hereby providing a mechanism by which this energy-demanding process is facilitated (Ford et al., 2002; Kweon et al., 2006). While the ENTH domain induces the membrane curvature, the COOH-terminal region of epsin is essential for recruiting clathrin coat components, driving the clathrin-assembly and sorting of cargo (Legendre-Guillemin et al., 2004; Wendland, 2002). Whereas epsin plays a fundamental role in clathrinmediated endocytosis at the plasma membrane, epsinR is involved in clathrin-mediated budding from internal compartments, more specifically it functions in vesicular trafficking from the trans-Golgi network to the endosomes. The phospholipid interaction is mediated by the ENTH domain of epsinR, but unlike the ENTH domain of the classical epsins, the ENTH domain of epsinR has different lipid specificity (Ford et al., 2002; Mills et al., 2003). Besides its established function in curvature formation, the ENTH domain may also be involved in actin dynamics. Endocytic sites are composed of patches of actin interacting with a complex series of endocytic regulatory proteins. Several of the endocytic proteins contain independently folded protein modules, such as the epsin ENTH domain. A recent study by Aguilar et al. has demonstrated that ENTH domains use a surface area, distinct from the lipid binding pocket, to interact with GTPase activating proteins (GAPs) interacting with Cdc42, such as RalBP1/RLIP76. This interaction reveals an increased complexity of the ENTH module and provides a direct link between endocytosis and events downstream of Cdc42 activation including actin dynamics and cell polarity. In both yeast and mammalian cells, sites of polarized cell growth often coincide with regions of secretion and endocytosis. Therefore, by its ENTH domain, epsin may be a master regulator coupling membrane curvature during endocytosis and regulation of actin dynamics, both key cellular functions (Aguilar et al., 2006; Ritter & McPherson, 2006). Other domains of epsin are also involved in the endocytic process. Epsin fulfils an important role as adaptor protein in clathrin-mediated internalization. Via its UIM repeats, epsin is able to recognize ubiquitinated cargo, especially transmembrane proteins. In order to avoid proteasomal degradation of ubiquitin-labeled cargo, tight temporal intracellular regulation of (de)-ubiquitination of internalized cargo appears to be essential (Madshus, 2006). Several recent papers stress the importance of ubiquitination to secure high-avidity interactions with clathrin adaptor proteins containing UIM domains like epsin, subsequently initiating endocytosis of ubiquitin-
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tagged cargo from clathrin-coated pits (Barriere et al., 2006; Hawryluk et al., 2006). ENTH domain containing proteins engage additional protein-protein interactions. A study by Hyman et al. revealed the interaction of the ENTH domain with the promyelocytic leukemia zinc finger protein (PLZF), a transcription factor. The interaction of epsin with PLZF targets epsin to the nucleus. Therefore, epsin may function as a transcriptional regulator through its ENTH domain (Hyman et al., 2000; Legendre-Guillemin et al., 2004).
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cle. The authors would also like to apologise to a large number of colleagues whose important contributions have not been referenced or whose work has only been cited indirectly through reference to a review article. Caroline Horvath is supported by Institute for Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). Johannes Bogers is supported by the Fund for Scientific Research Flanders (FWO-Vlaanderen, G.0205.04) and the Belgian Cancer Foundation (Belgische Stichting tegen Kanker). Ga¨elle Boulet is supported by the Fund for Scientific Research Flanders.
5. Possible applications A major hurdle in identifying which particular endocytic pathway is utilized for the cellular uptake of a given ligand has been the development of an experimental strategy that could suppress one route exclusively. As approximately half of all known ligands are internalized through clathrin-coated pits, this internalization pathway is an attractive candidate to be specifically shut down. Recently, RNA interference has successfully been exploited to generate a cell line constitutively silencing epsin expression, displaying a firmly reduced uptake of clathrin-associated marker proteins such as the transferrin receptor and the epidermal growth factor receptor (Vanden Broeck & De Wolf, 2006). Studies in progress are exploiting these epsin-deficient cells to investigate the internalization route of a number of ligands including cholera toxin, human papillomavirus and several others. In general, this epsin-deficient cell line can also be a most promising tool for multiple industrial or scientific settings. 6. Concluding remarks The interest in epsin is growing fast. It is now well established that epsin is a key accessory protein assigned with a central role in clathrin-mediated endocytosis. Not only does epsin stimulate clathrin-assembly and may act as initial anchor protein onto which the clathrin cage can be assembled, but it also induces curvature formation through its ENTH domain. In addition, besides its function in recruiting and bridging vesicle coat components other than clathrin, epsin could well have a role not only in forming the container in which cargo is transported but also in the recognition and transport of the cargo itself. Acknowledgements We are grateful to Prof. Dr. Emeritus Albert Lagrou for helpful discussions and carefully reading the arti-
References Aguilar, R. C., Longhi, S. A., Shaw, J. D., Yeh, L. Y., Kim, S., Schon, A., et al. (2006). Epsin N-terminal homology domains perform an essential function regulating Cdc42 through binding Cdc42 GTPase-activating proteins. Proc. Natl. Acad. Sci., 11, 4116–4121. Barriere, H., Nemes, C., Lechardeur, D., Khan-Mohammad, M., Fruh, K., & Lukacs, G. L. (2006). Molecular basis of oligoubiquitindependent internalization of membrane proteins in mammalian cells. Traffic, 7, 282–297. Chen, H., Fre, S., Slepnev, V. I., Capua, M. R., Takei, K., Butler, M. H., et al. (1998). Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis. Nature, 394, 793–797. Chen, H., Slepnev, V. I., Di Fiore, P. P., & De Camilli, P. (1999). The interaction of epsin and Eps15 with the clathrin adaptor AP-2 is inhibited by mitotic phosphorylation and enhanced by stimulationdependent dephosphorylation in nerve terminals. J. Biol. Chem., 274, 3257–3260. De Camilli, P., Chen, H., Hyman, J., Panepucci, E., Bateman, A., & Brunger, A. T. (2002). The ENTH domain. FEBS Lett., 513, 11–18. Ford, M. G., Mills, I. G., Peter, B. J., Vallis, Y., Praefcke, G. J., Evans, P. R., et al. (2002). Curvature of clathrin-coated pits driven by epsin. Nature, 419, 361–366. Hawryluk, M. J., Keyel, P. A., Mishra, S. K., Watkins, S. C., Heuser, J. E., & Traub, L. M. (2006). Epsin 1 is a polyubiquitin-selective clathrin-associated sorting protein. Traffic, 7, 262–281. Hurley, J. H., & Wendland, B. (2002). Driving membranes around the bend. Cell, 111, 143–146. Hyman, J., Chen, H., Di Fiore, P. P., De Camilli, P., & Brunger, A. T. (2000). Epsin 1 undergoes nucleocytosolic shuttling and its Eps15 interactor NH2 -terminal homology (ENTH) domain, structurally similar to Armadillo and HEAT repeats, interacts with the transcription factor promyelocytic leukaemia Zn2+ -finger protein (PLFZ). J. Cell Biol., 149, 537–546. Kalthoff, C., Groos, S., Kohl, R., Mahrhold, S., & Ungewickell, E. J. (2002). Clint: A novel clathrin-binding ENTH-domain protein at the Golgi. Mol. Biol. Cell, 13, 4073–4660. Kweon, D. H., Shin, Y. K., Shin, J. Y., Lee, J. H., Lee, J. B., Seo, J. H., et al. (2006). Membrane topology of helix 0 of the Epsin N-terminal homology domain. Mol. Cells, 30, 428–435. Legendre-Guillemin, V., Wasiak, S., Hussain, N. K., Angers, A., & McPherson, P. S. (2004). ENTH/ANTH proteins and clathrinmediated membrane budding. J. Cell Sci., 117, 9–18. Madshus, I. H. (2006). Ubiquitin binding in endocytosis–How tight should it be and where does it happen? Traffic, 7, 258–261.
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Mills, I. G., Praefcke, G. J., Vallis, Y., Peter, B. J., Olesen, L. E., Gallop, J. L., et al. (2003). EpsinR: An AP1/clathrin interacting protein involved in vesicle trafficking. J. Cell Biol., 160, 213–222. Pelham, H. R. B. (2004). Membrane traffic: GGAs sort ubiquitin. Curr. Biol., 14, 357–359. Ritter, B., & McPherson, P. S. (2006). There’s a GAP in the ENTH domain. Proc. Natl. Acad. Sci., 103, 3953–3954. Rosenthal, J. A., Chen, H., Slepnev, V. I., Pellegrini, L., Salcini, A. E., Di Fiore, P. P., et al. (1999). The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein module. J. Biol. Chem., 274, 33959–33965. Salcini, A. E., Chen, H., Iannolo, G., De Camilli, P., & Di Fiore, P. P. (1999). Epidermal growth factor pathway substrate 15, Eps15. Int. J. Biochem. Cell Biol., 31, 805–809.
Spradling, K. D., McDaniel, A. E., Lohi, J., & Pilcher, B. K. (2001). Epsin 3 is a novel extracellular matrix-induced transcript specific to wounded epithelia. J. Biol. Chem., 276, 29257– 29267. Vanden Broeck, D., & De Wolf, M. J. S. (2006). Selective blocking of clathrin-mediated endocytosis by RNA interference: Epsin as a target protein. Biotechniques, 41, 475–484. Wasiak, S., Legendre-Guillemin, V., Puertollano, R., Blondeau, F., Girard, M., de Heuvel, E., et al. (2002). Enthoprotin: A novel clathrin-associated protein identified through subcellular proteomics. J. Cell Biol., 158, 855–862. Wendland, B. (2002). Epsins: Adaptors in endocytosis? Nat. Rev. Mol. Cell Biol., 3, 971–977.
Molecules in focus
Epsin: Inducing membrane curvature Caroline A.J. Horvath a,1 , Davy Vanden Broeck b,∗,1 , Ga¨elle A.V. Boulet a , Johannes Bogers a , Marc J.S. De Wolf b a
UA-Laboratory of Cell Biology and Histology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium b UA-Laboratory of Human Biochemistry, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium Received 16 November 2006; received in revised form 8 December 2006; accepted 8 December 2006 Available online 17 January 2007
Abstract Epsin was originally discovered by virtue of its binding to another accessory protein, Eps15. Members of the epsin family play an important role as accessory proteins in clathrin-mediated endocytosis. Epsin isoforms have been described that differ in intracellular site of action and/or in tissue distribution, although all epsins essentially contribute to membrane deformation. Besides inducing membrane curvature, epsin also plays a key function as adaptor protein, coupling various components of the clathrin-assisted uptake and fulfils an important role in selecting and recognizing cargo. Furthermore, epsin possesses the ability to block vesicle formation during mitosis. To perform all these functions, epsin, apart from interacting with PtdIns(4,5)P2 via its ENTH domain, also engages in several protein interactions with different components of the clathrin-mediated endocytic system. Recently, RNA interference has successfully been exploited to generate a cell line constitutively silencing epsin expression, which can be used to study internalization of multiple ligands. © 2007 Elsevier Ltd. All rights reserved. Keywords: Epsin; ENTH; Membrane curvature; Clathrin-endocytosis
1. Introduction Epsin (epsin1) is one of the best characterized members of a family of accessory proteins involved in coated pit formation. The protein is inextricably bound up with the clathrin-mediated internalization pathway, where its primary function is to add bending stress to the lipid bilayer, hereby inducing membrane curvature, followed by vesicle formation (Hurley & Wendland, 2002). Epsin was initially discovered in 1998 by virtue of its binding to another auxiliary protein factor, Eps15, and ∗ Corresponding author. Tel.: +32 3 265 33 25; fax: +32 3 265 33 26. E-mail address: [email protected] (D. Vanden Broeck). 1 These authors contributed equally to this work.
1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2006.12.004
hence was named the Eps15 Interacting protein or epsin for short (Chen et al., 1998). Subsequently, epsin and its isoforms (epsin2a, epsin2b, epsin3) were shown to be expressed in all vertebrates, were they are found in many tissues and cells, particularly in actively secreting cells. Recently, another epsin-like protein was identified, variously named epsinR (for epsin-related protein) (Mills et al., 2003), enthoprotin (Wasiak et al., 2002) and Clint (Kalthoff, Groos, Kohl, Mahrhold, & Ungewickell, 2002). Evolutionary, epsin is a well conserved protein with many homologues in lower species. A Xenopus epsin was identified as a mitotic phosphoprotein in oocytes (MP90), while the epsin orthologue, liquid facets (LqF), is present in Drosophila. Two characterized epsin homologues, Ent1 and Ent2, are found in Saccharomyces cerevisiae (De Camilli et al., 2002).
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2. Structure The members of the epsin family contain several conserved domains, as shown in Fig. 1(A and B) for
interaction with a variety of binding partners. A critically important feature of epsin, at the primary structure level, is an evolutionary conserved domain of ∼150 amino acids located at the NH2 -terminus and therefore
Fig. 1. Structure of different isoforms of the epsin superfamily. Panel (A) Schematic representation of epsins 1–3 and epsinR proteins. These proteins share a similar modular organization, with a NH2 -terminal ENTH domain. Immediately carboxyl terminal to this domain several ubiquitin-interacting motifs (EEElqLqlAlamSkE) are situated, responsible for interaction with ubiquitinated proteins. The central region of epsin is characterized by the presence of multiple DPW motifs, essential for AP2 binding. These motifs are flanked by clathrin boxes (LLDLD/LIELE) involved in clathrin binding. The COOH-terminal part of these proteins comprises NPF repeats, required for interaction with EH domain bearing proteins, such as Eps15, POB1 and intersectin. In epsinR, additional AP/GGA binding motifs are present, with a role in AP1 recruitment. Moreover, epsinR contains a methonine-rich domain with a not yet clearly defined function. Remarkably, no UIM and NPF repeats are observed in epsinR. Panel (B) Sequence alignment of the ENTH domain of epsins 1–3 and epsinR. The NH2 -terminal region of epsin proteins is known to be highly conserved, concerning its constitutive role in inducing membrane curvature. Amino acid residues, equal in all human epsin isoforms, including epsinR, are depicted in green, with a similarity of ∼40%. In addition, epsins 1–3, termed classical epsins, were found to share ∼70% homology (shaded in grey), clearly discriminating epsinR as a more distantly related ENTH-bearing protein. Panel (C) Ribbon diagram of the ENTH domain of human epsin, bound ˚ The ENTH domain displays an all ␣-helical structure and is composed of 7 to PtdIns(4,5)P2 , as dissolved by X-ray diffraction (resolution: 1.70 A). ␣-helices (Swiss-Model repository, UniProt AC: Q9Y6I3).
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known as the Epsin N-Terminal Homology (ENTH) domain (Chen et al., 1998; Wendland, 2002). Adjacent to the ENTH domain several ubiquitin-interacting motifs (UIMs) are situated, responsible for ubiquitin recognition. The central part of epsin is characterized by the presence of multiple DPW (Asp-Pro-Trp) motifs, flanked by clathrin boxes. This region functions as binding site for various components of the endocytic machinery, including AP2 adaptor proteins and clathrin (Legendre-Guillemin, Wasiak, Hussain, Angers, & McPherson, 2004; Wendland, 2002). The COOH-terminal region of the protein comprises NPF (Asn-Pro-Phe) repeats required for binding EH domain bearing proteins, such as Eps15, POB1 and intersectin (De Camilli et al., 2002; Salcini, Chen, Iannolo, De Camilli, & Di Fiore, 1999). The ENTH domain has been proposed to represent a separately folded protein module displaying a super helix of seven ␣-helices (Fig. 1C) with a supplementary phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2 )induced ␣0 helix misaligned with the super helical axis (Chen et al., 1998). The most highly conserved amino acids fall roughly into two classes: (i) internal residues, implicated in packing and therefore necessary for structural integrity; (ii) solvent accessible residues most likely involved in protein-protein interactions (De Camilli et al., 2002). The module most related to the ENTH domain is the ANTH (AP180 N-Terminal Homology) domain, found at the NH2 -termini of AP180, CALM and HIP (Ford et al., 2002). A common feature among many E/ANTH domain-bearing proteins is that their COOH-termini contain peptide motifs, including clathrin and clathrin adaptor protein-binding elements, indicative for a functional role in clathrin-mediated budding (De Camilli et al., 2002). EpsinR is more distantly related to epsins 1–3 having no Eps15 binding motifs (NPF repeats) and therefore not a classical epsin (Fig. 1A and B). On the other hand, this protein possesses the conserved ENTH domain, but the lipid specificity is predicted to be different (Ford et al., 2002). Furthermore, epsinR does not have UIMs, while the clathrin/adaptor binding domains are conserved, although the motifs are different (Mills et al., 2003). Recent insights in sorting at the trans-Golgi network revealed a possible link between ubiquitin signals and epsinR. It has been shown that epsinR interacts with GGA proteins (GGA: Golgi-localized, Gammaear-containing, Arf-binding proteins), a class of proteins interacting with ubiquitin (Pelham, 2004), thus forming a potential connection between ubiquitinated cargo and epsinR.
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3. Expression and activation Depending on the cell type, different isoforms of epsin are expressed. Epsin1 is abundantly expressed throughout most cell types, but is found to be enriched in brain (Rosenthal et al., 1999). Similar expression and localization patterns are described for epsin2a, epsin2b and epsinR (Kalthoff et al., 2002; Rosenthal et al., 1999; Wasiak et al., 2002). In contrast, epsin3 is exclusively associated with keratinocytes of wounded epithelia (Spradling, McDaniel, Lohi, & Pilcher, 2001). The gene encoding epsin1, EPN1 (GenBank accession no. NM013333), comprises 11 exons and encodes a protein composed of 551 amino acids. This gene is located on chromosome 19q13 and to date little is known about its regulation or expression pattern. However, based on the observation that epsin is present in all cell types investigated and regarding its function in a constitutively operating system, a widespread and persistent expression without specific spatiotemporal regulation can be inferred. Subcellular localization studies of epsin point to a general cytoplasmic distribution with a pronounced accumulation in puncta on the plasma membrane. Epsin colocalizes with AP2, clathrin, Eps15, and dynamin, suggesting that the puncta may represent endocytically incompetent coated pits (Ford et al., 2002). By blocking the nucleocytosolic export pathway, epsin has also been proven to occur inside the nucleus, indicating that epsin may also play a role in a signalling pathway, linking the endocytic machinery to the regulation of nuclear function (Hyman, Chen, Di Fiore, De Camilli, & Brunger, 2000). To assure blocking of clathrin-mediated endocytosis during cell mitosis, epsin is subjected to serinethreonine phosphorylation, which can be reversed by a stimulus-induced dephosphorylation. Dephosphorylation promotes the interaction of epsin with AP2, hereby facilitating a burst of clathrin-mediated uptake. On the other hand, mitotic phosphorylation of epsin by Cdc2 (Cdk1) results in a reduced interaction of the protein with AP2 and as such presumably contributes to the arrest of clathrin-mediated endocytosis during mitosis (Chen, Slepnev, Di Fiore, & De Camilli, 1999). 4. Biological function Epsin and its isoforms participate in clathrinmediated endocytosis in mammalian cells. This biological function depends on their localization at endocytic sites and the presence of the ENTH domain and other motifs in the unstructured COOH-terminal part.
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Fig. 2. Schematic overview of epsin function in membrane bending. (A) Certain microdomains of the plasma membrane are enriched in PtdIns(4,5)P2 , hotspots for clathrin-mediated endocytosis. Binding of PtdIns(4,5)P2 to the ENTH domain of epsin is the first step in bending the membrane. Upon binding, unstructured residues at the NH2 -terminus form an amphipathic ␣ helix, called ␣0, misaligned with the super helical axis of the ENTH domain. (B) The ␣0 helix inserts in the inner leaflet of the plasma membrane, deforming the lipid structure, hereby relieving the bending stress. (C) Insertion of the ␣0 helix induces membrane curvature, providing a mechanism which facilitates this energy demanding process.
This region is involved in interactions with clathrin and other components of the endocytic machinery (Ritter & McPherson, 2006). Epsin plays an important role in driving membrane deformation (Fig. 2). The creation of membrane curvature has been considered the major function of the ENTH domain (Ford et al., 2002; Ritter & McPherson, 2006). This domain has a compact ␣helical structure and binds PtdIns(4,5)P2 , which is found to be enriched in certain patches of the plasma membrane. Binding of PtdIns(4,5)P2 to the ENTH domain induces the formation of the NH2 -terminal amphipathic ␣0-helix that inserts into the inner leaflet of the lipid
bilayer inducing membrane curvature, hereby providing a mechanism by which this energy-demanding process is facilitated (Ford et al., 2002; Kweon et al., 2006). While the ENTH domain induces the membrane curvature, the COOH-terminal region of epsin is essential for recruiting clathrin coat components, driving the clathrin-assembly and sorting of cargo (Legendre-Guillemin et al., 2004; Wendland, 2002). Whereas epsin plays a fundamental role in clathrinmediated endocytosis at the plasma membrane, epsinR is involved in clathrin-mediated budding from internal compartments, more specifically it functions in vesicular trafficking from the trans-Golgi network to the endosomes. The phospholipid interaction is mediated by the ENTH domain of epsinR, but unlike the ENTH domain of the classical epsins, the ENTH domain of epsinR has different lipid specificity (Ford et al., 2002; Mills et al., 2003). Besides its established function in curvature formation, the ENTH domain may also be involved in actin dynamics. Endocytic sites are composed of patches of actin interacting with a complex series of endocytic regulatory proteins. Several of the endocytic proteins contain independently folded protein modules, such as the epsin ENTH domain. A recent study by Aguilar et al. has demonstrated that ENTH domains use a surface area, distinct from the lipid binding pocket, to interact with GTPase activating proteins (GAPs) interacting with Cdc42, such as RalBP1/RLIP76. This interaction reveals an increased complexity of the ENTH module and provides a direct link between endocytosis and events downstream of Cdc42 activation including actin dynamics and cell polarity. In both yeast and mammalian cells, sites of polarized cell growth often coincide with regions of secretion and endocytosis. Therefore, by its ENTH domain, epsin may be a master regulator coupling membrane curvature during endocytosis and regulation of actin dynamics, both key cellular functions (Aguilar et al., 2006; Ritter & McPherson, 2006). Other domains of epsin are also involved in the endocytic process. Epsin fulfils an important role as adaptor protein in clathrin-mediated internalization. Via its UIM repeats, epsin is able to recognize ubiquitinated cargo, especially transmembrane proteins. In order to avoid proteasomal degradation of ubiquitin-labeled cargo, tight temporal intracellular regulation of (de)-ubiquitination of internalized cargo appears to be essential (Madshus, 2006). Several recent papers stress the importance of ubiquitination to secure high-avidity interactions with clathrin adaptor proteins containing UIM domains like epsin, subsequently initiating endocytosis of ubiquitin-
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tagged cargo from clathrin-coated pits (Barriere et al., 2006; Hawryluk et al., 2006). ENTH domain containing proteins engage additional protein-protein interactions. A study by Hyman et al. revealed the interaction of the ENTH domain with the promyelocytic leukemia zinc finger protein (PLZF), a transcription factor. The interaction of epsin with PLZF targets epsin to the nucleus. Therefore, epsin may function as a transcriptional regulator through its ENTH domain (Hyman et al., 2000; Legendre-Guillemin et al., 2004).
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cle. The authors would also like to apologise to a large number of colleagues whose important contributions have not been referenced or whose work has only been cited indirectly through reference to a review article. Caroline Horvath is supported by Institute for Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). Johannes Bogers is supported by the Fund for Scientific Research Flanders (FWO-Vlaanderen, G.0205.04) and the Belgian Cancer Foundation (Belgische Stichting tegen Kanker). Ga¨elle Boulet is supported by the Fund for Scientific Research Flanders.
5. Possible applications A major hurdle in identifying which particular endocytic pathway is utilized for the cellular uptake of a given ligand has been the development of an experimental strategy that could suppress one route exclusively. As approximately half of all known ligands are internalized through clathrin-coated pits, this internalization pathway is an attractive candidate to be specifically shut down. Recently, RNA interference has successfully been exploited to generate a cell line constitutively silencing epsin expression, displaying a firmly reduced uptake of clathrin-associated marker proteins such as the transferrin receptor and the epidermal growth factor receptor (Vanden Broeck & De Wolf, 2006). Studies in progress are exploiting these epsin-deficient cells to investigate the internalization route of a number of ligands including cholera toxin, human papillomavirus and several others. In general, this epsin-deficient cell line can also be a most promising tool for multiple industrial or scientific settings. 6. Concluding remarks The interest in epsin is growing fast. It is now well established that epsin is a key accessory protein assigned with a central role in clathrin-mediated endocytosis. Not only does epsin stimulate clathrin-assembly and may act as initial anchor protein onto which the clathrin cage can be assembled, but it also induces curvature formation through its ENTH domain. In addition, besides its function in recruiting and bridging vesicle coat components other than clathrin, epsin could well have a role not only in forming the container in which cargo is transported but also in the recognition and transport of the cargo itself. Acknowledgements We are grateful to Prof. Dr. Emeritus Albert Lagrou for helpful discussions and carefully reading the arti-
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