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EpsinR: an AP1/clathrin interacting protein involved in vesicle trafficking.

Mills IG, Praefcke GJ, Vallis Y, Peter BJ, Olesen LE, Gallop JL, Butler PJ, Evans PR, McMahon HT - J. Cell Biol. (2003)

Bottom Line: Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively.Thus, potentially, two AP1 complexes can bind to one epsinR.This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.

ABSTRACT
EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

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Clathrin and AP1 adaptors bind to epsinR. (A) Epsin1 and epsinR constructs were tested for binding to adaptors, clathrin, and AP180 in rat brain extract. Epsin1, Eps15 and AP180 all bind to α- and β-adaptins but only epsinR binds to γ-adaptin (asterisk). All constructs were GST-tagged, and +/− indicates the presence/absence of brain extract. (B) Truncations of N1 narrowed the major clathrin/adaptor-binding domain to 291–426 (N3), and further truncations abolish clathrin and then adaptor binding (Coomassie-stained gel). GGAs also bind to the N3 construct, and the first deletion affects the interaction (blot). Point mutagenesis shows that D422R primarily affects clathrin binding over adaptor binding while AP2 adaptors are affected by the D349 mutation. AP1 adaptors are affected by D349 and D371 mutations. GGA binding does not follow the same pattern as clathrin or the multimeric adaptors. (C) Full-length Myc-epsin1 and Myc-epsinR expressed in COS cells bind to the GST-appendage domains of α-, β-, and γ-adaptin, but not the L762E γ-adaptin appendage. (D) GST constructs of GGA1 and GGA2 appendage domains and the N3 construct of epsinR bind to full-length epsinR in brain extract. (E) Clathrin assembly is not promoted by the N3 domain of epsinR at neutral pH. Controls with β-adaptin appendage + hinge domain and full-length AP180 both show different extents of assembly. Clathrin in pellet (P) and supernatant (S) fractions after spinning are shown.
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fig4: Clathrin and AP1 adaptors bind to epsinR. (A) Epsin1 and epsinR constructs were tested for binding to adaptors, clathrin, and AP180 in rat brain extract. Epsin1, Eps15 and AP180 all bind to α- and β-adaptins but only epsinR binds to γ-adaptin (asterisk). All constructs were GST-tagged, and +/− indicates the presence/absence of brain extract. (B) Truncations of N1 narrowed the major clathrin/adaptor-binding domain to 291–426 (N3), and further truncations abolish clathrin and then adaptor binding (Coomassie-stained gel). GGAs also bind to the N3 construct, and the first deletion affects the interaction (blot). Point mutagenesis shows that D422R primarily affects clathrin binding over adaptor binding while AP2 adaptors are affected by the D349 mutation. AP1 adaptors are affected by D349 and D371 mutations. GGA binding does not follow the same pattern as clathrin or the multimeric adaptors. (C) Full-length Myc-epsin1 and Myc-epsinR expressed in COS cells bind to the GST-appendage domains of α-, β-, and γ-adaptin, but not the L762E γ-adaptin appendage. (D) GST constructs of GGA1 and GGA2 appendage domains and the N3 construct of epsinR bind to full-length epsinR in brain extract. (E) Clathrin assembly is not promoted by the N3 domain of epsinR at neutral pH. Controls with β-adaptin appendage + hinge domain and full-length AP180 both show different extents of assembly. Clathrin in pellet (P) and supernatant (S) fractions after spinning are shown.

Mentions: 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.


EpsinR: an AP1/clathrin interacting protein involved in vesicle trafficking.

Mills IG, Praefcke GJ, Vallis Y, Peter BJ, Olesen LE, Gallop JL, Butler PJ, Evans PR, McMahon HT - J. Cell Biol. (2003)

Clathrin and AP1 adaptors bind to epsinR. (A) Epsin1 and epsinR constructs were tested for binding to adaptors, clathrin, and AP180 in rat brain extract. Epsin1, Eps15 and AP180 all bind to α- and β-adaptins but only epsinR binds to γ-adaptin (asterisk). All constructs were GST-tagged, and +/− indicates the presence/absence of brain extract. (B) Truncations of N1 narrowed the major clathrin/adaptor-binding domain to 291–426 (N3), and further truncations abolish clathrin and then adaptor binding (Coomassie-stained gel). GGAs also bind to the N3 construct, and the first deletion affects the interaction (blot). Point mutagenesis shows that D422R primarily affects clathrin binding over adaptor binding while AP2 adaptors are affected by the D349 mutation. AP1 adaptors are affected by D349 and D371 mutations. GGA binding does not follow the same pattern as clathrin or the multimeric adaptors. (C) Full-length Myc-epsin1 and Myc-epsinR expressed in COS cells bind to the GST-appendage domains of α-, β-, and γ-adaptin, but not the L762E γ-adaptin appendage. (D) GST constructs of GGA1 and GGA2 appendage domains and the N3 construct of epsinR bind to full-length epsinR in brain extract. (E) Clathrin assembly is not promoted by the N3 domain of epsinR at neutral pH. Controls with β-adaptin appendage + hinge domain and full-length AP180 both show different extents of assembly. Clathrin in pellet (P) and supernatant (S) fractions after spinning are shown.
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Related In: Results  -  Collection

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fig4: Clathrin and AP1 adaptors bind to epsinR. (A) Epsin1 and epsinR constructs were tested for binding to adaptors, clathrin, and AP180 in rat brain extract. Epsin1, Eps15 and AP180 all bind to α- and β-adaptins but only epsinR binds to γ-adaptin (asterisk). All constructs were GST-tagged, and +/− indicates the presence/absence of brain extract. (B) Truncations of N1 narrowed the major clathrin/adaptor-binding domain to 291–426 (N3), and further truncations abolish clathrin and then adaptor binding (Coomassie-stained gel). GGAs also bind to the N3 construct, and the first deletion affects the interaction (blot). Point mutagenesis shows that D422R primarily affects clathrin binding over adaptor binding while AP2 adaptors are affected by the D349 mutation. AP1 adaptors are affected by D349 and D371 mutations. GGA binding does not follow the same pattern as clathrin or the multimeric adaptors. (C) Full-length Myc-epsin1 and Myc-epsinR expressed in COS cells bind to the GST-appendage domains of α-, β-, and γ-adaptin, but not the L762E γ-adaptin appendage. (D) GST constructs of GGA1 and GGA2 appendage domains and the N3 construct of epsinR bind to full-length epsinR in brain extract. (E) Clathrin assembly is not promoted by the N3 domain of epsinR at neutral pH. Controls with β-adaptin appendage + hinge domain and full-length AP180 both show different extents of assembly. Clathrin in pellet (P) and supernatant (S) fractions after spinning are shown.
Mentions: 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.

Bottom Line: Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively.Thus, potentially, two AP1 complexes can bind to one epsinR.This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.

ABSTRACT
EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.

Show MeSH
Related in: MedlinePlus