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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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Subcellular localization of epsinR. As a convention throughout the figure, myc-epsinR and mutants are labeled green and the endogenous proteins are labeled red. (A) Endogenous epsinR shows a perinuclear enrichment (Ai) with a very different distribution to Myc-epsin1 (green in Aii), but it colocalizes with overexpressed Myc-epsinR (green in Aiii). Colocalization is orange/yellow. We observed that the epsinR perinuclear compartment was frequently enlarged, and in extreme examples much of the overexpressed epsinR was accumulated there (Aiv). Endogenous epsinR is also accumulated in this compartment (Aiv). (B) Colocalization of Myc-epsinR with endogenous AP1 (ii and v), transferrin (iii and x), clathrin (vi), GGA (vii), cation-independent mannose-6-phosphate receptors (M6P, viii), and TGN46 (ix). Panels v–x are closer views of the perinuclear regions of cells stained for overexpressed epsinR and the indicated marker. There is much orange/yellow color in the perinuclear region of the cell implying a great deal of colocalization, but precise colocalization is hard to define in this region because of the accumulation of so many compartments. Transferrin (panel x) is labeled blue and colocalization with AP1 is cyan. (C and D) Colocalization of various markers with the D34G + R67L and the L10E mutants of epsinR, respectively. Labeling is the same as in B. Links to original images and extra data can be found at http://www.jcb.org/cgi/content/full/jcb.200208023/DC1.
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fig6: Subcellular localization of epsinR. As a convention throughout the figure, myc-epsinR and mutants are labeled green and the endogenous proteins are labeled red. (A) Endogenous epsinR shows a perinuclear enrichment (Ai) with a very different distribution to Myc-epsin1 (green in Aii), but it colocalizes with overexpressed Myc-epsinR (green in Aiii). Colocalization is orange/yellow. We observed that the epsinR perinuclear compartment was frequently enlarged, and in extreme examples much of the overexpressed epsinR was accumulated there (Aiv). Endogenous epsinR is also accumulated in this compartment (Aiv). (B) Colocalization of Myc-epsinR with endogenous AP1 (ii and v), transferrin (iii and x), clathrin (vi), GGA (vii), cation-independent mannose-6-phosphate receptors (M6P, viii), and TGN46 (ix). Panels v–x are closer views of the perinuclear regions of cells stained for overexpressed epsinR and the indicated marker. There is much orange/yellow color in the perinuclear region of the cell implying a great deal of colocalization, but precise colocalization is hard to define in this region because of the accumulation of so many compartments. Transferrin (panel x) is labeled blue and colocalization with AP1 is cyan. (C and D) Colocalization of various markers with the D34G + R67L and the L10E mutants of epsinR, respectively. Labeling is the same as in B. Links to original images and extra data can be found at http://www.jcb.org/cgi/content/full/jcb.200208023/DC1.

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)

Subcellular localization of epsinR. As a convention throughout the figure, myc-epsinR and mutants are labeled green and the endogenous proteins are labeled red. (A) Endogenous epsinR shows a perinuclear enrichment (Ai) with a very different distribution to Myc-epsin1 (green in Aii), but it colocalizes with overexpressed Myc-epsinR (green in Aiii). Colocalization is orange/yellow. We observed that the epsinR perinuclear compartment was frequently enlarged, and in extreme examples much of the overexpressed epsinR was accumulated there (Aiv). Endogenous epsinR is also accumulated in this compartment (Aiv). (B) Colocalization of Myc-epsinR with endogenous AP1 (ii and v), transferrin (iii and x), clathrin (vi), GGA (vii), cation-independent mannose-6-phosphate receptors (M6P, viii), and TGN46 (ix). Panels v–x are closer views of the perinuclear regions of cells stained for overexpressed epsinR and the indicated marker. There is much orange/yellow color in the perinuclear region of the cell implying a great deal of colocalization, but precise colocalization is hard to define in this region because of the accumulation of so many compartments. Transferrin (panel x) is labeled blue and colocalization with AP1 is cyan. (C and D) Colocalization of various markers with the D34G + R67L and the L10E mutants of epsinR, respectively. Labeling is the same as in B. Links to original images and extra data can be found at http://www.jcb.org/cgi/content/full/jcb.200208023/DC1.
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Related In: Results  -  Collection

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fig6: Subcellular localization of epsinR. As a convention throughout the figure, myc-epsinR and mutants are labeled green and the endogenous proteins are labeled red. (A) Endogenous epsinR shows a perinuclear enrichment (Ai) with a very different distribution to Myc-epsin1 (green in Aii), but it colocalizes with overexpressed Myc-epsinR (green in Aiii). Colocalization is orange/yellow. We observed that the epsinR perinuclear compartment was frequently enlarged, and in extreme examples much of the overexpressed epsinR was accumulated there (Aiv). Endogenous epsinR is also accumulated in this compartment (Aiv). (B) Colocalization of Myc-epsinR with endogenous AP1 (ii and v), transferrin (iii and x), clathrin (vi), GGA (vii), cation-independent mannose-6-phosphate receptors (M6P, viii), and TGN46 (ix). Panels v–x are closer views of the perinuclear regions of cells stained for overexpressed epsinR and the indicated marker. There is much orange/yellow color in the perinuclear region of the cell implying a great deal of colocalization, but precise colocalization is hard to define in this region because of the accumulation of so many compartments. Transferrin (panel x) is labeled blue and colocalization with AP1 is cyan. (C and D) Colocalization of various markers with the D34G + R67L and the L10E mutants of epsinR, respectively. Labeling is the same as in B. Links to original images and extra data can be found at http://www.jcb.org/cgi/content/full/jcb.200208023/DC1.
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