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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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Disruption of CCV trafficking from the TGN. (A) Overexpression of Myc-epsinR caused mistargeting of pro-cathepsin D. COS cells, either untransfected (UTC) or transfected with WT Myc-epsinR or Myc γ-appendage domain were pulse-chased with 35S, and both-cell associated (C) and -secreted (S) cathepsin D were immunoprecipitated and gels were phosphorimaged. The secreted forms are indicated by the asterisks. (B) Reduced incorporation of the cation-independent M6P receptor into purified CCVs in epsinR transfected COS cells.
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fig7: Disruption of CCV trafficking from the TGN. (A) Overexpression of Myc-epsinR caused mistargeting of pro-cathepsin D. COS cells, either untransfected (UTC) or transfected with WT Myc-epsinR or Myc γ-appendage domain were pulse-chased with 35S, and both-cell associated (C) and -secreted (S) cathepsin D were immunoprecipitated and gels were phosphorimaged. The secreted forms are indicated by the asterisks. (B) Reduced incorporation of the cation-independent M6P receptor into purified CCVs in epsinR transfected COS cells.

Mentions: 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.


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)

Disruption of CCV trafficking from the TGN. (A) Overexpression of Myc-epsinR caused mistargeting of pro-cathepsin D. COS cells, either untransfected (UTC) or transfected with WT Myc-epsinR or Myc γ-appendage domain were pulse-chased with 35S, and both-cell associated (C) and -secreted (S) cathepsin D were immunoprecipitated and gels were phosphorimaged. The secreted forms are indicated by the asterisks. (B) Reduced incorporation of the cation-independent M6P receptor into purified CCVs in epsinR transfected COS cells.
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fig7: Disruption of CCV trafficking from the TGN. (A) Overexpression of Myc-epsinR caused mistargeting of pro-cathepsin D. COS cells, either untransfected (UTC) or transfected with WT Myc-epsinR or Myc γ-appendage domain were pulse-chased with 35S, and both-cell associated (C) and -secreted (S) cathepsin D were immunoprecipitated and gels were phosphorimaged. The secreted forms are indicated by the asterisks. (B) Reduced incorporation of the cation-independent M6P receptor into purified CCVs in epsinR transfected COS cells.
Mentions: 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.

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