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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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EpsinR, a ubiquitous protein enriched in CCVs. (A) Purification of rat liver and brain CCVs. A stronger adaptor band is seen by Coomassie staining in the liver CCV preparation, although by electron microscopy, the liver CCVs are not as pure as the brain CCVs (not depicted). (B) Brain fractions were blotted for various CCV-enriched and -nonenriched proteins, and these were compared with epsin1 and epsinR in the same preparation. Blots for epsinR and the cation-independent M6P receptor are also shown for liver fractions. (C) Brain-specific distribution of epsin1 compared with the ubiquitous distribution of epsinR. Equal concentrations of rat tissue were loaded.
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fig3: EpsinR, a ubiquitous protein enriched in CCVs. (A) Purification of rat liver and brain CCVs. A stronger adaptor band is seen by Coomassie staining in the liver CCV preparation, although by electron microscopy, the liver CCVs are not as pure as the brain CCVs (not depicted). (B) Brain fractions were blotted for various CCV-enriched and -nonenriched proteins, and these were compared with epsin1 and epsinR in the same preparation. Blots for epsinR and the cation-independent M6P receptor are also shown for liver fractions. (C) Brain-specific distribution of epsin1 compared with the ubiquitous distribution of epsinR. Equal concentrations of rat tissue were loaded.

Mentions: EpsinR has a ubiquitous tissue distribution both on Northern blots and by Western blots (Fig. 3 C; refer to Materials and methods for antibody used). Epsin1 is brain-enriched and colocalizes with plasma membrane clathrin and AP2 adaptors in cells, but is not enriched in CCVs (Chen et al., 1998; Ford et al., 2002). In contrast, epsinR showed a good enrichment in both brain and liver CCVs (Fig. 3, A and B). There are numerous possible explanations for different enrichments of epsins in brain CCVs, one being the activity of the major brain lipid phosphatase, synaptojanin (McPherson et al., 1996). Synaptojanin activity in CCVs would result in the loss of the 5′ phosphate from PtdIns(4,5)P2, which would only lead to the release of epsin1 and the AP2 complex, while not having as profound an effect on epsinR and AP1. We also found endogenous and overexpressed epsinR enrichment in Golgi fractions using the Balch purification procedure (Balch et al., 1984) and a de-enrichment of the lipid-binding mutant epsinR-D34G (Fig. S1). On the basis of these combined results, we can conclude that epsinR is a widely distributed protein enriched in both CCVs and Golgi fractions.


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)

EpsinR, a ubiquitous protein enriched in CCVs. (A) Purification of rat liver and brain CCVs. A stronger adaptor band is seen by Coomassie staining in the liver CCV preparation, although by electron microscopy, the liver CCVs are not as pure as the brain CCVs (not depicted). (B) Brain fractions were blotted for various CCV-enriched and -nonenriched proteins, and these were compared with epsin1 and epsinR in the same preparation. Blots for epsinR and the cation-independent M6P receptor are also shown for liver fractions. (C) Brain-specific distribution of epsin1 compared with the ubiquitous distribution of epsinR. Equal concentrations of rat tissue were loaded.
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Related In: Results  -  Collection

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fig3: EpsinR, a ubiquitous protein enriched in CCVs. (A) Purification of rat liver and brain CCVs. A stronger adaptor band is seen by Coomassie staining in the liver CCV preparation, although by electron microscopy, the liver CCVs are not as pure as the brain CCVs (not depicted). (B) Brain fractions were blotted for various CCV-enriched and -nonenriched proteins, and these were compared with epsin1 and epsinR in the same preparation. Blots for epsinR and the cation-independent M6P receptor are also shown for liver fractions. (C) Brain-specific distribution of epsin1 compared with the ubiquitous distribution of epsinR. Equal concentrations of rat tissue were loaded.
Mentions: EpsinR has a ubiquitous tissue distribution both on Northern blots and by Western blots (Fig. 3 C; refer to Materials and methods for antibody used). Epsin1 is brain-enriched and colocalizes with plasma membrane clathrin and AP2 adaptors in cells, but is not enriched in CCVs (Chen et al., 1998; Ford et al., 2002). In contrast, epsinR showed a good enrichment in both brain and liver CCVs (Fig. 3, A and B). There are numerous possible explanations for different enrichments of epsins in brain CCVs, one being the activity of the major brain lipid phosphatase, synaptojanin (McPherson et al., 1996). Synaptojanin activity in CCVs would result in the loss of the 5′ phosphate from PtdIns(4,5)P2, which would only lead to the release of epsin1 and the AP2 complex, while not having as profound an effect on epsinR and AP1. We also found endogenous and overexpressed epsinR enrichment in Golgi fractions using the Balch purification procedure (Balch et al., 1984) and a de-enrichment of the lipid-binding mutant epsinR-D34G (Fig. S1). On the basis of these combined results, we can conclude that epsinR is a widely distributed protein enriched in both CCVs and Golgi fractions.

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