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Gamma-synergin: an EH domain-containing protein that interacts with gamma-adaptin.

Page LJ, Sowerby PJ, Lui WW, Robinson MS - J. Cell Biol. (1999)

Bottom Line: It binds directly to the ear domain of gamma-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the gamma-adaptin binding site.In cells expressing alpha-adaptin with the gamma-adaptin ear, a construct that goes mainly to the plasma membrane, much of the gamma-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there.The presence of an EH domain suggests that gamma-synergin links the AP-1 complex to another protein or proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry and Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, England.

ABSTRACT
The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in clathrin-coated vesicles. To identify other proteins that participate in the clathrin-coated vesicle cycle at the TGN, we have carried out a yeast two- hybrid library screen using the gamma-adaptin subunit of the AP-1 complex as bait. Two novel, ubiquitously expressed proteins were found: p34, which interacts with both gamma-adaptin and alpha-adaptin, and gamma-synergin, an alternatively spliced protein with an apparent molecular mass of approximately 110-190 kD, which only interacts with gamma-adaptin. gamma-Synergin is associated with AP-1 both in the cytosol and on TGN membranes, and it is strongly enriched in clathrin-coated vesicles. It binds directly to the ear domain of gamma-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the gamma-adaptin binding site. In cells expressing alpha-adaptin with the gamma-adaptin ear, a construct that goes mainly to the plasma membrane, much of the gamma-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there. The presence of an EH domain suggests that gamma-synergin links the AP-1 complex to another protein or proteins.

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γ-Synergin colocalizes with γ-adaptin by immunofluorescence. MDBK cells were double labeled with anti–γ-synergin (a, c, and e) and anti–γ-adaptin (b, d, and f) either under control conditions (a and b), after incubating with brefeldin A for 2 min (c and d), or after incubating with brefeldin A for 30 min and allowing the cells to recover for 2 min (e and f). Bar, 20 mm.
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Figure 5: γ-Synergin colocalizes with γ-adaptin by immunofluorescence. MDBK cells were double labeled with anti–γ-synergin (a, c, and e) and anti–γ-adaptin (b, d, and f) either under control conditions (a and b), after incubating with brefeldin A for 2 min (c and d), or after incubating with brefeldin A for 30 min and allowing the cells to recover for 2 min (e and f). Bar, 20 mm.

Mentions: The association between γ-synergin and γ-adaptin was confirmed by immunofluorescence microscopy. Double labeling of MDBK cells with anti–γ-synergin and anti–γ-adaptin revealed a striking degree of colocalization of the two proteins (Fig. 5, a and b). Next, we investigated whether the membrane association of g-synergin is affected by the drug brefeldin A (BFA). This drug causes ARF to dissociate rapidly from membranes (Donaldson et al. 1992; Helms and Rothman 1992), leading to the dissociation of other peripheral membrane proteins whose membrane association is ARF-dependent, such as the AP-1 adaptor complex (Robinson and Kreis 1992). In Fig. 5c and Fig. d, MDBK cells were treated with BFA for 2 min, and then double labeled for γ-synergin (c) and γ-adaptin (d). Both proteins can be seen to have redistributed to the cytoplasm. To examine the behavior of the two proteins upon BFA washout, we treated cells with the drug for 30 min, and then allowed them to recover for 2 min (Fig. 5e and Fig. f). Both proteins can be seen to have reassociated with the membrane, which now has a more tubular appearance as a result of the BFA treatment. Thus, γ-synergin, like the AP-1 adaptor complex, appears to associate with the TGN membrane in an ARF-dependent manner.


Gamma-synergin: an EH domain-containing protein that interacts with gamma-adaptin.

Page LJ, Sowerby PJ, Lui WW, Robinson MS - J. Cell Biol. (1999)

γ-Synergin colocalizes with γ-adaptin by immunofluorescence. MDBK cells were double labeled with anti–γ-synergin (a, c, and e) and anti–γ-adaptin (b, d, and f) either under control conditions (a and b), after incubating with brefeldin A for 2 min (c and d), or after incubating with brefeldin A for 30 min and allowing the cells to recover for 2 min (e and f). Bar, 20 mm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169493&req=5

Figure 5: γ-Synergin colocalizes with γ-adaptin by immunofluorescence. MDBK cells were double labeled with anti–γ-synergin (a, c, and e) and anti–γ-adaptin (b, d, and f) either under control conditions (a and b), after incubating with brefeldin A for 2 min (c and d), or after incubating with brefeldin A for 30 min and allowing the cells to recover for 2 min (e and f). Bar, 20 mm.
Mentions: The association between γ-synergin and γ-adaptin was confirmed by immunofluorescence microscopy. Double labeling of MDBK cells with anti–γ-synergin and anti–γ-adaptin revealed a striking degree of colocalization of the two proteins (Fig. 5, a and b). Next, we investigated whether the membrane association of g-synergin is affected by the drug brefeldin A (BFA). This drug causes ARF to dissociate rapidly from membranes (Donaldson et al. 1992; Helms and Rothman 1992), leading to the dissociation of other peripheral membrane proteins whose membrane association is ARF-dependent, such as the AP-1 adaptor complex (Robinson and Kreis 1992). In Fig. 5c and Fig. d, MDBK cells were treated with BFA for 2 min, and then double labeled for γ-synergin (c) and γ-adaptin (d). Both proteins can be seen to have redistributed to the cytoplasm. To examine the behavior of the two proteins upon BFA washout, we treated cells with the drug for 30 min, and then allowed them to recover for 2 min (Fig. 5e and Fig. f). Both proteins can be seen to have reassociated with the membrane, which now has a more tubular appearance as a result of the BFA treatment. Thus, γ-synergin, like the AP-1 adaptor complex, appears to associate with the TGN membrane in an ARF-dependent manner.

Bottom Line: It binds directly to the ear domain of gamma-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the gamma-adaptin binding site.In cells expressing alpha-adaptin with the gamma-adaptin ear, a construct that goes mainly to the plasma membrane, much of the gamma-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there.The presence of an EH domain suggests that gamma-synergin links the AP-1 complex to another protein or proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry and Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, England.

ABSTRACT
The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in clathrin-coated vesicles. To identify other proteins that participate in the clathrin-coated vesicle cycle at the TGN, we have carried out a yeast two- hybrid library screen using the gamma-adaptin subunit of the AP-1 complex as bait. Two novel, ubiquitously expressed proteins were found: p34, which interacts with both gamma-adaptin and alpha-adaptin, and gamma-synergin, an alternatively spliced protein with an apparent molecular mass of approximately 110-190 kD, which only interacts with gamma-adaptin. gamma-Synergin is associated with AP-1 both in the cytosol and on TGN membranes, and it is strongly enriched in clathrin-coated vesicles. It binds directly to the ear domain of gamma-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the gamma-adaptin binding site. In cells expressing alpha-adaptin with the gamma-adaptin ear, a construct that goes mainly to the plasma membrane, much of the gamma-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there. The presence of an EH domain suggests that gamma-synergin links the AP-1 complex to another protein or proteins.

Show MeSH
Related in: MedlinePlus