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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 is associated with AP-1 in the cytosol. Rat liver cytosol was immunoprecipitated with either anti–γ-adaptin or anti–α-adaptin, and Western blots were probed as indicated with either anti–γ-synergin, anti–γ-adaptin, or anti–α-adaptin. The lower molecular mass band (∼165 kD) labeled with the anti–γ-synergin antibody is probably a breakdown product of the protein.
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Figure 6: γ-Synergin is associated with AP-1 in the cytosol. Rat liver cytosol was immunoprecipitated with either anti–γ-adaptin or anti–α-adaptin, and Western blots were probed as indicated with either anti–γ-synergin, anti–γ-adaptin, or anti–α-adaptin. The lower molecular mass band (∼165 kD) labeled with the anti–γ-synergin antibody is probably a breakdown product of the protein.

Mentions: The immunofluorescence data demonstrate that γ-synergin is associated with AP-1 on TGN membranes. To find out whether the two proteins are also associated in the cytosol, immunoprecipitations were carried out under nondenaturing conditions (Fig. 6). Rat liver cytosol was immunoprecipitated with anti–γ-adaptin followed by protein A–Sepharose, and Western blots were probed with anti–γ-synergin or anti–γ-adaptin. As a control, cytosol was also immunoprecipitated with anti–a-adaptin. Cytosolic γ-synergin was found to coprecipitate with γ-adaptin but not with α-adaptin. Thus, γ-synergin is associated with AP-1 in the cytosol as well as on membranes.


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 is associated with AP-1 in the cytosol. Rat liver cytosol was immunoprecipitated with either anti–γ-adaptin or anti–α-adaptin, and Western blots were probed as indicated with either anti–γ-synergin, anti–γ-adaptin, or anti–α-adaptin. The lower molecular mass band (∼165 kD) labeled with the anti–γ-synergin antibody is probably a breakdown product of the protein.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169493&req=5

Figure 6: γ-Synergin is associated with AP-1 in the cytosol. Rat liver cytosol was immunoprecipitated with either anti–γ-adaptin or anti–α-adaptin, and Western blots were probed as indicated with either anti–γ-synergin, anti–γ-adaptin, or anti–α-adaptin. The lower molecular mass band (∼165 kD) labeled with the anti–γ-synergin antibody is probably a breakdown product of the protein.
Mentions: The immunofluorescence data demonstrate that γ-synergin is associated with AP-1 on TGN membranes. To find out whether the two proteins are also associated in the cytosol, immunoprecipitations were carried out under nondenaturing conditions (Fig. 6). Rat liver cytosol was immunoprecipitated with anti–γ-adaptin followed by protein A–Sepharose, and Western blots were probed with anti–γ-synergin or anti–γ-adaptin. As a control, cytosol was also immunoprecipitated with anti–a-adaptin. Cytosolic γ-synergin was found to coprecipitate with γ-adaptin but not with α-adaptin. Thus, γ-synergin is associated with AP-1 in the cytosol as well as on membranes.

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