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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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Related in: MedlinePlus

SDS-PAGE and Western blot probed with anti–γ-synergin. The samples, from left to right, are: pig brain homogenate, rat liver homogenate, rat liver high speed supernatant (cytosol), rat liver high speed pellet, rat liver TGN-enriched fraction, and rat liver clathrin-coated vesicles. The blot shows that γ-synergin is a peripheral membrane protein that is highly enriched in clathrin-coated vesicles. The different patterns seen in brain and liver may be due to alternative splicing.
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Figure 4: SDS-PAGE and Western blot probed with anti–γ-synergin. The samples, from left to right, are: pig brain homogenate, rat liver homogenate, rat liver high speed supernatant (cytosol), rat liver high speed pellet, rat liver TGN-enriched fraction, and rat liver clathrin-coated vesicles. The blot shows that γ-synergin is a peripheral membrane protein that is highly enriched in clathrin-coated vesicles. The different patterns seen in brain and liver may be due to alternative splicing.

Mentions: To learn more about the function of γ-synergin, the original clone identified in the two-hybrid library screen was expressed as a fusion protein for antibody production. Fig. 4 shows a Western blot of equal protein loadings of homogenate from both brain and liver as well as various subcellular fractions from liver probed with the affinity-purified antibody. Two bands are labeled in the brain of ∼110 and ∼150 kD, whereas in the liver, a single band is labeled of ∼190 kD. This is consistent with the Northern blot (Fig. 2 a) in which a single band was labeled in liver, whereas two bands were labeled in brain, indicating that the different protein species might represent different spliced variants, although we cannot rule out the possibility that the differences might also be due to proteolysis. γ-Synergin is found in both a high speed supernatant and membrane-containing pellet, indicating that it is peripherally associated with membranes. It is somewhat enriched in a TGN-enriched fraction from liver and it is strongly enriched in liver clathrin-coated vesicles.


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

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

SDS-PAGE and Western blot probed with anti–γ-synergin. The samples, from left to right, are: pig brain homogenate, rat liver homogenate, rat liver high speed supernatant (cytosol), rat liver high speed pellet, rat liver TGN-enriched fraction, and rat liver clathrin-coated vesicles. The blot shows that γ-synergin is a peripheral membrane protein that is highly enriched in clathrin-coated vesicles. The different patterns seen in brain and liver may be due to alternative splicing.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: SDS-PAGE and Western blot probed with anti–γ-synergin. The samples, from left to right, are: pig brain homogenate, rat liver homogenate, rat liver high speed supernatant (cytosol), rat liver high speed pellet, rat liver TGN-enriched fraction, and rat liver clathrin-coated vesicles. The blot shows that γ-synergin is a peripheral membrane protein that is highly enriched in clathrin-coated vesicles. The different patterns seen in brain and liver may be due to alternative splicing.
Mentions: To learn more about the function of γ-synergin, the original clone identified in the two-hybrid library screen was expressed as a fusion protein for antibody production. Fig. 4 shows a Western blot of equal protein loadings of homogenate from both brain and liver as well as various subcellular fractions from liver probed with the affinity-purified antibody. Two bands are labeled in the brain of ∼110 and ∼150 kD, whereas in the liver, a single band is labeled of ∼190 kD. This is consistent with the Northern blot (Fig. 2 a) in which a single band was labeled in liver, whereas two bands were labeled in brain, indicating that the different protein species might represent different spliced variants, although we cannot rule out the possibility that the differences might also be due to proteolysis. γ-Synergin is found in both a high speed supernatant and membrane-containing pellet, indicating that it is peripherally associated with membranes. It is somewhat enriched in a TGN-enriched fraction from liver and it is strongly enriched in liver clathrin-coated vesicles.

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