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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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Identification of binding sites on g-synergin and γ-adaptin. (a, left to right) Rat liver cytosol was incubated with GST alone, with a GST fusion protein containing the α-adaptin ear domain, or with a GST fusion protein containing the γ-adaptin ear domain, followed by glutathione-Sepharose. The Western blot was probed with anti–γ-synergin. The γ-synergin can be seen to bind specifically to the ear domain of γ-adaptin. (b, left to right) Rat liver cytosol was incubated with GST alone, with a GST fusion protein containing the EH domain of γ-synergin, with a GST fusion protein containing amino acids 168–517 of γ-synergin (but missing amino acids 197–274, presumably because of alternative splicing) (GST-γs1), with a GST fusion protein containing amino acids 385–661 of g-synergin (GST-γs2), or with a GST fusion protein containing amino acids 518–786 of γ-synergin (GST-γs3), followed by glutathione-Sepharose. The Western blot was probed with anti–γ-adaptin. Only the GST-γs3 construct pulls down γ-adaptin. (c) Two histidine-tagged constructs, one containing the same domain of γ-synergin as the fusion protein γs3 (His-γs3) and a control construct, histidine-tagged DHFR (His-control), were subjected to SDS-PAGE followed by Western blotting. The blots were probed with either GST followed by anti-GST, GST coupled to the γ-adaptin ear domain (GST-gear) followed by anti-GST, or anti–γ-synergin. A band of ∼45 kD, corresponding to His-γs3, can be seen to be labeled in the GST-γear overlay.
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Figure 7: Identification of binding sites on g-synergin and γ-adaptin. (a, left to right) Rat liver cytosol was incubated with GST alone, with a GST fusion protein containing the α-adaptin ear domain, or with a GST fusion protein containing the γ-adaptin ear domain, followed by glutathione-Sepharose. The Western blot was probed with anti–γ-synergin. The γ-synergin can be seen to bind specifically to the ear domain of γ-adaptin. (b, left to right) Rat liver cytosol was incubated with GST alone, with a GST fusion protein containing the EH domain of γ-synergin, with a GST fusion protein containing amino acids 168–517 of γ-synergin (but missing amino acids 197–274, presumably because of alternative splicing) (GST-γs1), with a GST fusion protein containing amino acids 385–661 of g-synergin (GST-γs2), or with a GST fusion protein containing amino acids 518–786 of γ-synergin (GST-γs3), followed by glutathione-Sepharose. The Western blot was probed with anti–γ-adaptin. Only the GST-γs3 construct pulls down γ-adaptin. (c) Two histidine-tagged constructs, one containing the same domain of γ-synergin as the fusion protein γs3 (His-γs3) and a control construct, histidine-tagged DHFR (His-control), were subjected to SDS-PAGE followed by Western blotting. The blots were probed with either GST followed by anti-GST, GST coupled to the γ-adaptin ear domain (GST-gear) followed by anti-GST, or anti–γ-synergin. A band of ∼45 kD, corresponding to His-γs3, can be seen to be labeled in the GST-γear overlay.

Mentions: To identify the domain on γ-adaptin that binds to γ-synergin and the domain on γ-synergin that binds to γ-adaptin, two approaches were used: yeast two-hybrid analysis and GST pulldown experiments. The NH2-terminal domain construct of γ-adaptin, which was found to interact with p34 in the two-hybrid system (Fig. 1 c), did not interact with γ-synergin nor did a γ-adaptin construct with the a-adaptin ear, indicating that g-synergin binds to the ear domain of γ-adaptin (data not shown). This was confirmed using GST fusion proteins to isolate binding partners in rat liver cytosol, followed by Western blotting and probing with anti–γ-synergin. Fig. 7 a shows that GST fused to the γ-adaptin ear binds γ-synergin, whereas GST alone or GST fused to the α-adaptin ear do not.


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

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

Identification of binding sites on g-synergin and γ-adaptin. (a, left to right) Rat liver cytosol was incubated with GST alone, with a GST fusion protein containing the α-adaptin ear domain, or with a GST fusion protein containing the γ-adaptin ear domain, followed by glutathione-Sepharose. The Western blot was probed with anti–γ-synergin. The γ-synergin can be seen to bind specifically to the ear domain of γ-adaptin. (b, left to right) Rat liver cytosol was incubated with GST alone, with a GST fusion protein containing the EH domain of γ-synergin, with a GST fusion protein containing amino acids 168–517 of γ-synergin (but missing amino acids 197–274, presumably because of alternative splicing) (GST-γs1), with a GST fusion protein containing amino acids 385–661 of g-synergin (GST-γs2), or with a GST fusion protein containing amino acids 518–786 of γ-synergin (GST-γs3), followed by glutathione-Sepharose. The Western blot was probed with anti–γ-adaptin. Only the GST-γs3 construct pulls down γ-adaptin. (c) Two histidine-tagged constructs, one containing the same domain of γ-synergin as the fusion protein γs3 (His-γs3) and a control construct, histidine-tagged DHFR (His-control), were subjected to SDS-PAGE followed by Western blotting. The blots were probed with either GST followed by anti-GST, GST coupled to the γ-adaptin ear domain (GST-gear) followed by anti-GST, or anti–γ-synergin. A band of ∼45 kD, corresponding to His-γs3, can be seen to be labeled in the GST-γear overlay.
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Related In: Results  -  Collection

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Figure 7: Identification of binding sites on g-synergin and γ-adaptin. (a, left to right) Rat liver cytosol was incubated with GST alone, with a GST fusion protein containing the α-adaptin ear domain, or with a GST fusion protein containing the γ-adaptin ear domain, followed by glutathione-Sepharose. The Western blot was probed with anti–γ-synergin. The γ-synergin can be seen to bind specifically to the ear domain of γ-adaptin. (b, left to right) Rat liver cytosol was incubated with GST alone, with a GST fusion protein containing the EH domain of γ-synergin, with a GST fusion protein containing amino acids 168–517 of γ-synergin (but missing amino acids 197–274, presumably because of alternative splicing) (GST-γs1), with a GST fusion protein containing amino acids 385–661 of g-synergin (GST-γs2), or with a GST fusion protein containing amino acids 518–786 of γ-synergin (GST-γs3), followed by glutathione-Sepharose. The Western blot was probed with anti–γ-adaptin. Only the GST-γs3 construct pulls down γ-adaptin. (c) Two histidine-tagged constructs, one containing the same domain of γ-synergin as the fusion protein γs3 (His-γs3) and a control construct, histidine-tagged DHFR (His-control), were subjected to SDS-PAGE followed by Western blotting. The blots were probed with either GST followed by anti-GST, GST coupled to the γ-adaptin ear domain (GST-gear) followed by anti-GST, or anti–γ-synergin. A band of ∼45 kD, corresponding to His-γs3, can be seen to be labeled in the GST-γear overlay.
Mentions: To identify the domain on γ-adaptin that binds to γ-synergin and the domain on γ-synergin that binds to γ-adaptin, two approaches were used: yeast two-hybrid analysis and GST pulldown experiments. The NH2-terminal domain construct of γ-adaptin, which was found to interact with p34 in the two-hybrid system (Fig. 1 c), did not interact with γ-synergin nor did a γ-adaptin construct with the a-adaptin ear, indicating that g-synergin binds to the ear domain of γ-adaptin (data not shown). This was confirmed using GST fusion proteins to isolate binding partners in rat liver cytosol, followed by Western blotting and probing with anti–γ-synergin. Fig. 7 a shows that GST fused to the γ-adaptin ear binds γ-synergin, whereas GST alone or GST fused to the α-adaptin ear do not.

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