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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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Characterization of p34. (a) Protein sequence deduced from the cDNA sequence. The identification of the initiator methionine is based on two observations: first, it is preceded by a Kozak sequence; and second, none of the clones, either from the original library screen or from a subsequent screen of a λgt10 cDNA library, extended any further in the 5′ direction. (b) Northern blot. A rat multiple tissue Northern blot was probed with the insert from one of the clones isolated in the two-hybrid library screen, which contains the complete coding sequence for p34. (c) p34 interacts with both γ and α in the two-hybrid system. The NH2-terminal domains of both γ-adaptin and α-adaptin were cloned into the two-hybrid bait vector pGBT9 and coexpressed with either the prey vector pGAD424 with no insert (control) or with the prey vector pGAD10 containing the coding sequence for p34. Colonies were assayed for the expression of β-galactosidase. Cells expressing p34 in pGAD10 alone produced no detectable β-galactosidase.
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Figure 1: Characterization of p34. (a) Protein sequence deduced from the cDNA sequence. The identification of the initiator methionine is based on two observations: first, it is preceded by a Kozak sequence; and second, none of the clones, either from the original library screen or from a subsequent screen of a λgt10 cDNA library, extended any further in the 5′ direction. (b) Northern blot. A rat multiple tissue Northern blot was probed with the insert from one of the clones isolated in the two-hybrid library screen, which contains the complete coding sequence for p34. (c) p34 interacts with both γ and α in the two-hybrid system. The NH2-terminal domains of both γ-adaptin and α-adaptin were cloned into the two-hybrid bait vector pGBT9 and coexpressed with either the prey vector pGAD424 with no insert (control) or with the prey vector pGAD10 containing the coding sequence for p34. Colonies were assayed for the expression of β-galactosidase. Cells expressing p34 in pGAD10 alone produced no detectable β-galactosidase.

Mentions: The first of the two unknown cDNAs was isolated with very high frequency in the two-hybrid screen, accounting for more than half of all the clones. A representative clone with an insert of ∼2.5 kb was sequenced and was found to encode a protein of 315 amino acids with a deduced size of ∼34 kD (p34) (Fig. 1 a). There are several mammalian ESTs in the database encoding p34, but no homologues were found that might help to establish the protein's function. Northern blotting demonstrated that p34 is expressed ubiquitously and that the mRNA has a size of ∼2.75 kb (Fig. 1 b). Unlike most of the other proteins identified in the screen, p34 was found to interact not only with γ-adaptin but also with α-adaptin in the two-hybrid system, and this interaction was mapped to the NH2-terminal domains of the two adaptins (Fig. 1 c). Attempts were made to raise antisera against p34, but unfortunately the protein proved to be a very poor antigen. Thus, although two different domains were expressed as fusion proteins for antibody production, and although the resulting antisera were affinity-purified, all of the antisera labeled multiple bands on Western blots. However, one of the antisera labeled a band of around the expected size (∼37 kD), and this protein could be immunoprecipitated in substoichiometric amounts with cytosolic AP-1 and AP-2, suggesting that the interactions detected in the two-hybrid system are physiologically relevant (data not shown). But because we were looking for proteins that interact specifically with the AP-1 complex, p34 was not characterized further.


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

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

Characterization of p34. (a) Protein sequence deduced from the cDNA sequence. The identification of the initiator methionine is based on two observations: first, it is preceded by a Kozak sequence; and second, none of the clones, either from the original library screen or from a subsequent screen of a λgt10 cDNA library, extended any further in the 5′ direction. (b) Northern blot. A rat multiple tissue Northern blot was probed with the insert from one of the clones isolated in the two-hybrid library screen, which contains the complete coding sequence for p34. (c) p34 interacts with both γ and α in the two-hybrid system. The NH2-terminal domains of both γ-adaptin and α-adaptin were cloned into the two-hybrid bait vector pGBT9 and coexpressed with either the prey vector pGAD424 with no insert (control) or with the prey vector pGAD10 containing the coding sequence for p34. Colonies were assayed for the expression of β-galactosidase. Cells expressing p34 in pGAD10 alone produced no detectable β-galactosidase.
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Related In: Results  -  Collection

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Figure 1: Characterization of p34. (a) Protein sequence deduced from the cDNA sequence. The identification of the initiator methionine is based on two observations: first, it is preceded by a Kozak sequence; and second, none of the clones, either from the original library screen or from a subsequent screen of a λgt10 cDNA library, extended any further in the 5′ direction. (b) Northern blot. A rat multiple tissue Northern blot was probed with the insert from one of the clones isolated in the two-hybrid library screen, which contains the complete coding sequence for p34. (c) p34 interacts with both γ and α in the two-hybrid system. The NH2-terminal domains of both γ-adaptin and α-adaptin were cloned into the two-hybrid bait vector pGBT9 and coexpressed with either the prey vector pGAD424 with no insert (control) or with the prey vector pGAD10 containing the coding sequence for p34. Colonies were assayed for the expression of β-galactosidase. Cells expressing p34 in pGAD10 alone produced no detectable β-galactosidase.
Mentions: The first of the two unknown cDNAs was isolated with very high frequency in the two-hybrid screen, accounting for more than half of all the clones. A representative clone with an insert of ∼2.5 kb was sequenced and was found to encode a protein of 315 amino acids with a deduced size of ∼34 kD (p34) (Fig. 1 a). There are several mammalian ESTs in the database encoding p34, but no homologues were found that might help to establish the protein's function. Northern blotting demonstrated that p34 is expressed ubiquitously and that the mRNA has a size of ∼2.75 kb (Fig. 1 b). Unlike most of the other proteins identified in the screen, p34 was found to interact not only with γ-adaptin but also with α-adaptin in the two-hybrid system, and this interaction was mapped to the NH2-terminal domains of the two adaptins (Fig. 1 c). Attempts were made to raise antisera against p34, but unfortunately the protein proved to be a very poor antigen. Thus, although two different domains were expressed as fusion proteins for antibody production, and although the resulting antisera were affinity-purified, all of the antisera labeled multiple bands on Western blots. However, one of the antisera labeled a band of around the expected size (∼37 kD), and this protein could be immunoprecipitated in substoichiometric amounts with cytosolic AP-1 and AP-2, suggesting that the interactions detected in the two-hybrid system are physiologically relevant (data not shown). But because we were looking for proteins that interact specifically with the AP-1 complex, p34 was not characterized further.

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