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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 γ-synergin at the nucleic acid level. (a) Northern blot. A rat multiple tissue Northern blot was probed with the insert from the clone isolated in the two-hybrid library screen. Different sized bands are labeled in different tissues, indicative of alternative splicing. (b) Diagram of the protein sequence of γ-synergin, showing the open reading frame, the three human ESTs used in the assembly of the full-length sequence, the partial genomic sequence (after splicing), the original clone isolated in the two-hybrid library screen, and the three clones isolated from a subsequent screen of a rat brain λgt10 cDNA library. The rat clones isolated differ from the human sequence near their NH2 termini in missing amino acids 197–274. Although there is no genomic sequence available for this region, this difference is likely to be due to alternative splicing. The 3′ end of rat clone 1 has sequence homologous to the human intron between exons 1 and 2 (see c) and is, therefore, either an incompletely spliced precursor or an alternatively spliced mRNA. Rat clone 3 uses the alternative splice site between exons 1 and 2 indicated in c with an additional four amino acids that could be the result of an additional exon or a difference between the rat and human sequence at splice junctions. (c) Diagram of the human γ-synergin gene. Exon and intron junctions are shown for the 3′ region where the genomic sequence is known. Hatched regions represent sequence derived solely from cDNA clones. The sizes of the predicted introns (in kb) are indicated. The two alternative polyadenylation sites were determined from human ESTs in the database. (d) Deduced protein sequence of human γ-synergin. Amino acid 1,123 is ambiguous because of a single base difference between the submitted genomic sequence and our own cDNA sequence, and is either an E in the former or a K in the latter. The identification of the putative initiator methionine is based on the observation that it is preceded by a Kozak sequence and on the finding that there are three short (<500 bp), internally primed γ-synergin sequences in the EST database, none of which extends any further in the upstream direction than EST 510825. The EH domain of the protein is highlighted. The rat and human sequences have 85% amino acid identity overall, and 98% identity in the EH domain. These sequence data are available from GenBank/EMBL/DDBJ under accession numbers AF169548 (human sequence) and AF169549 (rat sequence).
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Figure 2: Characterization of γ-synergin at the nucleic acid level. (a) Northern blot. A rat multiple tissue Northern blot was probed with the insert from the clone isolated in the two-hybrid library screen. Different sized bands are labeled in different tissues, indicative of alternative splicing. (b) Diagram of the protein sequence of γ-synergin, showing the open reading frame, the three human ESTs used in the assembly of the full-length sequence, the partial genomic sequence (after splicing), the original clone isolated in the two-hybrid library screen, and the three clones isolated from a subsequent screen of a rat brain λgt10 cDNA library. The rat clones isolated differ from the human sequence near their NH2 termini in missing amino acids 197–274. Although there is no genomic sequence available for this region, this difference is likely to be due to alternative splicing. The 3′ end of rat clone 1 has sequence homologous to the human intron between exons 1 and 2 (see c) and is, therefore, either an incompletely spliced precursor or an alternatively spliced mRNA. Rat clone 3 uses the alternative splice site between exons 1 and 2 indicated in c with an additional four amino acids that could be the result of an additional exon or a difference between the rat and human sequence at splice junctions. (c) Diagram of the human γ-synergin gene. Exon and intron junctions are shown for the 3′ region where the genomic sequence is known. Hatched regions represent sequence derived solely from cDNA clones. The sizes of the predicted introns (in kb) are indicated. The two alternative polyadenylation sites were determined from human ESTs in the database. (d) Deduced protein sequence of human γ-synergin. Amino acid 1,123 is ambiguous because of a single base difference between the submitted genomic sequence and our own cDNA sequence, and is either an E in the former or a K in the latter. The identification of the putative initiator methionine is based on the observation that it is preceded by a Kozak sequence and on the finding that there are three short (<500 bp), internally primed γ-synergin sequences in the EST database, none of which extends any further in the upstream direction than EST 510825. The EH domain of the protein is highlighted. The rat and human sequences have 85% amino acid identity overall, and 98% identity in the EH domain. These sequence data are available from GenBank/EMBL/DDBJ under accession numbers AF169548 (human sequence) and AF169549 (rat sequence).

Mentions: The other unknown cDNA came up with the least frequency in the library screen, accounting for only one of the clones. Northern blotting showed that the transcript is expressed ubiquitously and has a size of ∼4.4–5.6 kb, with bands of different mobilities labeled in different tissues (Fig. 2 a). The original clone contained an insert of only ∼1.6 kb and the sequence appeared to be all open reading frame, so this clone was used as a probe to screen a rat brain λgt10 cDNA library to try to obtain a full-length sequence. Three additional clones were isolated and sequenced (Fig. 2 b). A comparison of the four sequences revealed that the mRNA is alternatively spliced, consistent with the heterogeneity seen on the Northern blot. A putative start was identified in one of the clones, but none of the clones had an in-frame stop codon at the 3′ end. However, when the EST database was searched with the rat sequences, three human sequences were found, and from the corresponding cDNAs it was possible to assemble a contiguous human open reading frame. The nonredundant database was also searched with both the rat and the human sequences, and the human genomic sequence encoding the 3′ end of the mRNA was found. Fig. 2 c shows the genomic structure, including the alternative splice sites.


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 γ-synergin at the nucleic acid level. (a) Northern blot. A rat multiple tissue Northern blot was probed with the insert from the clone isolated in the two-hybrid library screen. Different sized bands are labeled in different tissues, indicative of alternative splicing. (b) Diagram of the protein sequence of γ-synergin, showing the open reading frame, the three human ESTs used in the assembly of the full-length sequence, the partial genomic sequence (after splicing), the original clone isolated in the two-hybrid library screen, and the three clones isolated from a subsequent screen of a rat brain λgt10 cDNA library. The rat clones isolated differ from the human sequence near their NH2 termini in missing amino acids 197–274. Although there is no genomic sequence available for this region, this difference is likely to be due to alternative splicing. The 3′ end of rat clone 1 has sequence homologous to the human intron between exons 1 and 2 (see c) and is, therefore, either an incompletely spliced precursor or an alternatively spliced mRNA. Rat clone 3 uses the alternative splice site between exons 1 and 2 indicated in c with an additional four amino acids that could be the result of an additional exon or a difference between the rat and human sequence at splice junctions. (c) Diagram of the human γ-synergin gene. Exon and intron junctions are shown for the 3′ region where the genomic sequence is known. Hatched regions represent sequence derived solely from cDNA clones. The sizes of the predicted introns (in kb) are indicated. The two alternative polyadenylation sites were determined from human ESTs in the database. (d) Deduced protein sequence of human γ-synergin. Amino acid 1,123 is ambiguous because of a single base difference between the submitted genomic sequence and our own cDNA sequence, and is either an E in the former or a K in the latter. The identification of the putative initiator methionine is based on the observation that it is preceded by a Kozak sequence and on the finding that there are three short (<500 bp), internally primed γ-synergin sequences in the EST database, none of which extends any further in the upstream direction than EST 510825. The EH domain of the protein is highlighted. The rat and human sequences have 85% amino acid identity overall, and 98% identity in the EH domain. These sequence data are available from GenBank/EMBL/DDBJ under accession numbers AF169548 (human sequence) and AF169549 (rat sequence).
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Related In: Results  -  Collection

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Figure 2: Characterization of γ-synergin at the nucleic acid level. (a) Northern blot. A rat multiple tissue Northern blot was probed with the insert from the clone isolated in the two-hybrid library screen. Different sized bands are labeled in different tissues, indicative of alternative splicing. (b) Diagram of the protein sequence of γ-synergin, showing the open reading frame, the three human ESTs used in the assembly of the full-length sequence, the partial genomic sequence (after splicing), the original clone isolated in the two-hybrid library screen, and the three clones isolated from a subsequent screen of a rat brain λgt10 cDNA library. The rat clones isolated differ from the human sequence near their NH2 termini in missing amino acids 197–274. Although there is no genomic sequence available for this region, this difference is likely to be due to alternative splicing. The 3′ end of rat clone 1 has sequence homologous to the human intron between exons 1 and 2 (see c) and is, therefore, either an incompletely spliced precursor or an alternatively spliced mRNA. Rat clone 3 uses the alternative splice site between exons 1 and 2 indicated in c with an additional four amino acids that could be the result of an additional exon or a difference between the rat and human sequence at splice junctions. (c) Diagram of the human γ-synergin gene. Exon and intron junctions are shown for the 3′ region where the genomic sequence is known. Hatched regions represent sequence derived solely from cDNA clones. The sizes of the predicted introns (in kb) are indicated. The two alternative polyadenylation sites were determined from human ESTs in the database. (d) Deduced protein sequence of human γ-synergin. Amino acid 1,123 is ambiguous because of a single base difference between the submitted genomic sequence and our own cDNA sequence, and is either an E in the former or a K in the latter. The identification of the putative initiator methionine is based on the observation that it is preceded by a Kozak sequence and on the finding that there are three short (<500 bp), internally primed γ-synergin sequences in the EST database, none of which extends any further in the upstream direction than EST 510825. The EH domain of the protein is highlighted. The rat and human sequences have 85% amino acid identity overall, and 98% identity in the EH domain. These sequence data are available from GenBank/EMBL/DDBJ under accession numbers AF169548 (human sequence) and AF169549 (rat sequence).
Mentions: The other unknown cDNA came up with the least frequency in the library screen, accounting for only one of the clones. Northern blotting showed that the transcript is expressed ubiquitously and has a size of ∼4.4–5.6 kb, with bands of different mobilities labeled in different tissues (Fig. 2 a). The original clone contained an insert of only ∼1.6 kb and the sequence appeared to be all open reading frame, so this clone was used as a probe to screen a rat brain λgt10 cDNA library to try to obtain a full-length sequence. Three additional clones were isolated and sequenced (Fig. 2 b). A comparison of the four sequences revealed that the mRNA is alternatively spliced, consistent with the heterogeneity seen on the Northern blot. A putative start was identified in one of the clones, but none of the clones had an in-frame stop codon at the 3′ end. However, when the EST database was searched with the rat sequences, three human sequences were found, and from the corresponding cDNAs it was possible to assemble a contiguous human open reading frame. The nonredundant database was also searched with both the rat and the human sequences, and the human genomic sequence encoding the 3′ end of the mRNA was found. Fig. 2 c shows the genomic structure, including the alternative splice sites.

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