γ-Synergin: An Eh Domain–Containing Protein That Interacts with γ-Adaptin

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 γ-adaptin subunit of the AP-1 complex as bait. Two novel, ubiquitously expressed proteins were found: p34, which interacts with both γ-adaptin and α-adaptin, and γ-synergin, an alternatively spliced protein with an apparent molecular mass of ∼110–190 kD, which only interacts with γ-adaptin. γ-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 γ-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the γ-adaptin binding site. In cells expressing α-adaptin with the γ-adaptin ear, a construct that goes mainly to the plasma membrane, much of the γ-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 γ-synergin links the AP-1 complex to another protein or proteins.