The adaptor appendage domains are believed to act as binding platforms for coated vesicle accessory proteins. Using glutathione S-transferase pulldowns from pig brain cytosol, we find three proteins that can bind to the appendage domains of both the AP-1 γ subunit and the GGAs: γ-synergin and two novel proteins, p56 and p200. p56 elicited better antibodies than p200 and was generally more tractable. Although p56 and γ-synergin bind to both GGA and γ appendages in vitro, immunofluorescence labeling of nocodazole-treated cells shows that p56 colocalizes with GGAs on TGN46-positive membranes, whereas γ-synergin colocalizes with AP-1 primarily on a different membrane compartment. Furthermore, in AP-1–deficient cells, p56 remains membrane-associated whereas γ-synergin becomes cytosolic. Thus, p56 and γ-synergin show very strong preferences for GGAs and AP-1, respectively, in vivo. However, the GGA and γ appendages share the same fold as determined by x-ray crystallography, and mutagenesis reveals that the same amino acids contribute to their binding sites. By overexpressing wild-type GGA and γ appendage domains in cells, we can drive p56 and γ-synergin, respectively, into the cytosol, suggesting a possible mechanism for selectively disrupting the two pathways.
展开▼
