CLATHRIN ASSEMBLY PROTEIN AP-3 IS PHOSPHORYLATED AND GLYCOSYLATED ON THE 50-KDA STRUCTURAL DOMAIN

AP-3 (AP180) in rat sympathetic neurons maintained in culture was anal yzed by pulse-chase labeling with [S-35]methionine to look for post-tr anslational modifications. At early times, two lower molecular weight precursors of the mature species were detected. By 10 min, all of the AP-3 was found in the mature form which is stable for at least 9 h. me show here that at least one of these processing events is due to the addition of O-linked N-acetylglucosamine (GlcNAc) which is present on the mature form of the protein. Wheat germ agglutinin, a GlcNAc-specif ic probe, bound to AP-3 and the binding was blocked by excess GlcNAc b ut not by excess mannose. Purified AP-3, and AP-3 in coated vesicles d erived from bovine brain. served as substrates for beta-D-galactosyltr ansferase which is specific for terminal GlcNAc residues. Analysis of the disaccharide released by beta-elimination indicated that single Gl cNAc residues are attached to AP-3 through an O-glycosidic linkage to threonine or serine residues. In vivo P-32-labeled AP-3, the result of serine phosphorylation (Keen J. H., and Black, M. M. (1986) J. Cell B iol. 102, 1325-1333), bound to wheat germ agglutinin-Sepharose indicat ing that phosphorylation and glycosylation can occur simultaneously on the same molecule. Both modifications have been mapped to the central 50-kDa structural domain that is responsible for the anomalous migrat ion of AP-3. Consistent with localization to the nonclathrin binding d omain, the O-GlcNAc modification does not play a discernible role in t he interaction of AP-3 with clathrin.