Clathrin assembly protein AP-3 is phosphorylated and glycosylated on the 50-kDa structural domain

• Published in: The Journal of biological chemistry 1994-08, Vol.269 (33), p.21346-21352
• Main Authors: MURPHY, J.-E, HANOVER, J. A, FROEHLICH, M, DUBOIS, G, KEEN, J. H
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - metabolism; Adaptor Proteins, Vesicular Transport; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Cattle; Cells, Cultured; Clathrin - metabolism; Fundamental and applied biological sciences. Psychology; Galactose - metabolism; Glycosylation; Miscellaneous; Molecular and cellular biology; Molecular genetics; Monomeric Clathrin Assembly Proteins; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - metabolism; Phosphoproteins - chemistry; Phosphoproteins - metabolism; Phosphorylation; Protein Conformation; Protein Processing, Post-Translational; Proteins; Rats; Translation. Translation factors. Protein processing
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)31968-3

AP-3 (AP180) in rat sympathetic neurons maintained in culture was analyzed by pulse-chase labeling with [35S]methionine to look for post-translational 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. We 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-specific probe, bound to AP-3 and the binding was blocked by excess GlcNAc but not by excess mannose. Purified AP-3, and AP-3 in coated vesicles derived from bovine brain, served as substrates for beta-D-galactosyltransferase which is specific for terminal GlcNAc residues. Analysis of the disaccharide released by beta-elimination indicated that single GlcNAc residues are attached to AP-3 through an O-glycosidic linkage to threonine or serine residues. In vivo 32P-labeled AP-3, the result of serine phosphorylation (Keen, J. H., and Black, M.M. (1986) J. Cell Biol. 102, 1325-1333), bound to wheat germ agglutinin-Sepharose indicating 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 migration of AP-3. Consistent with localization to the nonclathrin binding domain, the O-GlcNAc modification does not play a discernible role in the interaction of AP-3 with clathrin.