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 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.
Murphy
J E
JE
Laboratory of Biochemistry and Metabolism, NIDDKD, National Institutes of Health, Bethesda, Maryland 20892.
Hanover
J A
JA
Froehlich
M
M
DuBois
G
G
Keen
J H
JH
D000117:Acetylglucosamine / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D006031:Glycosylation; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D051381:Rats
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; C439023:clathrin assembly protein AP180; D000117:Acetylglucosamine; D005690:Galactose