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...

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Veröffentlicht in:The Journal of biological chemistry 1994-08, Vol.269 (33), p.21346-21352
Hauptverfasser: MURPHY, J.-E, HANOVER, J. A, FROEHLICH, M, DUBOIS, G, KEEN, J. H
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Sprache:eng
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Zusammenfassung: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(17)31968-3