title: Identification of a UDP-GlcNAc:Skp1-hydroxyproline GlcNAc-transferase in the cytoplasm of Dictyostelium.
abstract: Skp1 is a cytoplasmic and nuclear protein required for the ubiquitination of cell cycle regulatory proteins and transcriptional factors. In Dictyostelium, Skp1 is modified by a linear pentasaccharide, Galalpha1-6Galalpha1-Fucalpha1-2Galbeta1-3Glc NAc, attached to a hydroxyproline (HyPro) residue at position 143. To study the formation of the GlcNAc-HyPro linkage, an assay was developed for the transfer of [(3)H]GlcNAc from UDP-[(3)H]GlcNAc to Skp1-HyPro-143 or a synthetic Skp1 4-HyPro peptide. The cytosolic but not the particulate fraction of the cell mediated transfer in a time-, concentration-, and HyPro-dependent fashion. Incorporated radioactivity was alkali-resistant and was recovered as GlcNH(2) after acid hydrolysis, consistent with linkage of GlcNAc to HyPro. The GlcNAc-transferase activity was purified 130,000-fold as a single component with a recovery of 5%. Key to the purification was the synthesis of a novel affinity resin linking UDP-GlcNAc at its 5-uridyl position. The purified activity had an apparent M(r) of approximately 45,000 by gel filtration, required dithiothreitol and a divalent cation, and consisted predominantly of a M(r) 51,000 band after SDS-polyacrylamide gel electrophoresis that was photoaffinity labeled with 5-(125)I-[3-(p-azidosalicylamido)-1-propenyl-UDP-GlcNAc in a UDP-GlcNAc-sensitive fashion. Its apparent K(m) values for UDP-GlcNAc and Skp1 were submicromolar. The presence of the enzyme in the cytosolic fraction, its dependence on a reducing environment, and its high affinity for UDP-GlcNAc strongly suggest that Skp1 is glycosylated by a HyPro GlcNAc-transferase that resides in the cytoplasm.
authors.affiliation: Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610-0235, USA.
authors.forename: P
authors.initials: P
authors.lastname: Teng-Umnuay
authors.forename: H
authors.initials: H
authors.lastname: van der Wel
authors.forename: C M
authors.initials: CM
authors.lastname: West
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D004023:Dictyostelium / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D044786:S-Phase Kinase-Associated Proteins
chemical_list: D018797:Cell Cycle Proteins; D044786:S-Phase Kinase-Associated Proteins; D017351:N-Acetylglucosaminyltransferases; C403138:UDP-N-acetylglucosamine - SKP1-hydroxyproline N-acetylglucosaminyltransferase