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J Biol Chem, Vol. 274, Issue 51, 36392-36402, December 17, 1999

Identification of a UDP-GlcNAc:Skp1-Hydroxyproline GlcNAc-transferase in the Cytoplasm of Dictyostelium^*

Patana Teng-umnuay, Hanke van der Wel, and Christopher M. West^§

From the Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610-0235

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, Gal1-6Gal1-Fuc1-2Gal1-3GlcNAc, 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 [³H]GlcNAc from UDP-[³H]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 GlcNH₂ 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 ~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 5-¹²⁵I-[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.

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^* This work was supported in part by National Institutes of Health Grant RO1-GM37539 and funds from the University of Florida.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Pathology and Laboratory Medicine, Emory University, 1364 Clifton Rd. N.E., H-185, Atlanta, GA 30322.

^§ To whom correspondence should be addressed: Box 100235, 1600 SW Archer Rd., Gainesville, FL 32610-0235. Tel.: 352-392-3329, Fax: 352-392-3305; E-mail: westcm@college.med.ufl.edu.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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