Evolutionary and functional implications of the complex glycosylation of Skp1, a cytoplasmic/nuclear glycoprotein associated with polyubiquitination.

Protein degradation is regulatory for the cell cycle, signal transduction and gene transcription. A critical step is the selective marking of the target protein, resulting in polyubiquitination by one of a large number of E3-ubiquitin ligases. Both target marking and E3-ubiquitin ligase activity are associated with common as well as unusual posttranslational modifications. For example, hydroxylation of Pro-residues and modification of Asn-residues by high-mannose sugar chains can target the modified proteins for rapid polyubiquitination in the mammalian cytoplasm. Both prolyl hydroxylation and glycosylation also occur on Skp1, a subunit of the SCF class of E3-ubiquitin ligases, from Dictyostelium. In this case, a pentasaccharide containing Gal, Fuc and N-acetyl-D-glucosmine (GlcNAc) is attached to the HyPro-residue. The sugars are added sequentially by enzymes that reside in the cytoplasm rather than the secretory pathway. Two of the glycosyltransferases appear to be positioned in ancient evolutionary lineages that bridge prokaryotes and eukaryotes. The first, which attaches GlcNAc to HyPro, is related to enzymes that form alpha-GalNAc- and alpha-GlcNAc-Ser/Thr linkages in the Golgi. GlcNAc is extended by a bifunctional glycosyltransferase that mediates the ordered addition of beta1,3-linked Gal and alpha1,2-linked Fuc, using an architecture resembling that of two-domain prokaryotic glycosyltransferases involved in glycosaminoglycan synthesis. Mutational and pharmacological perturbation of glycosylation alters the subcellular localization of Skpl and growth properties ofcells. Prolyl hydroxylation and complex O-glycosylation provide the cell with new options for epigenetic regulation of protein turnover in its cytoplasmic and nuclear compartments.

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