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

Abstract

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.

Initiation of Mucin-type O-Glycosylation in Dictyostelium Is Homologous to the Corresponding Step in Animals and Is Important for Spore Coat Function

“…A Similar Enzyme in Other Unicellular Eukaryotes?-A pp ␣-GlcNAc-T2-like enzyme activity has been reported in Golgi preparations from T. cruzi (6,12) and a pp ␣-GlcNAc-T2-like sequence is present in the T. cruzi genome 3 (26). This sequence is more similar to Dictyostelium pp ␣-GlcNAc-T2 than to pp ␣-GlcNAc-T1 or the pp ␣-GalNAc-Tases.…”

“…3 of Ref. 26), a diatom, and a soybean stem and root rot, 3 suggesting that a similar O-glycosylation step exists in the secretory organelles of these organisms.…”

“…3 of Ref. 26). Although the first 285 amino acids of this putative domain exhibit very low sequence identity with the pp ␣-GalNAc-Tases (ϳ14% compared with murine pp ␣-GalNAc-T1), they have 41% similarity, allowing for conservative substitutions, and key motifs are shared.…”

“…Although this linkage involves a distinct acceptor hydroxyamino acid and the enzyme resides in the cytoplasm rather than the Golgi apparatus, its sequence exhibits homology with those of the animal pp ␣-GalNAc-Tases (25,26). Here we report the identification and characterization of a related Dictyostelium pp ␣-GlcNAc-Tase that is be expressed in the secretory pathway and initiates the O-glycosylation of SP29 and the spore coat protein SP85/PsB.…”

Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions

“…Vpu expressed in the ER interacts with a membrane-proximal domain of the cytoplasmic tail of CD4 and links it to h-βTrCP [5], a member of the F-box protein family first characterized as components of ubiquitin-ligase complexes [6]. The CD4-Vpu-h-βTrCP ternary complex then recruits SKP1, another member of the ubiquitination machinery [7]. As a result, CD4 is ubiquitinated and targeted to proteasomes for degradation.…”