Glycosylation of Skp1 Affects Its Conformation and Promotes Binding to a Model F-Box Protein

Abstract

In the social amoeba Dictyostelium, Skp1 is hydroxylated on proline 143 and further modified by three cytosolic glycosyltransferases to yield an O-linked pentasaccharide that contributes to O2 regulation of development. Skp1 is an adapter in the Skp1/cullin1/F-box protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent proteasomal degradation. To investigate the biochemical consequences of glycosylation, untagged full-length Skp1 and several of its posttranslationally modified isoforms were expressed and purified to near homogeneity using recombinant and in vitro strategies. Interaction studies with the soluble mammalian F-box protein Fbs1/Fbg1/OCP1 revealed preferential binding to the glycosylated isoforms of Skp1. This difference correlated with the increased α-helical and decreased β-sheet content of glycosylated Skp1s based on circular dichroism and increased folding order based on small-angle X-ray scattering. A comparison of the molecular envelopes of fully glycosylated Skp1 and the apoprotein indicated that both isoforms exist as an antiparallel dimer that is more compact and extended in the glycosylated state. Analytical gel filtration and chemical cross-linking studies showed a growing tendency of less modified isoforms to dimerize. Considering that regions of free Skp1 are intrinsically disordered and Skp1 can adopt distinct folds when bound to F-box proteins, we propose that glycosylation, which occurs adjacent to the F-box binding site, influences the spectrum of energetically similar conformations that vary inversely in their propensity to dock with Fbs1 or another Skp1. Glycosylation may thus influence Skp1 function by modulating F-box protein binding in cells.

Glycosylation of Skp1 Promotes Formation of Skp1–Cullin-1–F-box Protein Complexes in Dictyostelium

“…Previously, we showed that hydroxylation and glycosylation of Dictyostelium Skp1 affect its conformation and promote binding to a soluble FBP, guinea pig Fbs1, in studies of purified proteins (16). Here we show that Dictyostelium Skp1 is indeed a subunit of a canonical SCF complex, as expected.…”

“…We recently showed that a model soluble FBP, guinea pig Fbs1, binds preferentially to GGFGGn-Skp1 and Gn-Skp1 relative to HO-Skp1 and Skp1 in vitro in the absence of other proteins (16). This suggests that glycosylation might affect Skp1 directly to promote its interactions with FBPs.…”

“…A cytosolic extract was prepared via ultracentrifugation (100,000 ϫ g for 60 min) at 4°C and chromatographed on a Superose 12 gel filtration column at 22°C pre-equilibrated in 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM DTT, and 10% glycerol. Fractions were analyzed via SDS-PAGE and Western blotting for Skp1 using mAb 4E1, and they were quantitated densitometrically as previously described (13,16). phyA -cells were strain HW288, gnt1 -cells were HW503, pgtA -cells were HW260, and agtA -cells were HW420.…”

“…Mutant gnt1 -cells were used to prevent glycosylation of HO-Skp1, which blocks recognition by pAb UOK85. pAb UOK85 is highly specific for HO-Skp1 based on Western blotting (13,16) and ELISA assays (24), and no […”

Generating Isoform-Specific Antibodies: Lessons from Nucleocytoplasmic Glycoprotein Skp1

“…In protists such as the social amoeba Dictyostelium (West et al 2010) and the apicomplexan human pathogen Toxoplasma gondii (Xu et al 2012a), a novel posttranslational modification of the Skp1 subunit may represent an additional level of CRL1-specific control (Wang et al 2011;Sheikh et al 2014). The modification is initiated by 4-hydroxylation of Pro143, followed by step-wise addition of five sugars (Fig.…”