Potential for the Terminal SKP1 Glycosyltransferase to Exert Non-Enzymatic Control of SKP1 in Toxoplasma gondii

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The SKP1/CUL1/F-Box protein (SCF) family of E3 ubiquitin ligases is responsible for targeting multiple proteins for degradation by the 26S proteosome. Within the family, multiple F-box proteins (FBPs) associate with the SKP1 adaptor which in turn docks to CUL1. In the human parasite Toxoplasma gondii, SKP1 is subject to oxygen-dependent regulation. Under normoxic conditions, the prolyl hydroxylase PHYa hydroxylates SKP1 priming it for modification by five SKP1-specific glycosyltransferase activities. Glycosylation weakens competing homodimerization of SKP1, and because SKP1 is limiting in cells, this affects the profile of bound FBPs. A previous study noted that the terminal SKP1 glycosyltransferase, GAT1, is a stable member of the SKP1 interactome regardless of its glycosylation status. Furthermore, gat1-knockout cells exhibit a unique repertoire of FBPs bound to SKP1 relative to normal and other glycosylation-defective mutants. Utilizing sedimentation velocity analytical ultracentrifugation, we demonstrate that the native GAT1 homodimer complexes with SKP1 monomers with affinity and stoichiometry dictated by its glycostate. Computational modeling validated by mutational probing shows that GAT1 competes with the core hydrophobic interface also utilized by FBPs and the SKP1 homodimer. This interface is complemented by varying, transient fuzzy-like interactions contributed by the intrinsically disordered C-terminal region (CTR) of SKP1, that are in turn constrained by the glycan. Furthermore, GAT1 substoichiometrically monomerizes SKP1 in a CTR-dependent manner. Thus, GAT1 is specific for monomeric SKP1, and the mechanism by which this is achieved also modifies the dimer/monomer equilibrium of unmodified SKP1 with downstream effects on the steady-state profile of FBP/SKP1 complexes in cells.

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