The SKP1/Cul1/F-Box (SCF) complex is an E3 ubiquitin ligase responsible for targeting a range of proteins for degradation by the 26S proteosome. Within this complex, a variety of F-box proteins (FBPs) link to the SCF complex via the SKP1 adaptor protein allowing for differential substrate recognition. In the intracellular 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 plays an important role in controlling SKP1 by weakening the tight SKP1 homodimer and affecting the profile of bound FBPs in cells. However, the presence of the terminal SKP1 glycosyltransferase, GAT1, in the SKP1 interactome regardless of its glycosylation status is atypical for an enzyme. 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 same core hydrophobic interface 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, substoichiometric levels of GAT1 mediate monomerization of SKP1 in a CTR-dependent manner, indicating that GAT1 has the kinetic potential to promote SKP1 monomer availability, with consequences on its FBP-binding preference in cells.
Cantrell, D. A., Gas-Pascual, E., West, C. M.
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