Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is uniquely modified in protists by an O2-dependent prolyl hydroxylase that generates the attachment site for a defined pentasaccharide. Previous studies demonstrated the importance of the core glycan for growth of the parasite Toxoplasma gondii in fibroblasts, but the significance of the non-reducing terminal sugar was unknown. Here, we find that a homolog of glycogenin, an enzyme that can initiate and prime glycogen synthesis in yeast and animals, is required to catalyze the addition of an α-galactose in 3-linkage to the subterminal glucose to complete pentasaccharide assembly in cells. A strong selectivity of the enzyme (Gat1) for Skp1 in extracts is consistent with other evidence that Skp1 is the sole target of the glycosyltransferase pathway. gat1 -disruption results in slow growth attesting to the importance of the terminal sugar. Molecular dynamics simulations provide an explanation for this finding and confirm the potential of the full glycan to control Skp1 organization as in the amoebozoan Dictyostelium despite the different terminal disaccharide assembled by different glycosyltransferases. Though Gat1 also exhibits low α-glucosyltransferase activity like glycogenin, autoglycosylation is not detected and gat1 -disruption reveals no effect on starch accumulation. A crystal structure of the ortholog from the crop pathogen Pythium ultimum explains the distinct substrate preference and regiospecificity relative to glycogenin. A phylogenetic analysis suggests that Gat1 is related to the evolutionary progenitor of glycogenin, and acquired a role in glycogen formation following the ancestral disappearance of the underlying Skp1 glycosyltransferase prior to amoebozoan emergence.*   DBA    :   Dolichos biflorus lectin    Dd    :   Dictyostelium discoideum     FBP    :   F-box protein    GaGlFGaGn–    :   Galα1,3Glcα1,3Fucα1,2Galβ1,3GlcNAcα1–    GalT    :   galactosyl transferase    GT    :   glycosyltransferase    Hyp    :   ( 2S , 4R )-4-hydroxy-l-proline    mAb    :   monoclonal antibody    P4H    :   prolyl 4-hydroxylase    pAb    :   polyclonal antibody    pNP    :   para-nitrophenol    Pu    :   Pythium ultimum     RHΔΔ    :   RHΔ ku80 Δ hxgprt     SCF    :   Skp1/Cullin-1/F-box protein subcomplex of E3 Cullin-RING-1 ubiquitin ligases    Tg    :   Toxoplasma gondii     Ub    :   ubiquitin

A glycogenin homolog controls Toxoplasma gondii growth via glycosylation of an E3 ubiquitin ligase