O-fucosylation affects abundance but not localization of select nucleocytoplasmic proteins in toxoplasma gondii

Abstract

Toxoplasma gondii is a highly successful intracellular mammalian and avian pathogen that must adapt to a wide range of intracellular and extracellular environments. A mechanism that may support this is the modification of hydroxyamino acid rich sequences of nucleocytoplasmic proteins with O-fucose. O-fucosylation of possibly hundreds of proteins is mediated by a single highly conserved nucleocytoplasmic enzyme. Deletion of the SPY O-fucosyltransferase gene is tolerated but inhibits parasite proliferation in fibroblasts and their accumulation in mouse brains. A prior ectopic expression study suggested that O-fucose is required to detect proteins considered essential. To distinguish whether the SPY requirement was specific to the method or for protein expression per se, GPN1, an RNA polymerase chaperone, was epitope-tagged at its endogenous locus in both normal and SPYΔ strains. GPN1 was shown to be substantially and quantitatively O-fucosylated and exhibited a modest 24% reduction in level in SPYΔ cells. Proteomic analysis of its interactome indicated that fucosylation did not affect its association with RNA polymerase subunits. GPN1 was mostly cytoplasmic based on super-resolution immunofluorescence microscopy, and this localization was not affected by O-Fuc. A fusion of its O-fucosylated serine-rich domain to yellow fluorescent protein behaved similarly. In comparison, the abundance of a Zn-finger containing protein also depended on SPY, whereas the abundance and localization of ERK7 were not affected nor were levels of two other proteins. Thus O-fucose directly but modestly promotes the accumulation of select targets, but it does not enforce their localization in nuclear assemblies that are highlighted by immunofluorescence studies.

Keywords: GPN-loop GTPase; O-fucose; Toxoplasma gondii; nucleocytoplasmic glycosylation; spindly.