Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIF␣ prolyl hydroxylases is required for optimal parasite proliferation, especially at low O 2 . We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O 2 -dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.Toxoplasma is a worldwide obligate intracellular apicomplexan parasite that infects most nucleated cells of warm-blooded animals (1). Toxoplasmosis, the disease caused by Toxoplasma, is an opportunistic infection in AIDS and other immune-suppressed patients (2). In addition, in utero infections can cause mental retardation, blindness, and death (3). Toxoplasma is transmitted by digesting parasites from feline feces (as oocysts) or undercooked meat (as tissue cysts). Once in the host, parasites convert to the tachyzoite form that disseminates to peripheral tissues (e.g. brain, retina, and muscle). The resulting immune response and/or drugs can control tachyzoite replication, but the parasite survives by converting into slow growing bradyzoites that encyst. Cysts sporadically burst, and the released parasites convert to tachyzoites whose unabated growth, as can occur in immune suppressed hosts, results in cell and tissue damage (4). Currently, no Toxoplasma vaccine exists; anti-toxoplasmosis drugs have severe side effects, and resistance to these drugs is occurring.Recently, disruption of the gene for PhyA, the prolyl 4-hydroxylase that hydroxylates Pro-154 in Skp1, was observed to reduce tachyzoite proliferation in cell culture and fitness in a competition assay (5). Skp1 is an adaptor in the Skp1/Cullin-1/ F-box protein (SCF) 2 class of E3 ubiq...

The interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and virions through a poorly characterized mechanism. Mutation of proline rich transmembrane protein 2 (PRRT2), a regulator of neurotransmitter release, at glycine-305 was previously linked to paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG motif. Mutation of glycine-95 (and to a lesser extent, glycine-91) disrupted IFITM3 oligomerization and reduced its antiviral activity against Influenza A virus. An oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-dependent and amphipathic helix-dependent manner. Furthermore, a compound which counteracts virus inhibition by IFITM3, amphotericin B, prevented the IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 oligomers inhibit virus-cell fusion by promoting membrane rigidity.

Skp1 is a subunit of the SCF (kp1/ullin 1/-box protein) class of E3 ubiquitin ligases that are important for eukaryotic protein degradation. Unlike its animal counterparts, Skp1 from is hydroxylated by an O-dependent prolyl-4-hydroxylase (PhyA), and the resulting hydroxyproline can subsequently be modified by a five-sugar chain. A similar modification is found in the social amoeba , where it regulates SCF assembly and O-dependent development. Homologous glycosyltransferases assemble a similar core trisaccharide in both organisms, and a bifunctional α-galactosyltransferase from CAZy family GT77 mediates the addition of the final two sugars in , generating Galα1, 3Galα1,3Fucα1,2Galβ1,3GlcNAcα1-. Here, we found that utilizes a cytoplasmic glycosyltransferase from an ancient clade of CAZy family GT32 to catalyze transfer of the fourth sugar. Catalytically active Glt1 was required for the addition of the terminal disaccharide in cells, and cytosolic extracts catalyzed transfer of [H]glucose from UDP-[H]glucose to the trisaccharide form of Skp1 in a -dependent fashion. Recombinant Glt1 catalyzed the same reaction, confirming that it directly mediates Skp1 glucosylation, and NMR demonstrated formation of a Glcα1,3Fuc linkage. Recombinant Glt1 strongly preferred the full core trisaccharide attached to Skp1 and labeled only Skp1 inΔ extracts, suggesting specificity for Skp1. -knock-out parasites exhibited a growth defect not rescued by catalytically inactive Glt1, indicating that the glycan acts in concert with the first enzyme in the pathway, PhyA, in cells. A genomic bioinformatics survey suggested that Glt1 belongs to the ancestral Skp1 glycosylation pathway in protists and evolved separately from related Golgi-resident GT32 glycosyltransferases.