Fig 2. Site-specific crosslinking of UPRT in T. gondii.

(A) The UPRT homodimer is stabilized by a β-arm (darker pink, structure adapted from PDB entry 1BD4). L92 and Y96 were chosen for Azi substitution based on orientation towards the other subunit in the crystal structure. (B) IFA showing cytoplasmic localization for parasites expressing the Y96 mutant UPRT-HA upon addition of Azi. Red, mouse anti-HA. Scale bar represents 2 μm. (C) Schematic for UPRT dimer formation using a second copy of UPRT with a Myc tag. UPRT proteins will assemble as either HA/HA or Myc/Myc homodimers, or a HA/Myc heterodimer. As the Myc-tagged monomers lack Azi, they should be crosslinked only when bound to an Azi-containing UPRT-HA partner. (D) Anti-HA western blot of UPRT crosslinking using strains expressing the synthetase/tRNA and either WT, L92, or Y96 UPRT-HA. Uncrosslinked UPRT migrates at 27 kDa (blue arrow). A small amount of UPRT is observed without Azi, indicating that nonspecific incorporation of other amino acids can occur, but this material is low abundance and lacks crosslinking ability. In the +Azi/+UV conditions, shifted products can be observed for both L92 and Y96 lines (red arrowheads), indicating successful crosslinking of a UPRT dimer. (E) Immunoblot of the UPRT crosslink samples with anti-Myc antibody verifies that the shifted products are covalently crosslinked UPRT homodimers. The Myc blot shows a lower relative efficiency of crosslinked to uncrosslinked material, presumably because the Myc-tagged monomers can only crosslink as the heterodimer, while the HA-tagged UPRT can crosslink as both the heterodimer and an HA/HA homodimer. Azi, p-azidophenylalanine; HA, hemagglutinin; IB, immunoblot; IFA, immunofluorescence assay; PDB, Protein Data Bank; UPRT, uracil phosphoribosyltransferase; WT, wild-type.