Fig 3. Site-specific crosslinking of ILP1 reveals multiple potential binding partners.

(A) Diagram of ILP1 showing an N-terminal putative EF-hand domain (gray) followed by a coiled-coil domain (yellow). Also shown is the JPred secondary structure prediction of ILP1 revealing alpha-helices (black bars) and beta-strands (white arrows), as well as buried residue prediction used for choosing likely exposed residues for amber substitution [34]. Also noted are the potentially myristoylated glycine at position two (teal) and tandem cysteine prenylation/palmitoylation motifs internally and at the extreme C terminus (purple). Fourteen residues in ILP1 were chosen for amber mutagenesis to test for Azi-mediated crosslinking (green). (B) COILS prediction of ILP1; the window size refers to the number of residues used in the analysis. (C) Representative IFA of the Q168 ILP1 mutant containing Azi, which localizes properly to the parasite periphery. Red, rabbit anti-HA antibody; green, mouse anti-Ty1 antibody. Scale bar represents 2 μm. (D) Western blot of the ILP1 Azi mutants after UV irradiation reveals three major crosslinked species (red arrowheads). A smaller upshift (approximately 65 kDa) is observed for residues Y160 and Q168, with weak similar products for T152 and Q170. Residues T184, T187, and I188 exhibit a major band at approximately 200 kDa. E209 and K212 form a third upshift at approximately 140 kDa. Uncrosslinked ILP1 is denoted by the blue arrows (approximately 35 kDa). Azi, p-azidophenyalanine; E2AziRS, Azi-tRNA synthetase; HA, hemagglutinin; IB, immunoblot; IFA, immunofluorescence assay; ILP1, IMC localizing protein 1.