Fig 5. The C-terminal region of IMC3 is required for binding to ILP1 at T187.

(A) Diagram and IFA of IMC3 truncations expressed in Toxoplasma to determine which region of IMC3 binds to ILP1 T187. In endogenously tagged IMC3-3xMyc parasites, regions corresponding to alveolin only (112–279, IMC3_A) and alveolin plus C terminus (112–538, IMC3_AC) were tagged with V5 and localized. IMC3_A partially mislocalizes to the maternal cytoplasm, suggesting that although the alveolin domain plays a role in IMC targeting, the inclusion of the C-terminal region of the protein improves IMC targeting similarly to wild-type IMC3. Red, mouse anti-V5 antibody; green, rabbit anti-Myc antibody. Scale bar represents 2 μm. (B) Western blot showing the high molecular weight product corresponding to a crosslinked full-length IMC3 in parasites expressing either IMC3_A or IMC3_AC (black asterisk). However, another smaller band (red arrowhead) is seen in the IMC3_AC lysate, likely representing an ILP1/IMC3_AC crosslinked product. The uncrosslinked ILP1 Azi mutant is denoted with a blue arrow. (C) ILP1-3xHA T187 denaturing IP shows the same ILP1 shifted products seen in whole cell lysates (first panel, black asterisk and red arrowhead), but an anti-V5 blot now clearly labels a band migrating at the same position as the new smaller anti-HA upshifted product (second panel, red arrowhead), demonstrating that this species corresponds to ILP1 T187 crosslinked to the IMC3_AC. The light chain (lc) signal seen at 25 kDa obscures detection of residual uncrosslinked IMC3_A. However, detecting the original ILP1 T187/IMC3 full length species in the anti-Myc blot (third panel, black asterisk) shows that IP in both IMC3_A and IMC3_AC conditions was successful. Uncrosslinked proteins are denoted with blue arrows. (D) Western blot analysis of anti-V5 denaturing IP performed with the same strains. Both IMC3_A and IMC3_AC are robustly enriched (second panel, blue arrows), but crosslinked ILP1 is only obtained in the IMC3_AC condition (first panel, red arrowhead). Some uncrosslinked IMC3-3xMyc is seen in the IP, likely reflecting interactions of this abundant alveolin in the lysate (third panel). Uncrosslinked proteins are denoted with blue arrows, and both heavy chains (hc) and lc are seen as extra signal in the anti-V5 blot. A, alveolin domain only; AC, alveolin domain and C-terminus; Azi, p-azidophenylalanine; HA, hemagglutinin; hc, heavy chain; IFA, immunofluorescence assay; ILP1, IMC localizing protein 1; IP, immunoprecipitation; lc, light chain; trunc., truncation.