PI(4)P directly interacts with an arginine motif in Smo C-tail.
(A) Smo interaction with phospholipids in a protein:lipid overlay assay. Lipid-dotted strips were incubated with Myc-SmoWT (left) or Myc-SmoΔC (middle) purified from S2 cells, and bound Smo detected using the anti-Myc antibody. The PIP-Strip was dotted with 15 different lipids at 100 pmol per spot. LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphinogosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. Input Myc-SmoWT and Myc-SmoΔC proteins are shown in the right panel. (B) Schematic drawing of full-length Smo with the sequences of the four arginine motifs shown underneath. Arginine residues were mutated as indicated. (C) A WT wing disc stained for dpp-lacZ expression. (D–G) Wing discs expressing Myr-Smo730-1035 (D–E) or Myr-Smo764-1035 (F–G), with or without the coexpression of Myr-PHOSBP by MS1096-Gal4, were stained for dpp-lacZ expression. Arrow indicates the elevated dpp-lacZ expression. (H) The protein:lipid overlay assay was used as in (A) to examine GST-SmoWT (left) or GST-SmoRA4 (middle) binding lipids on the lipid-dotted strip. Input GST or GST-Smo proteins are shown in the right panel. (I) The indicated GST or GST-Smo proteins were bacterially expressed, purified, and incubated with PI(4)P beads, followed by western blot analysis using the anti-GST antibody. Input for the incubation is shown in the lower panel. (J) S2 cells were transfected with either Myc-SmoWT or Myc-SmoRA4 and immunoprecipitated with the anti-Myc antibody. Purified proteins were then incubated with PI(4)P beads, followed by western blot with the anti-Myc antibody to examine the bound Myc-tagged proteins. (K) S2 cells were transfected with the indicated constructs and treated with Hh-conditioned medium and/or PI(4)P, followed by immunoprecipitation with the anti-Myc antibody to detect Smo-bound Cos2 and Fu. Cell lysates were analyzed by western blot to examine the protein expression. (L–M) Wing discs containing smo mutant clones and expressing tub-SmoRA4 were immunostained to show En, Ptc, and GFP. Mutant clones are recognized by the lack of GFP expression and indicated by arrows. (N–R) Wing discs expressing the indicated Smo variants driven by MS1096-Gal4 were stained for dpp-lacZ or En. Arrow in (N) indicates the ectopic expression of dpp-lacZ induced by SmoWT. Arrow in (P) indicates the elevated dpp-lacZ expression compared to arrow in (N). Arrow in (R) indicates the reduction of En expression compared to (Q).