LST-4 interacts with PtdIns(4,5)P2, PtdIns3P, and MTM-1. (A and B) Binding of His-tagged LST-4 to the indicated liposomes was detected by Coomassie blue staining (A) or Western blotting (B). The lipid–protein complex is recovered in the pellet (P) but not the supernatant (S). The final concentration of LST-4 and liposomes used in each experiment is indicated. Three (A) and six (B) independent experiments were performed. Data (shown as mean ± SD) were compared by one-way ANOVA with Tukey’s post-test. *, P < 0.05; **, P < 0.0001. (C) His-tagged LST-4 is pulled down by GST::MTM-1::FLAG and GST::MTM-1(C378S)::FLAG but not GST::FLAG. Purified GST fusion proteins stained by Coomassie blue (arrows) are shown underneath. (D) Full-length LST-4 and the PX-BAR region, but not the SH3 domain, are coprecipitated from HEK293T cells by MTM-1. (E) mCHERRY::MTM-1(C378S) is coprecipitated by LST-4::GFP in lysates prepared from mix-staged worms coexpressing both fusion proteins. (F) Proposed model of coregulation of phagosomal sealing and PI conversion by the PtdIns(4,5)P2–PtdIns3P–MTM-1–LST-4 module in apoptotic cell clearance. Dashed arrows indicate regulations that need further investigation. IB, immunoblotting; IP, immunoprecipitation.
