Fig. S5.

The R46H mutation affects the binding of AtPH1 to PI3P in vitro. (A) The structure of the AtPH1 PH domain was modeled using the structure of the PH domain of DAPP1 with inositol 1,3,4,5-tetrakisphosphate (IP₄) as template (99.9% confidence). The position of the Arg46 lateral chain, corresponding to DAPP1 Arg184, is displayed (ProQ2 score = 0.00). (B) A close-up view of the area outlined in white in A. AtPH1 Arg46 is predicted to establish a hydrogen bond with the three-phosphate group of the IP4 inositol ring. (C) GST-AtPH1 and GST-AtPH1R46H proteins were purified from E. coli extracts. (D) GST-AtPH1 and GST-AtPH1R46H lipid overlay assay using a membrane spotted with serial dilutions of phospholipids (from 100–1.56 pmol per spot). A short (Upper) and a long (Lower) exposure are presented.