Fig. 7. The PX domains of Vps5p and Vps17p, respectively, bind specifically to PtdIns3P. (A) The ability of the PX domains of Vps5p and Vps17p to bind lipids in vitro was tested by a protein-lipid overlay assay using GST-PX domain fusion proteins and nitrocellulose-immobilized phospholipid strips. PI, phosphatidylinositol; PC, phosphatidylcholine. (B) Serial dilutions of PtdIns3P (100 pmol, 50 pmol and 25 pmol) were spotted onto nitrocellulose membranes and incubated with 10 ng ml^-1 of purified GST-Vps5 PX, GST-Vps17 PX or Vam7 PX domain fusion protein. (C) Localization of Vps17-GFP in PtdIns-kinase mutant strains defective for synthesis of PtdIns(3,5)P₂ (fab1, PBY116), PtdIns (4)P (pik1^tsf, PBY114; stt4^tsf, PBY115), and PtdIns (4,5)P₂ (mss4^tsf, PBY117). The strains were incubated at non-permissive temperature (37°C) as indicated.