Fig. 8. The Vps5 PX domain is responsible for Vps5p targeting and functionality. Point mutations in conserved PX domain residues of Vps5p were introduced as described in Materials and Methods (Y322A, R360A). (A) Localization of wild-type Vps5-GFP (pPB2) or mutant vps5^Y322A,R360A_ GFP fusion protein (pPB3) in vps5 cells (BHY152). (B) Wild-type (SEY6210), vps5 (BHY152) and vps5 cells carrying plasmid pPB4 (vps5^Y322A,R360A) were analyzed by pulse-chase labeling and immunoprecipitation with antibodies against CPY. The migration positions of ER-modified (p1), Golgi-modified precursors (p2) and mature (m) CPY are shown. (C) Protein-lipid overlay assay using nitrocellulose-immobilized phospholipid strips. The strips were incubated with 10 ng ml^-1 of purified wild-type GST-Vps5 PX (left panel) or mutated GST-vps5^Y322A,R360A PX domain fusion protein (right panel). PI, phosphatidylinositol; PC, phosphatidylcholine.