Figure 3. **A unique property of SNX9 subfamily in binding two phosphoinositides using two adjacent lipid-binding domains.** (A) Mutations in the various proposed lipid-binding sites within the PX and BAR domains of *Xenopus* SNX9. K511E/K517E, which are residues that make up a positively charged patch within the SNX9 BAR domain, and Y276A/K302A, which is the phosphoinositide lipid–binding pocket within the PX domain. The structure shown is 2RAI, downloaded from the Protein Data Bank and visualized in the CCP4mg molecular graphics package with the *Xenopus* numbering inferred from a sequence alignment performed in Lasergene software. (B) The liposome compositions used were 5% PI(4,5)P_{2}, 5% PI(3)P, or 4% PI(4,5)P_{2}/1% PI(3)P with 65% PC and 30% PS. Mutagenesis shows that binding of SNX9 to 100-nm PI(4,5)P_{2} liposomes is driven by positively charged patches on the BAR domain binding to PI(4,5)P_{2}. The pocket on the PX domain is important for binding PI(4,5)P_{2} and PI(3)P. Data are the mean of three independent liposome batches and sedimentation assays. One-way ANOVA between mutants is not significant for PI(3)P: P = 0.0594. One-way ANOVA between mutants is significant for PI(4,5)P_{2}: P = 0.0133; and PI(4,5)P_{2}/PI(3)P: P = 9.25 × 10^{−5}. By post-hoc Tukey's honest significant difference (HSD) test, the difference between WT and Y276A/K302A for PI(4,5)P_{2}/PI(3)P: **, P = 0.00223; for PI(4,5)P_{2}: P = 0.864; and for PI(3)P: P = 0.0730. For the WT compared with K511E/K517E mutant with PI(4,5)P_{2}/PI(3)P: **, P = 0.00101; for PI(4,5)P_{2}: *, P = 0.0161; and for PI(3)P: P = 0.098. (C) Phosphoinositides and SNX-BAR proteins in the endosomal network. Schematic diagram showing putative double phosphoinositide compositions for trafficking between compartments where SNX PX–BAR domains are implicated. (D) Sedimentation assay of SNX2, 4, 5, or 8 on liposomes that contain various double phosphoinositide lipid compositions. The liposome compositions were 5% of each phosphoinositide with 65% PC and 30% or 25% PS. Data are the mean of three independent liposome batches and sedimentation assays. No binding is above background suggesting that double phosphoinositide binding is a property specific to the SNX9 subfamily. (E) Sedimentation assay of extracts with liposomes that contain 4% PI(4,5)P_{2}; 1% PI(3)P; or 4% PI(4,5)P_{2}/1% PI(3)P and 65% PC; and 30, 34, or 31% PS with SNX9, 18, and 33 detected by Western blotting. Quantification from three independent liposome batches and Western blots. Error bars are the SEM. One-way ANOVA for SNX9 gives P = 3.113 × 10^{−8} with Tukey's post-hoc HSD test between PI(4,5)P_{2} and PI(4,5)P_{2}/PI(3)P: **, P = 0.00100. One-way ANOVA for SNX18 gives P = 0.0013 with Tukey's post-hoc HSD test between PI(4,5)P_{2} and PI(4,5)P_{2}/PI(3)P: *, P = 0.0112. One-way ANOVA for SNX33 gives P = 10^{−6} with Tukey's post-hoc HSD test between PI(4,5)P_{2} and PI(4,5)P_{2}/PI(3)P: **, P = 0.00101.