Figure 1. PI(3)P-dependent lipid binding... | Rockefeller University Press

Figure 1.

$Figure 1. PI(3)P-dependent lipid binding of hSNX16 CC domain mutants. (A) Schematic of hSNX16 constructs. (B) Location of hSNX16 CC mutations on hSNX16 PX-CC dimer (PDB accession number 5GW0; Xu et al., 2017). CC domains are shown in green and glutamates in magenta. (C–F) Liposome cosedimentation assays. Purified hSNX16 variants were incubated with liposomes of the indicated composition and pelleted. Representative Coomassie staining of supernatant (S) and pellet (P) fractions is shown in C and E. (C and D) Liposomes composed of 80% DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), 15% DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 5% DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), and PI(3)P (0.5%, 1%, 2.5%, 5%, and 10%; with a corresponding decrease in DOPC). PI(3)P-dependent lipid binding of purified hSNX16 CC domain mutants is not significantly different from wild-type hSNX16. Y145A mutsation abolishes PI(3)P binding of hSNX16 as previously reported (Choi et al., 2004b). (E and F) Binding of wild-type hSNX16 is more salt sensitive than hSNX163A. Liposomes (70% DOPC, 15% DOPE, 5% DOPS, and 10% PI(3)P) were incubated for 45 min with purified hSNX16 and the indicated NaCl concentrations before pelleting. In the last condition, hSNX16 and liposomes were incubated in 100 mM NaCl for 30 min, and then NaCl was added to a final concentration of 400 mM for 15 min before pelleting. Quantification is a result of three independent experiments, analyzed using a Kruskal–Wallis test followed by a Dunn’s multiple comparisons test. Data are presented as mean ± SEM. *, P < 0.05. ns, not significant.$

PI(3)P-dependent lipid binding of hSNX16 CC domain mutants. (A) Schematic of hSNX16 constructs. (B) Location of hSNX16 CC mutations on hSNX16 PX-CC dimer (PDB accession number 5GW0; Xu et al., 2017). CC domains are shown in green and glutamates in magenta. (C–F) Liposome cosedimentation assays. Purified hSNX16 variants were incubated with liposomes of the indicated composition and pelleted. Representative Coomassie staining of supernatant (S) and pellet (P) fractions is shown in C and E. (C and D) Liposomes composed of 80% DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), 15% DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 5% DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), and PI(3)P (0.5%, 1%, 2.5%, 5%, and 10%; with a corresponding decrease in DOPC). PI(3)P-dependent lipid binding of purified hSNX16 CC domain mutants is not significantly different from wild-type hSNX16. Y145A mutsation abolishes PI(3)P binding of hSNX16 as previously reported (Choi et al., 2004b). (E and F) Binding of wild-type hSNX16 is more salt sensitive than hSNX16^3A. Liposomes (70% DOPC, 15% DOPE, 5% DOPS, and 10% PI(3)P) were incubated for 45 min with purified hSNX16 and the indicated NaCl concentrations before pelleting. In the last condition, hSNX16 and liposomes were incubated in 100 mM NaCl for 30 min, and then NaCl was added to a final concentration of 400 mM for 15 min before pelleting. Quantification is a result of three independent experiments, analyzed using a Kruskal–Wallis test followed by a Dunn’s multiple comparisons test. Data are presented as mean ± SEM. *, P < 0.05. ns, not significant.