Fig. 3.

The N terminus of ATP13A2 interacts with PI(3,5)P2 and PA. (A–C) Lipid–protein overlay with GST-N-Ma on membrane lipid strips (A), PIP strips (B), and sphingolipid strips (C) spotted with 30 different lipids. Labels along the left refer to the leftmost column of spots in each panel, and labels to the right refer to the second column in each panel. CHL, cholesterol; CL, cardiolipin; CM, ceramide; DAG, diacylglycerol; GD3, disiaganglioside-GD3; GM1, monosialoganglioside-GM1; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; MS, myriosine; PA, phosphatidic acid; PC, phosphatidylcholine; PCH, psychosine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PHS, phytosphingosine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; SPC, sphingosylphosphorylcholine; SPN, sphingosine; S1P, sphingosine-1-phosphate; TG, triglyceride. (D–F) Liposome floatation assay. LUVs of different lipid compositions were subjected to a sucrose gradient. N-NBD-PE fluorescence was used as a control for vesicle floating in the sucrose fractions [0%, 25%, 30%, and pellet (R)] (D). LUVs were incubated with MultiPIPgrip (E) and GST-N-Ma and GST (F). Data are represented as average ± SD (n = 8). Immunoblot of PIP grip in the 0%, 25%, and 30% wt/vol sucrose fractions of LUVs with PC or PC with 5 mol% PI(3,5)P2 (E). Immunoblot of GST-N-Ma in 0%, 25%, and 30% wt/vol sucrose fractions of LUVs containing PC with or without 5 mol% PA or PI(3,5)P2 (F, Upper panel). Purified GST was used as a negative control (F, Lower panel). (G) Bar graph representing the flotation GST-N-Ma depicted in panel F. *Statistical difference between PC versus PC/PA or PC/PI(3,5)P2 (1 mark, P < 0.01; 3 marks, P < 0.001) (n = 8) (ANOVA with Bonferroni post hoc test).