The N17 region of NME3 is critical for mitochondrial and PA binding. (A) N17 of NME3 is sufficient for mitochondria localization. Helical wheel projections of N17 and mutants are derived from HeliQuest (https://heliquest.ipmc.cnrs.fr/). (B) The GFP-tagged N17 and mutant constructs were co-transfected with mito-BFP, and their localization onto mitochondria are imaged, compared, and shown in B. Scale bar, 10 μm. (C) The N17 region of NME3 is essential for PA binding. 1 μM NME3-His with indicated mutations were incubated with PA liposome to examine their lipid binding ability. (D) NME3^WT prefers to bind liposomes with higher membrane curvature. 1 μM NME3^WT was incubated with 300 μM liposome composed of 10% DOPA:89% DOPC:1% rhodamine-PE but extruded with indicated membrane pore size. The larger the extrusion membrane pore size, the lower the liposome membrane curvature. (E) The proportion of NME3^WT in top fraction was quantified and compared. (F) NME3^WT binds to sonicated, small unilamellar vesicle (SUV) composed of 100% PC. (G) The proportion of NME3^WT in the top fraction was quantified and compared in G. Data are expressed as mean ± SD of three independent experiments and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. Source data are available for this figure: SourceData F2.