Fig. 1: NPH3 binds to polyacidic phospholipids via its C-terminal domain.
a Representative confocal microscopy images of leaf epidermal cells from dark-adapted N. benthamiana transiently co-expressing GFP:NPH3 and either MAP:mCherry:SAC1 or MAP:mCherry:SAC1DEAD. Expression was driven by the 35S promoter. Z-stack projection of GFP:NPH3 co-expressed with MAP:mCherry:SAC1 is shown. Single expression of GFP:NPH3 is shown as control. Scale bars, 25 μm. b Lipid overlay assay performed with either in vitro-transcribed and -translated HA:NPH3 and HA:NPH3ΔC51 or purified GST and GST:NPH3-C51. Immunodetection was conducted by using anti-HA or anti-GST antibodies, respectively. See main text for abbreviations of lipids. c Liposome-binding assay performed with purified GST or GST:NPH3-C51 and large unilamellar liposomes containing the neutral phospholipids PE and PC mixed with either the polyacidic PI4P or PA as specified. Anti-GST immunoblot of pellet and supernatant is shown (lower band corresponds to GST, upper band to GST:NPH3-C51). d Representative confocal microscopy images of leaf epidermal cells from dark-adapted N. benthamiana transiently expressing RFP:NPH3ΔC51 (Z-stack projection) or RFP:NPH3ΔN54. Expression was driven by the 35S promoter. RFP:NPH3 is shown as control. Scale bars, 25 μm. All experiments were performed at least three times with similar results.
