Fig. 2: An amphipathic helix within the C-terminal domain is required for NPH3 phospholipid binding, membrane association, and plasma membrane localization. | Nature Communications

Fig. 2: An amphipathic helix within the C-terminal domain is required for NPH3 phospholipid binding, membrane association, and plasma membrane localization.

From: Light-triggered and phosphorylation-dependent 14-3-3 association with NON-PHOTOTROPIC HYPOCOTYL 3 is required for hypocotyl phototropism

Fig. 2

a Domain structure and primary sequence of NPH3 showing the two putative BH domains (amphipathic helix and R-rich motif) within the C-terminal region. Stars depict residues substituted by alanine (A) in the NPH3 variants, blue circle depicts the 14-3-3-binding site (see Fig. 3). b Lipid overlay assay performed with in vitro-transcribed and -translated HA:NPH3 and HA:NPH3 variants characterized by the substitutions depicted in a. Immunodetection was conducted by using the anti-HA antibody. c Liposome-binding assay performed with purified GST and GST:NPH3-C51 variants (referred to as C51) characterized by the substitutions depicted in a. Large unilamellar liposomes containing the neutral phospholipids PE and PC mixed with the polyacidic PA were used. Anti-GST immunoblot of the pellet (see Fig. 1c) is shown. d Representative confocal microscopy images of leaf epidermal cells from dark-adapted N. benthamiana transiently expressing RFP:NPH3 variants characterized by the substitutions depicted in a as well as RFP:NPH3ΔC28. Expression was driven by the 35S promoter. Z-stack projections of RFP:NPH3-4K/A and RFP:NPH3-4WLM/A are shown. RFP:NPH3 is shown as control. Scale bars, 25 μm. Experiments b–d were performed at least three times with similar results.

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