PILS-Nir1 shows specificity for PA and PIP2in vitro, based on a novel PABD structure. (A) Representative SDS-PAGE gel and quantification showing PILS-Nir1 binding of various PM lipids in POPC liposomes. Lipids indicated were mixed with POPC to produce a 2 mM solution, and then, 50 μl of the resulting liposome mixture was incubated with 50 μl of protein at ∼1 mg/ml to produce 1 mM lipids in the assay. Supernatant (S) and pellet (P) lanes were quantified using ImageJ to determine percent protein bound. The protein-only control pellet was used as a baseline (input). (B) Representative SDS-PAGE gel and quantification showing PILS-Nir1–binding affinity for PA. Liposomes were made of 80 mol% PC and 20 mol% PA, and the total concentration of lipids was increased to achieve the indicated [PA]. Supernatant (S) and pellet (P) lanes were quantified using ImageJ to determine percent protein bound. The protein-only control pellet was used as a baseline (input). A nonlinear fit was produced using an equation for a one-site specific binding with background (see Materials and methods). Background was constrained to the minimum value of 9.504. Bmax was constrained to <100. The Kd is as shown, with a 95% C.I. = 110.1–789.9 µM. Degrees of freedom = 3 and R2 = 0.9715. (C) Representative SDS-PAGE gel and quantification of PILS-Nir1 and PABD-Spo20 binding of various PM lipids in POPC liposomes. Statistics were determined using a two-way ANOVA with Šídák’s multiple comparisons test. DF = 1, MS = 1,567, F(1,14) = 7.750, P = 0.0146. Solutions were prepared, and binding was quantified as in A. (D) Representations of the AlphaFold predicted domain architecture of PILS-Nir1. It includes an amphipathic alpha helix spanning residues 613–630 (purple) and a large, structured domain at residues 631–894 (orange), which contains the SIDGS motif (pink) that is conserved with the lipin/Pah active site. The nearby K820 residue (green) is predicted to help stabilize PA within the domain. (E) Isolating either region of the PILS-Nir1 domain or introducing a K820E mutation destroyed the ability of PILS-Nir1 to respond to 100 nM PMA in HEK293A cells. The graph shows the grand means ± SEM of 3–4 experiments (35–42 total cells). (F) Representative SDS-PAGE gel and quantification for WT PILS-Nir1 and PILS-Nir1-K820E binding of various PM lipids in POPC liposomes. Statistics were determined using a two-way ANOVA with Šídák’s multiple comparisons test. DF = 1, MS = 256, F(1,16) = 45.36, P < 0.0001. Solutions were prepared, and binding was quantified as in A. Source data are available for this figure: SourceData F2.
Figure 2.

PILS-Nir1 shows specificity for PA and PIP 2 in vitro, based on a novel PABD structure. (A) Representative SDS-PAGE gel and quantification showing PILS-Nir1 binding of various PM lipids in POPC liposomes. Lipids indicated were mixed with POPC to produce a 2 mM solution, and then, 50 μl of the resulting liposome mixture was incubated with 50 μl of protein at ∼1 mg/ml to produce 1 mM lipids in the assay. Supernatant (S) and pellet (P) lanes were quantified using ImageJ to determine percent protein bound. The protein-only control pellet was used as a baseline (input). (B) Representative SDS-PAGE gel and quantification showing PILS-Nir1–binding affinity for PA. Liposomes were made of 80 mol% PC and 20 mol% PA, and the total concentration of lipids was increased to achieve the indicated [PA]. Supernatant (S) and pellet (P) lanes were quantified using ImageJ to determine percent protein bound. The protein-only control pellet was used as a baseline (input). A nonlinear fit was produced using an equation for a one-site specific binding with background (see Materials and methods). Background was constrained to the minimum value of 9.504. Bmax was constrained to <100. The Kd is as shown, with a 95% C.I. = 110.1–789.9 µM. Degrees of freedom = 3 and R2 = 0.9715. (C) Representative SDS-PAGE gel and quantification of PILS-Nir1 and PABD-Spo20 binding of various PM lipids in POPC liposomes. Statistics were determined using a two-way ANOVA with Šídák’s multiple comparisons test. DF = 1, MS = 1,567, F(1,14) = 7.750, P = 0.0146. Solutions were prepared, and binding was quantified as in A. (D) Representations of the AlphaFold predicted domain architecture of PILS-Nir1. It includes an amphipathic alpha helix spanning residues 613–630 (purple) and a large, structured domain at residues 631–894 (orange), which contains the SIDGS motif (pink) that is conserved with the lipin/Pah active site. The nearby K820 residue (green) is predicted to help stabilize PA within the domain. (E) Isolating either region of the PILS-Nir1 domain or introducing a K820E mutation destroyed the ability of PILS-Nir1 to respond to 100 nM PMA in HEK293A cells. The graph shows the grand means ± SEM of 3–4 experiments (35–42 total cells). (F) Representative SDS-PAGE gel and quantification for WT PILS-Nir1 and PILS-Nir1-K820E binding of various PM lipids in POPC liposomes. Statistics were determined using a two-way ANOVA with Šídák’s multiple comparisons test. DF = 1, MS = 256, F(1,16) = 45.36, P < 0.0001. Solutions were prepared, and binding was quantified as in A. Source data are available for this figure: SourceData F2.

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