Fig. 3: The M-Lip domain reduces lipin 1 membrane association and PAP activity.

a Deletion of the M-Lip or M-Lip^xtal domain does not affect lipin 1 transcriptional co-activation. HEK293 cells were co-transfected with lipin 1 or lipin 1 M-Lip domain deletion constructs with the transcription factors PPARα and RXRα, and the transcriptional co-activator PGC1α. Transcriptional co-activation was quantitated by measuring firefly luciferase activity, which is under the control of three peroxisome-proliferation response elements (PPREs). Firefly luciferase activity was normalized to Renilla luciferase from a control plasmid that was included in each transfection. Data are presented as the mean values ± SDs. n = 3 independent experiments. 1-way ANOVA with correction for multiple comparisons as determined by Tukey HSD post-hoc test. Letters (a, b, c) denote groups that are not statistically significant within their group (p > 0.05) and statistically significant to other groups (p < 0.0001). b SDS-PAGE of purified lipin 1 and lipin 1 ΔM-Lip used in PAP activity and liposome sedimentation assays. Purifications independently replicated n = 3 with similar results. c PAP activity of lipin 1 and lipin 1 ΔM-Lip in Triton X-100 mixed micelles with 5 mol% NBD-PA. Data are presented as mean values ± SDs. n = 3 independent experiments. Statistical analysis by a paired two-tailed t test. ns, p = 0.1554. d PAP activity of lipin 1 and lipin 1 ΔM-Lip in 90 mol% PC or 70/20 mol% PC/PE liposomes with 10 mol% NBD-PA. PC phosphatidylcholine, PE phosphatidylethanolamine, PA phosphatidic acid. Data are presented as mean values ± SDs of n = 3 independent experiments analyzed by 2-way ANOVA with Tukey’s multiple comparison. **p = 0.0074; ****p < 0.0001. e SDS-PAGE analysis of a liposome sedimentation assay reveals lipin 1 preferentially associates with liposomes containing 20 mol% PA. Deletion of the M-Lip domain (lipin 1 ΔM-Lip) results in a reduction of membrane association. S supernatant, P pellet, MW molecular weight markers. Representative images from n = 3 independent experiments. f Quantification of liposome association for lipin 1 and lipin 1 ΔM-Lip. Data are presented as mean values  ± SDs. n = 3 independent experiments. Statistical analysis by 2-way ANOVA with Tukey’s multiple comparison. ns, p = 0.8424; **p = 0.0020; ***p < 0.001; ****p < 0.0001. g Confocal microscopy images of Cos-7 cells transiently transfected with monomeric enhanced GFP (mEGFP) fusions of either lipin 1 or lipin 1 ΔM-Lip (green) and the ER marker mApple-Sec61b (red). Hoechst stain (blue), nucleus. Scale bar: 20 μm. n = 1 with independent images (n = 5) shown in Supplementary Fig. 9. h Mander’s overlap coefficient calculated using JacoP plugin in ImageJ analyzed by 2-way ANOVA with Tukey’s multiple comparison. n = 9 cells. ns, p = 0.9342; **p = 0.0024. Source data are provided as a Source data file.