TANGO2 interacts with specific acyl chains. (a–f) LPLAT assays were performed in CHO-K1 cell lysates using C16:0-LPA, LPC, LPS, LPI, LPE, or LPG as acyl acceptors, and C16:0-CoA, C18:1-CoA, C18:2-CoA, C20:4-CoA, and C22:6-CoA as acyl donors. LPLAT activity was tested by measuring PA (a), PC (b), phosphatidylserine (PS; c), phosphatidylinositol (PI; d), phosphatidylethanolamine (PE; e), or phosphatidylglycerol (PG; f) molecule production in TANGO2-overexpressed (vermillion bars) compared with control cells (blue bars) and background noise (gray). The data are representative of three independent experiments with similar results. (g and h) Real-time NBD fluorescence intensity analysis of 2 µM 16-NBD-16:0-CoA or 18-NBD-18:1-CoA alone (0–300 s), incubated with 1 µM wild-type TANGO2 or mutant TANGO2.∆NRDE protein (300–600 s), and with 0.8% Tween-20 addition (1,200–1,800 s) at 24°C. All the reactions were performed in 150 μl of reaction buffer (Tris-HCl 20 mM, pH 7.4; black). (i and j) Real-time NBD fluorescence intensity analysis of 1 µM 16-NBD-16:0-CoA (i, black) or 18-NBD-18:1-CoA (j, black) incubated with 1 µM 16:0-CoA (vermillion), 18:1-CoA (blue), or CoA alone (green) for 5 min (0–300 s), and then with 1 µM wild-type TANGO2 protein (330–630 s) at 24°C. The NBD fluorescence intensity experiments are representative of at least two independent experiments with similar results. Data are shown as the mean ± SD. A. U. means arbitrary units.