Fig. 7: PA produced by AGPAT2 is crucial for ER tubulation.

a A structure of ER membrane-embedded AGPAT2 predicted by TmAlphaFold is presented. Mutations introduced in the three catalytic motifs of AGPAT2 are shown in red. b AGPAT2-KO MEFs expressing the ER marker (GFP-SEC61β) were transduced with lentiviruses carrying WT and mutant AGPAT2-HA. Cells were subjected to laser confocal immunofluorescence microscopy with anti-GFP and anti-HA antibodies. Boxed areas are enlarged. Cells are outlined by a dotted line. c Quantification of ER morphology. Mean ± SD (n = 3 experiments). d We developed an in vitro assay to measure lysophosphatidic acid acyltransferase activity. Fluorescently labeled lysophosphatidic acid (LPA-TopFluor) and acyl-coenzyme A (Acyl-CoA) were incubated with ER purified from WT and AGPAT2-KO MEFs. e PA-TopFluor was detected using thin layer chromatography followed by fluorescence imaging. f PA-TopFluor amounts were quantified and normalized relative to those made in WT ER. Mean ± SD (n = 3 experiments). g AGPAT2 makes PA from LPA, while LIPIN1 generates diacylglycerol from PA in the glycerophospholipid/triacylglycerol biosynthesis pathway. h AGPAT2-KO MEFs expressing mCherry-SEC61β were transduced with lentiviruses carrying AGPAT2-HA along with WT LIPIN1 and enzymatically-inactive LIPIN1 (D712A). Cells were subjected to laser confocal immunofluorescence microscopy with antibodies to mCherry, HA, and FLAG. Boxed areas are enlarged. Cells are outlined by a dotted line. i Quantification of ER morphology. Mean ± SD (n = 3 experiments). Statistical analysis was performed using one-way ANOVA with post-hoc Tukey (c, i) and two-tailed Student’s t-test (f). Source data are provided as a Source Data file.