Extended Data Fig. 7: PE regulates YME1L-mediated proteolysis.

a, b, HeLa cells pretreated with ethanolamine (Etn, 200 µM) for 24 h were treated with Torin1 for 4 h. Phospholipids in mitochondrial fractions were analysed by mass spectrometry (a) (n = 3 independent experiments). Cell lysates were analysed by immunoblotting and indicated protein levels were quantified (n = 5 independent experiments) (b). c, d, HEK293 cells were treated with Torin1 and/or 100 µM LPE for 4 h. Phospholipids in mitochondrial fractions were analysed by mass spectrometry (c) (n = 3 independent experiments). Cell lysates were analysed by immunoblotting and indicated protein levels were quantified (n = 4–5 independent experiments) (d). e, f, HeLa cells transfected with scrambled control (Scr) or Pisd siRNA were treated with 100 µM LPE for 24 h. Cell lysates were analysed by immunoblotting and indicated protein levels were quantified (n = 5 independent experiments) (e). PE levels in mitochondrial fractions were determined by mass spectrometry (n = 3 independent experiments) (f). g, h, YME1L (WT or E543Q) and OPA1ΔC (g) or TIMM17A (h) reconstituted in liposomes was incubated in the presence or absence of ATP at 37 °C for the indicated times. Samples were analysed by immunoblotting (representative data from n = 3 independent experiments). i, j, YME1L (WT or E543Q) and OPA1ΔC (i) or TIMM17A (j) reconstituted in liposomes containing different amounts of PE were incubated in the presence of ATP at 37 °C for the indicated times. Samples were analysed by immunoblotting (representative data from n = 4 independent experiments). Mean ± s.e.m.; two-way ANOVA with Tukey’s multiple comparisons test (a–d), with Holm-Sidak’s multiple comparisons test (e, f). fc, fold change.

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