Extended Data Figure 7: TFR1 is the mediator of C18:0 signalling to mitochondrial morphology.

a, C17:0–azide is a functional analogue of C18:0 in that it induces mitochondrial fusion in HeLa cells, whereas other C18:0 derivatives are not. Cn:0–azide = HO₂C(CH₂) _{n − 1}N₃; Cn:0–alkyne = HO₂C(CH₂) _{n − 1}CCH. b, TFR1 is the most enriched protein in a C17:0–azide pulldown, and it regulates mitochondrial morphology. HeLa cells were treated with C17:0–azide for 2 h, and covalently bound proteins were precipitated by lysing cells under denaturing conditions (8 M urea), and linking the C17:0–azide to an alkyne-labelled resin via click chemistry (left). Precipitated proteins were identified by mass spectrometry, and peptide counts were normalized to peptide counts in a negative control pulldown from cells not treated with C17:0–azide (n = 3) (right; column 2). Indicated proteins were also tested by siRNA-mediated knockdown for effects on mitochondrial morphology (column 3). c, TFR1 is covalently bound to the C18:0 derivative C17:0–azide in HeLa cells in a ZDHHC6-dependent manner. HeLa cells were treated with C17:0–azide for 2 h, and subsequently lysed in denaturing conditions (8 M urea). Similar to b, the C17:0–azide was ‘clicked’ onto a biotinylated alkyne, and the labelled proteins were pulled-down with streptavidin beads. After washing, immunoprecipitated proteins were eluted off beads in Laemmli buffer containing biotin, and analysed by immunoblotting. The palmitic acid analogue C15:0–azide was used as a positive control since TFR1 is known to also be palmitoylated. C17:0–azide pulls down more TFR1 than equal amounts of C15:0–azide, indicating that TFR1 palmitoylation cannot account for the C17:0 signal. The C17:0–azide–TFR1 interaction is completely blunted upon ZDHHC6 knockdown. See Supplementary Fig. 18 for image of the uncropped full western blot. d, TFR1 is required for C18:0 removal to induce mitochondrial fragmentation. HeLa cells were transfected with either control or TFR1 targeting siRNAs before treatment with medium containing delipidated serum plus or minus C18:0. Representative images are shown here and quantification of mitochondrial fragmentation is shown in Fig. 3h. n = 15.