Extended Data Figure 3: Mitofusin loss of function phenocopies Elovl6 mutation or removal of C18:0.

a, dMfn knockout larvae (1st instar) have fragmented mitochondria, visualized with mitoGFP. Representative of ten images. b, Endogenous dMfn runs as a main band plus a laddering of apparently increasing molecular weights on an SDS–PAGE gel. Specificity is controlled by blotting lysates from control and Mfn-knockout female larvae with anti-Mfn antibody. c, Homozygous mutation of dMfn is lethal. Mfn-knockout larvae survive for several days as small L1/L2 larvae and eventually die. Synchronized 1st instar larvae were grown on standard fly food and examined every 24 h for developmental stage and per cent survival (n = 30). d, dMfn-knockout animals have impaired oxygen consumption. Oxygen consumption of inverted, digitonin permeabilized, female larval tissues was measured with an Oroboros oxygraph chamber and normalized to tissue weight. Oxygen consumption was measured in the presence of the following substrates: GMN (glutamate and malate), GMD (glutamate, malate and ADP), GMcD (glutamate, malate, cytochrome c and ADP), GMScD (glutamate, malate, succinate, cytochrome c and ADP), ETS (glutamate, malate, cytochrome c, ADP and uncoupling reagent), and Sc(Rot)u (glutamate, malate, cytochrome c, ADP and rotenone). n = 5. e, Endogenous dMfn is post-translationally modified in a C18:0-dependent manner in Drosophila. dMfn from Elovl6 female mutants migrates in an SDS–PAGE gel differently, compared with Mfn2 from control animals. This is reversed by supplementing the diet with C18:0. All indicated bands are dMfn, since they disappear in lysates from dMfn-knockout animals (see Extended Data Fig. 3b). Flies were grown on antifungal-free food. f, Endogenous MFN2 is post-translationally modified in a C18:0-dependent manner in human HeLa cells. MFN2 immunoprecipitated from HeLa cells treated for 24 h with medium containing standard or delipidated serum, and then for 2 h in the absence or presence of C18:0 (100 µM), lysed in 8M urea (see Methods). g, C18:0 affects ubiquitination of MFN2. MFN2 is more heavily ubiquitinated in cells treated with delipidated serum than in control cells and this is reversed by supplementing the medium with C18. HeLa cells were cotransfected with tagged versions of MFN2 (myc) and ubiquitin (HA). Tagged MFN2 was immunoprecipitated and blots were probed with HA antibody to detect ubiquitination. Quantification of ubiquitination, normalized to Myc–MFN2 in the immunoprecipitate (IP) is shown below the lane. h, C18:0 removal destabilizes MFN2 protein. A cyclohexamide (CHX) chase experiment was performed to block de novo synthesis of MFN2, thereby looking at turnover of existing MFN2 protein in vivo. HeLa cells treated with medium containing delipidated serum plus or minus C18:0 were treated with 100 µM CHX and then lysed at the indicated time points to compare MFN2 protein levels. Bottom, densitometric quantification of the blots normalized to loading control. i, dMfn expression is upregulated in Elovl6⁻ flies compared with controls. dMfn transcript levels in 24 h female pre-wandering larvae were determined by quantitative polymerase chain reaction with reverse transcription (RT–PCR), normalized to rp49 (in triplicates). Scale bar, 10 μm. d, i, *P < 0.05, **P < 0.01, ***P < 0.001, not significant (NS) P ≥ 0.05, two-tailed t-test. Error bars represent s.d. See Supplementary Fig. 16 for images of the uncropped full western blots.