Fig 2: RNF34 is required for the clearance of damaged mitochondria upon viral infection AImmunoblot showing MAVS, IRF3‐p, LC3, TOM20, and HSP60 levels in siCtrl‐ or siRNF34‐1 cells infected with VSV and cultured in the presence of Z‐VAD (50 μM) for the indicated times. α‐Tubulin was used as a loading control.BImmunoblot showing MAVS, LC3, TOM20, and HSP60 levels in cells transfected with Flag‐RNF34 or its H342A mutant and subsequently infected with VSV for the indicated times.C, DRepresentative confocal images (C) and quantification (D) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl‐ or shRNF34‐1 cells infected with VSV for the indicated times. Red represents MAVS, and green represents NDP52. Scale bar, 10 μm.ETransmission electron microscopy images of shCtrl‐ and shRNF34‐1 HEK293T cells cultured in the presence or absence of VSV. Arrows indicate mitochondria engulfed in autophagosomes. Double arrows indicate swollen mitochondria with disrupted cristae. M, mitochondria and N, nucleus. 40,000× magnification.FRepresentative confocal images of the shift in the fluorescence emission of Keima from 458 to 543 nm. Scale bar, 10 μm.Data information: Cell‐based studies were performed independently at least three times with comparable results.Source data are available online for this figure.

Fig 3: Loss of MTFR1L leads to mitochondrial elongation.(A) Representative confocal images of mitochondrial morphology from WT and MTFR1L KO U2OS cells. Mitochondria were labeled with an anti-TOM20 antibody. Scale bar, 20 µm. (B) Immunoblot analysis of MTFR1L levels in MTFR1L KO U2OS clones. Vinculin and HSP60 were used as loading controls. (C) Quantification of mitochondrial morphology from (A). (D to F) Mitochondrial morphology was quantified as (D) mitochondrial number, (E) mean mitochondrial area, and (F) number of junctions, per ROI. (G) Live-cell imaging of WT and MTFR1L KO U2OS cells overexpressing the ornithine carbamoyl transferase (OCT)–photoactivable GFP (mt-PAGFP) probe and the mitochondrial marker TOM20-mCherry. Scale bar, 10 µm. (H) Quantification of the OCT-PAGFP probe diffusion from (G), calculated as a ratio between OCT-PAGFP–occupied area on total mitochondria network (TOM20-mCherry) at 0 and 5 min. (I) Representative TEM images from WT and MTFR1L KO U2OS cells. Scale bar, 1 µm. (J) Quantification of TEM images showing the distribution of mitochondrial length from (I). (K) Representative confocal images of mitochondrial morphology of WT and MTFR1L KO U2OS cells transiently overexpressing P2A-mCherry alone or MTFR1L-P2A-mCherry. Mitochondria were labeled with an anti-TOM20 antibody. Scale bar, 20 µm. (L) Quantification of mitochondrial morphology from (K). All values: mean ± SD; at least three independent experiments. See also figs. S2 and S3.

Fig 4: MAPL SUMOylates ABCD3 and modulates complex assembly HEK 293T‐REX (tetracycline‐regulatable expression) cells stably expressing an inducible Tet‐ON fusion construct MAPL‐Flag‐BirA, or Flag‐BirA only, were treated with Dox for 24 h in the presence of biotin, and biotinylated proteins were isolated with streptavidin beads for identification by mass spectrometry. Top hits, by peptide count, are shown.Starting material (SM) and MAPL immunoprecipitated (IP) fractions obtained from crude liver extracts were probed for ABCD3, MAPL and Hsp60.Cytosol and heavy membrane fractions isolated from MAPLf/f and MAPL−/− livers were solubilized and incubated with SIM beads, and elution fractions probed for ABCD3, DRP1, Hsp70 and MAPL (Cyt = cytosolic fraction, HM = heavy membrane fraction).Heavy membrane fractions isolated from livers of MAPLf/f and MAPL−/− animals' tail‐vein injected with adenovirus expressing MAPL‐Flag, MAPL‐ΔRING‐Flag or empty virus (Ad‐rtTA) were solubilized and incubated with SIM beads. Elution fractions were probed for ABCD3, SUMO1 and MAPL (top panel). Quantification from three independent experiments of ABCD3 signals of the heavy membrane SIM‐beads elution fraction from livers isolated from rescued mice (lower panel).Two hundred fifty microgram of solubilized protein (from MAPLf/f and MAPL−/− livers) were separated on a 10–50% (w/v) sucrose gradient. Twelve different fractions, as well as the resuspended pellet (P) were analyzed by immunoblot. ABCD3 signals from MAPLf/f and MAPL−/− livers in the different fractions were quantified and plotted as percentage of the total signal, from three biological replicates. *P < 0.05 in a one‐way ANOVA. Data are represented as mean ± SEM. Source data are available online for this figure.