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. 2023 Nov 14;24(12):e57972. doi: 10.15252/embr.202357972

Figure 1. MAPL SUMOylates ABCD3 and modulates complex assembly.

Figure 1

  1. 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.
  2. Starting material (SM) and MAPL immunoprecipitated (IP) fractions obtained from crude liver extracts were probed for ABCD3, MAPL and Hsp60.
  3. 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).
  4. 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).
  5. 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.