Fig. 6: Biochemical characterization of the WIPI–ULK1 mitophagy initiation complex.
a, Microscopy-based bead assay of GST-tagged WIPI2d or WIPI3 incubated with mCherry-tagged ATG13/101 complex, composed of FL ATG13 (mCh-ATG13/101), HORMA domain only (mCh-HORMA; ATG13 (residues 1–191)/101) or IDR only (mCh-IDR; ATG13 residues 191–517). b, As in a, but with GFP-tagged ATG13 IDR-coated beads, either as full IDR (residues 191–517) or fragments (residues 191–230; residues 191–205; residues 206–230), incubated with mCherry-tagged WIPI2d or WIPI3. c, As in a, but with GFP-tagged ATG13 IDR-coated beads, either as full-length IDR (191–517 aa) or with variants containing deletion fragments (Δ191–230 aa), (Δ191–205 aa) or (Δ206–230 aa), and incubated with mCherry-tagged WIPI2d or WIPI3. d, AlphaFold predicted structure of WIPI2d (orange) and ATG13 (green) plus ATG101 (blue) with zoom-in on the interaction interface. Note that the indicated residue numbers for WIPI2 correspond to their residue number in the WIPI2d sequence (which match Y113 and R143 in WIPI2b). Structures were trimmed for visual clarity. Displayed are ATG13 (residues 1–223), ATG101 (residues 1–218) and WIPI2d (residues 1–383). e, As in a, but with GFP-tagged ATG13 IDR (residues 191–517) coated beads and incubated with mCherry-tagged WIPI2d or WIPI3. The IDR is composed of either WT, 3x Ala mutant (3A) or 11x Ala mutant (11A) (as indicated). f, Mitophagy flux measured by flow cytometry of WT or ATG13 KO HeLa cells, rescued (as indicated) with ATG13 WT, ATG13 lacking residues 191–230 (Δ191–230), ATG13 lacking residues 191–205 (Δ191–205) or ATG13 lacking residues 206–230 (Δ206–230), left untreated or treated with DFP for 24 h. Data are presented as mean ± s.d. (n = 3 biologically independent experiments). Two-way ANOVA with Dunnett’s multiple comparisons test. ****P < 0.0001. NS, not significant. Results are representative of three independent replicates (a–c,e). Scale bars, 100 µm. Source numerical data, including exact P values, are available in the source data.
