Fig. 2: NIX and BNIP3 initiate mitophagy through WIPI2 and WIPI3.
a, Identification of interactors of NIX(1–182)-GST and BNIP3(1–158)-GST by pulldown from HeLa cell lysates and mass spectrometry. Tables represent the top hits for NIX (upper) and BNIP3 (lower). b, Validation of mass spectrometry data by pulldowns with SDS–PAGE and western blot. c,d, Microscopy-based bead assays of NIX-GST or BNIP3-GST incubated with mCherry-tagged WIPI1, WIPI2d, WIPI3 or WIPI4. e, AF2 predicted structure of NIX or BNIP3 and WIPI2d. Note that the indicated residue numbers for WIPI2 correspond to their residue number in the WIPI2d sequence. Conservation of the interaction interface between NIX and BNIP3 is displayed. f,g, As in c, but with NIX wild-type (WT), E72A/L75A/D77A/E81A mutant (4A) or W36A/L39A (ΔLIR) and mCherry-tagged WIPI2d WT or K87A/K88A mutant. h, As in c, but with BNIP3 WT or W18A/L21A mutant (ΔLIR) and mCherry-tagged WIPI3 or WIPI2d. i, AF2 Multimer predicted complex structure of WIPI2d and NIX (residues 30–82). The zoom highlights the interaction between the LIR of NIX and WIPI2d. The C-terminal intrinsically disordered region of WIPI2d is omitted for visual clarity. j, Number of backbone hydrogen bonds, nH-bonds, between the LIR of NIX and WIPI2d, insertion depth dTRP of NIX W36, and minimum heavy atom distance dpocket between WIPI2d F169 and I133 from three 1-μs MD simulations. k, Representative snapshots of W36 interacting with WIPI2d (top) and inserted into an initially closed pocket (bottom). The symbols in the lower left corner indicate the point in the trajectory in j from which the respective snapshots were extracted. l, Mitophagy flux measured by flow cytometry of WT or NIX/BNIP3 double-knockout (2KO) HeLa cells, rescued with V5-BNIP3, V5-NIX, V5-NIX ΔLIR (W36A/L39A mutant) or V5-NIX ΔWIPI2 (4A mutant; E72A/L75A/D77A/E81A), untreated or treated with DFP for 24 h. The percentage of non-induced cells (lower right) versus mitophagy-induced cells (upper left) is indicated. Representative fluorescence-activated cell sorting (FACS) plots are shown from one of three biological replicates, and data are presented as mean ± s.d. (n = 3 biologically independent experiments). Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. ****P < 0.0001. NS, not significant. Results are representative of three replicates (b–h). Scale bars, 100 µm. Source numerical data, including exact P values, and unprocessed blots are available in the source data.
