Extended Data Fig. 6: ANT mediates closure of TIM23 via TIM44.
From: The ADP/ATP translocase drives mitophagy independent of nucleotide exchange

a, Deletion of Ant1 or Ant2 does not affect expression of TIM or TOM proteins (right) or destabilize TIM and TOM complexes, as assessed by blue native PAGE (left). b, c, ANT1 and ANT2 bind to TIMM23 and TIMM44, as assessed by co-immunoprecipitation (b) and blue native PAGE (c). The ANT–TIM23 complex is marked with an asterisk. d, Wild-type ANT1 and the ADP/ATP binding double mutant (K43E/R244E) bind to the TIM23 complex component TIMM23, whereas disease-causing mutants (A90D, A123D) do not. e, Closure of TIM23 in response to CCCP treatment is impaired in the presence of disease-causing mutants (A90D, A123D), but is preserved in the presence of the ADP/ATP binding double mutant (K43E/R244E), as shown by import of SU9–GFP into mitochondria; n = 3 biological replicates per group. P values calculated by two-sided unpaired t-test relative to empty. Scale bar, 40 μm. f, ANT1 binds to both TIMM23 and TIMM44. g, Mitophagy is impaired in cells lacking TIMM44; n = 3 biological replicates per gRNA, P values calculated by two-sided unpaired t-test relative to NTC_1. h, PINK1 stabilization by CCCP treatment is abrogated in the absence of TIMM44. i, Rescue of mitophagy with wild-type ANT and ADP/ATP exchange mutants (K33Q, K43E/R244E), but not with known disease-causing mutants (A90D, A123D) and TIMM44-binding site mutant (G146E/K147D). Top left, schematic of ANT and sites of mutations. Bottom, western blotting demonstrating equivalent expression of ANT constructs. Right, quantification of mitophagy; n = 3 biological replicates per group, P values calculated by one-way ANOVA with post hoc Tukey test, **P < 0.01, ***P < 0.001. j, Mutation of the predicted ANT1 interaction site in TIMM44 abrogates binding to ANT1. k, Rescue of mitophagy with wild-type TIMM44, but not with binding site mutant (K282D); n = 3 biological replicates per group, P values calculated by one-way ANOVA with post hoc Tukey test, **P < 0.01, ***P < 0.001. l, Mutation in TIMM44 of the ANT1 interaction site does not abrogate TIMM44 binding to TIMM23. Data are mean ± s.d. Similar results were obtained in two biological replicates (a–d, f, h–j, l). For gel source data, see Supplementary Fig. 1.