Fig 2: CLIPT controls MCU degradation.a Doxycycline (doxy) treatment represses transcription of MCU-Flag. MCU-Flag persists after Complex I inhibition (rotenone) but not Complex III (antimycin A) or IV (sodium azide). b MCU-Flag stabilization induced by NTD peptide. c Design of rapamycin (Rapa)-induced dimerization experiment to test if MCU-Complex I interactions dictate MCU degradation. d Immunocytochemistry reveals co-localization of FRB-MCU, NDUFA10-FKBP, and mito-FKBP with mitochondrial markers CoxIV or ATP5F1. e With replacement of NTD with FRB, MCU only binds FKBP-tagged NDUFA10 in the presence of 100 nM rapamycin. Mito-FKBP-HA is a control. f FRB-MCU is stable in the absence or presence of rapamycin when cells co-express mito-FKBP-HA control, but rapidly degraded when rapamycin induces Complex I-binding in NDUFA10-FKBP-HA expressing cells. Images are representative of 3 (a, b, f) or 2 (d, e) separate trials. Source data are provided as a Source Data file.

Fig 3: Genetic interaction between MCU and Complex I in Drosophila.a, c, e Drosophila survival for the indicated genotypes and sex. b, d, f Quantification of dwell time in the island assay for indicated genotypes for female flies. a, b The muscle specific MHC-GAL4 was used to drive expression of NDUFB10RNAi, MCUWT, or MCU?NTD in wild-type or MCU1/MCU1 mutant flies, as indicated. c, d As in (a, b), except the dominant-negative pore mutant MCUDQEQ was expressed with MHC-GAL4. e, f As in (a, b), except the isolated NTD fragment was expressed with MHC-GAL4. *p < 0.05, **p < 0.01, ***p < 0.001. Statistics: (a, c, e) Fischer’s Exact Test with Bonferroni correction; (b, d, f) Log-rank (Mantel-Cox) test with Bonferroni correction. Source data are provided as a Source Data file.

Fig 4: Loss of OPTN impairs mitophagy flux in AML cells. (A) Representative flow cytometry plots of mt-mKEIMA in MV4–11 and THP1 cells after knockdown of OPTN and treatment with 0.5 mM deferiprone (DFP) for 16 h. (B) Quantification of mitophagy induction from (A) and Figure S2E. Data are normalized to untreated control cells (n = 3). (C) Representative flow cytometry plots of mt-mKEIMA in MV4–11 and THP1 cells after knockdown of OPTN and treatment with 10 µM oligomycin/antimycin a (OA) for 4 h. (D) Quantification of mitophagy induction from (C) and Figure 2E. Data are normalized to untreated control cells (n = 3). (E) Schematic illustration of colocalization experiments for early- and late-stage mitophagy using immunofluorescent staining of TIMM23 or COX4/COXIV in combination with LC3 or LAMP1. (F) Representative immunofluorescent staining of early-stage mitophagy (see E) of CTRL and OPTN-KD human MV4–11 cells after 16 h of 0.5 mM DFP treatment for mitochondrial and autophagosomal markers TIMM23 and LC3, respectively. Arrows indicate colocalization of both markers. DAPI (4‘,6-diamidino-2-phenylindole) was used as a nuclear stain. Scale bar: 10 µm. Original magnification × 63. (G) Pearson’s coefficient for colocalization of TIMM23 and LC3 was analyzed using ImageJ for MV4–11 and THP1 cells (n = 3 with > 50 cells per replicate). Pearson’s coefficient was calculated between TIMM23 staining (channel A) and LC3 staining (channel B) in single cells upon automatic thresholding using moments algorithm [23]. (H) Immunofluorescent staining of late-stage mitophagy (see E) of CTRL and OPTN-KD human THP1 cells after 16 h of 0.5 mM DFP treatment was performed with mitochondrial and lysosomal markers COX4 and LAMP1, respectively. Arrows indicate colocalization of both markers. DAPI (4‘,6-diamidino-2-phenylindole) was used as a nuclear stain. Scale bar: 10 µm. Original magnification × 63. (I) Pearson’s coefficient for colocalization of COX4 and LAMP1 was analyzed using ImageJ for THP1 cells (n = 3 with > 50 cells per replicate). Values are means ± SEM. Student‘s t-test was performed in (B, D, G, I). Note: *p < 0.05, **p < 0.01, ***p < 0.001.