Fig 2: Landscape of Parkin-Dependent Ubiquitylation and USP30-Dependent Deubiquitylation on Mitochondria in iNeurons(A) Venn diagram of overlapping diGLY sites observed in whole-cell lysates (WCLs) (6 h post-depolarization) from iNeurons and sites observed for purified mitochondria for HeLa cells 1 or 6 h post-depolarization. All peptides used were increased by at least 1-fold (log2 ratio > 1.0), with p < 0.05.(B) Venn diagram of overlapping diGLY sites observed in purified mitochondria (3 and/or 4 h post-depolarization) from iNeurons and sites observed for purified mitochondria for HeLa cells 1 or 6 h post-depolarization. All peptides used were increased by at least 1-fold (log2 ratio > 1.0), with p < 0.05.(C) Diagram showing the sites of ubiquitination in 41 proteins identified as high-confidence Parkin substrates in iNeurons and the corresponding identifications in previously reported data using HeLa cells ectopically expressing Parkin with a parallel TMT-MS3 platform (Rose et al., 2016). Residue numbers for diGLY modified Lys residues are shown. Black font and open hexagon, diGLY site found in purified mitochondria from iNeurons (3 and/or 4 h post-depolarization; Table S2) and in purified mitochondria from HeLa cells (1 or 6 h post-depolarization). Sites in black font also noted by the blue asterisk were also found in whole-cell lysates from iNeurons (6 h post-depolarization; Table S1). Red font and yellow hexagon, diGLY site found in purified mitochondria from iNeurons (3 and/or 4 h post-depolarization; Table S2) and in purified mitochondria from HeLa cells depolarized for 1 or 6 h. Blue font and blue hexagon, diGLY site found in whole-cell lysates from iNeurons (6 h post-depolarization; Table S1) and in purified mitochondria from HeLa cells depolarized for 1 or 6 h.(D) Model for USP30-dependent mitochondrial protein deubiquitylation.(E) Structural constraints for USP30-dependent removal of Ub from MOM proteins. Transmembrane segments, gray rectangles. Membrane proximal linkers, dotted lines. Sites of ubiquitylation (red) are those that are reversed by USP30 under depolarization conditions, while those in cyan are largely unchanged in USP30-/- cells. USP30 catalytic cysteine, yellow. TOMM70, modeled on S.c. Tom70, PDB: 2GW1; USP30 in complex with K6-di-Ub, PDB: 5OHP; CISD1 model, PDB: 3EW0; TOMM5 model, PDB: 3PRM; TOMM20 model, PDB: 1OM2.See also Figure S7 and Tables S1 and S3.

Fig 3: MTX1 interacted with CISD1 to co-regulate HCC cell resistance to sorafenib. (A) Protein interaction networks were obtained by GeneMANIA analysis performed on Cytoscape (Red lines). (B) Co-IP and western blot assays were performed using anti-Flag and Dynabeads Protein G to confirm semi-exogenous interaction between FLAG-MTX1 and CISD1. (C) Endogenous CISD1 antibody could pull down MTX1-FLAG protein through Co-IP assay. (D, E) Autophagy-related proteins were detected by western blot analysis when CISD1 was silenced or overexpressed. (F) CISD1 overexpression antagonized MTX1 overexpression-induced autophagy-related protein expression under the treatment of sofafenib. (G) CISD1 reduced cell viability induced by MTX1 overexpression. CCK-8 assay was used to monitor cell viability at 0 and 48 h after the treatment of sofafenib in HCC cells as indicated. Data are presented as mean ± SD from three experiments. NS: no significance, * P<0.05, ** P<0.01.

Fig 4: MITOL knockout attenuates Parkin recruitment to depolarized mitochondria and Parkin-catalyzed ubiquitylation. A, MITOL knockout HCT116 cells were generated using the CRISPR/Cas9 system. Intact HCT116 cells and MITOL knockout HCT116 cells stably expressing GFP–Parkin were treated with 15 µm CCCP for 60 min, and then the subcellular localization of GFP–Parkin was observed. Scale bars, 20 µm. B, statistical analysis of GFP–Parkin mitochondrial localization following 15 µm CCCP treatment for the indicated times in WT or MITOL knockout HCT116 cells. The percentage of cells with Parkin-positive mitochondria were determined using >100 cells. The numbers in the box-and-whisker plot are mean values across three independent experiments. Statistical significance was calculated using a one-tailed Student's t test. ***, p < 0.001. NS, not significant. C, MITOL depletion in MITOL knockout HCT116 cells was verified by immunoblotting (IB) with an anti-MITOL antibody. D, WT or MITOL knockout HCT116 cells stably expressing GFP–Parkin were treated with 15 µm CCCP for the indicated times and then immunoblotted with the indicated antibodies. The red bars indicate Parkin-catalyzed ubiquitylation (Ubn) of the endogenous substrates HKI, Tom20, and MitoNEET/CISD1.

Fig 5: Detection of PINK1-Dependent Mitophagy In Vitro and Detection of Endogenous PINK1 Protein at Basal Levels In Vivo(A) Primary MEF cultures established from Pink1+/+mito-QC+/+ and Pink1-/-mito-QC+/+ littermate embryos were transduced with retroviral HA-Parkin, and stimulated with DMSO (Control), CCCP, or DFP for 24 hr. Mitophagy (mCherry-only mitolysosomes) was visualized by confocal microscopy.(B) Mitophagy was readily observed in CCCP-treated WT and not in KO MEFs (two-way ANOVA, Bonferroni correction: ***p < 0.001, **p < 0.01; n.s., p > 0.05).(C) Immunoblot showing levels of exogenous HA-Parkin and endogenous PINK1 protein following CCCP treatment in Pink1 WT and KO mito-QC MEFs. Diminished levels of the Parkin substrate CISD1 and equal loading using vinculin are shown.(D) Pancreas sections from mCherry-GFP-LC3 mice and mito-QC animals from fasting experiments.(E) Elevated levels of general autophagy are observed following a 24-hr fasting period, compared with animals fed ad libitum (Student's t test; **p < 0.01). A corresponding induction of mitophagy is not observed upon starvation (n.s., p > 0.05).(F) IP-immunoblot showing biochemical detection of endogenous PINK1 protein in mouse brain homogenates, n = 2 animals per genotype. PINK1 protein is detectable at basal levels using enrichment by IP, but not in whole-cell lysates (inputs). Note the absence of the PINK1 band in the immunoglobulin G (IgG) control and KO samples.(G) Analytical validation of PINK1 biochemical experiments by mass spectrometry. Example of extracted-ion chromatograms detailing two unique PINK1 peptides present in IP's from WT, but not PINK1 KO cortex and cerebellum. Data are represented as mean ± SEM.Scale bars, 5 µm. See also Figure S1.