Fig 2: Landscape of the mitochondria-autophagosome synapse.(A) A schematic depicting a subset of proteins identified by proximity biotinylation is shown. OPTN, a Ub-binding receptor for mitophagy, associates with Ub via its C-terminal UBAN domain (dark blue) and is thought to interact minimally with a di-Ub moiety. OPTN is a coiled-coil dimeric protein and is expected to be ~20 nm in a fully extended conformation. Recruitment of APEX2-tagged OPTN to ubiquitylated PARK2 targets on the MOM allows the identification of proteins located nearby (within ~10 nm of the APEX2 domain). Biotinylated proteins identified by quantitative mass spectrometry are indicated with a “B.” The autophagosome/phagophore is recruited nearby the MOM (within ~25 to 50 nm). The approximate sizes of the TOMM40 component of the translocon, the VDAC proteins, and dimeric Ub are shown. (B) Schematic showing the potential role of HK2 as a protein that promotes assembly of PINK1 in a ~700-kDa complex with components of the translocon and PARK2. In the absence of HK2, there is reduced accumulation of PINK1 and PARK2 in the ~700-kDa complex and reduced levels of pS65-Ub.

Fig 3: TOM40 promotes the proliferation of epithelial ovarian cancer (EOC) cell lines and EOC xenografts. EOC cell lines (RMUS-S, TOV-112D, SK-OV-3, OVCA-429, and OVCA-433) that stably express sh-control and sh-TOM40 were established by lentivirus and selected with 3 µg/ml puromycin for 15 days. (A) Numbers of EOC cells that stably express sh-TOM40 were counted 48 hours after seeding with a LUNAIITM automated cell counter. Data are expressed as the mean ± minimum to maximum, n = 4; ** p < 0.01 and *** p < 0.001. TOM40 knockdown was examined by western blot analysis using an anti-TOM40 antibody (images to the right of each box plot). (B) The comparative growth rate of sh-control and sh-TOM40 ovarian cancer cell lines was measured daily with a crystal violet assay on days 1 through 4 post-seeding. Data are expressed as the mean ± Standard Deviation (S.D.)., n = 4; *** p < 0.001. (C) The long-term growth rates of EOC cells that stably express sh-TOM40 were evaluated via a colonization assay (RMUG-S, TOV-112D, and SK-OV-3 were evaluated 10 days post-seeding; OVCA-429 and OVCAR-433 were evaluated 11 days post-seeding). (Images, top half of panel) Representative images of crystal violet staining of EOC cells that stably express sh-control or sh-TOM40. (Bar graph, lower half of panel) Crystal violet intensities were measured at 595 nm using an ELISA reader. Data are expressed as the mean ± S.D., n = 4. (D) BALB/c nude mice were inoculated subcutaneously into both flanks with 3 × 106 RMUG-S cells that stably express sh-control or sh-TOM40. Six tumors were used in each group. Shown is an image of the aforementioned xenograft tumors, which were dissected from the mice 68 days after inoculation. The scale bar is 5 mm. (E) Tumor volume was monitored at 14, 26, 40, 54, and 68 days after inoculation with RMUG-S cells that stably express sh-control or sh-TOM40. Tumor growth is expressed as the mean tumor volume ± Standard Error (S.E.), n = 6; ** p < 0.01 and *** p < 0.001. (F) Final volume (left box plot) and final weight (right box plot) of tumors were measured after sacrifice at 68 days post-inoculation. Data are expressed as the mean ± minimum to maximum, n = 6; *** p < 0.001.

Fig 4: Accumulation of APP and stress responses in APPswe versus wild-type (WT) N2A cells. (a,b). Representative Western blot and quantification of amyloid ß precursor protein (APP) (n = 4–6) in total fraction (TF), microsomes (Micro), mitochondria (Mito), and mitochondrial-associated membranes (MAMs). (c–e). Representative Western blots and quantification of protein kinase R-like ER kinase (PERK) (n = 4–5) and inositol-requiring enzyme 1a (IRE1a) (n = 3–6) proteins in TF, Micro, and MAM. (f–i). Representative Western blots and quantification of endoplasmic reticulum oxidase 1 (ERO1a) (n = 5–9), proliferating cell nuclear antigen (PCNA) (n = 4–10) and outer mitochondrial membrane 40 (TOM40) (n = 4–9) proteins in TF, Micro, Mito, and MAM. (j,k). Representative Western blot and quantification for the chaperone heat shock protein 60 (Hsp60) (n = 4–10) in TF, Micro, Mito, and MAM. All data presented as mean ± SEM; p-values were obtained by using two-way ANOVA with Sidak’s multiple comparisons test for all proteins, except for (h) that was obtained using non-parametric Kruskal-Wallis with Dunn’s multiple comparisons test. * p = 0.05, ** p = 0.01, *** p = 0.001, and **** p = 0.0001 were considered significant.

Fig 5: Iron-induced Tom20 accumulation promotes pyroptosis. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM) for 2 h, followed by CCCP (20 µM), FeSO4 (100 µM), or CCCP/FeSO4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. a Top, addition of FeSO4 to CCCP induced Tom20 and Tom40 accumulation. Bottom, the effect of CCCP/FeSO4 on the expression levels of Tom20 and Tom40. Cells were treated with CCCP or CCCP/FeSO4 for the indicated times. b Knockdown of Tom20 rescued the cell viability in response to CCCP/FeSO4 stimulation. c–e Knockdown of Tom20 reversed the CCCP/FeSO4-induced cell morphology from pyroptosis to normal state, reduced LDH release and blocked GSDME cleavage (c), abolished cytochrome c release (detected in the cytosol fraction) and cleavage of caspase-3 and -9 (d), and blocked mitochondria aggregation (e). Hsp60 was used as a mitochondrial indicator, and DAPI was used to display the nuclei. f NAC and GSH attenuated the CCCP/FeSO4-induced Tom20 accumulation. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. ***P < 0.001