Human C17orf80 and its mouse ortholog D11Wsu47e colocalize with mtDNA. (A) The proteins interacting with mtDNA were screened by IP followed by MS identification. Isolated mitochondria from HEK-293T cells were subjected to IP using an anti-DNA antibody. The score number reflects the levels of protein credibility and abundance. The known mtDNA-interacting proteins (red) and the uncharacterized protein C17orf80 (blue) are marked. (B) Western blot confirmed the interaction between C17orf80 and mtDNA. Anti-DNA antibody was used for IP. TFAM served as the positive control and MCU (the core channel portion of mitochondrial Ca²⁺ uniporter holocomplex) as the negative control. (C) Western blot showing C17orf80 is highly enriched in mitochondria isolated from HEK-293T cells. ATP5A and β-actin served as the mitochondrial and cytosol markers, respectively. (D) Electron microscopy images of immunogold stained mitochondria in HEK-293T cells not expressing (CON) or stably expressing C17orf80-HA fusion protein (OE). Anti-HA antibody was used for immunogold staining. Left: Western blot showing expression of endogenous and C-terminal HA-tagged C17orf80 in CON and OE cells. Anti-β-actin served as the loading control. Middle: Representative electron microscopy images. Red arrowheads indicate immunogold particles. Scale bars: 100 nm. Right: Quantification of particle numbers. Data are mean ± SEM. n = 25 mitochondria per group. (E and F) Immunofluorescence staining shows that C17orf80 is distributed in a punctate pattern within the mitochondria of HeLa cells (E) and U2OS cells (F). Mitochondria were visualized with anti-ATPB immunofluorescence, and C-terminal Myc-tagged C17orf80 was stained with anti-Myc immunofluorescence. Scale bars are 5 μm in the large-view image and 1 µm in the zoom in image. (G) Colocalization of C17orf80 and mtDNA in HeLa cells. The C-terminal Flag-tagged C17orf80 was indicated by anti-Flag immunofluorescence and mtDNA was stained by Picogreen. Note that nuclear (N, marked by a dotted line) DNA was also stained by Picogreen. The pixel intensity plot of the white dashed line is shown in the right panel. Scale bars: 2 µm. AU, arbitrary unit. (H) Immunofluorescence staining showing colocalization between C17orf80 and mtDNA in HeLa cells. C17orf80-Flag and mtDNA were indicated by anti-Flag and anti-DNA immunofluorescence, respectively. The nucleus (N) was marked by a dotted line. The pixel intensity plot of the white dashed line is shown in the right panel. Scale bars: 2 µm. (I) Colocalization of mouse ortholog D11Wsu47e with mtDNA in MEF cells. D11Wsu47e was indicated by anti-HA immunofluorescence, mtDNA was stained by Picogreen, and mitochondria was indicated by anti-ATPB immunofluorescence. The nucleus (N) was marked by a dotted line. The right panel shows a pixel intensity plot of the white dashed line. Scale bars: 2 µm. (J) Pearson’s correlation coefficients are shown for C17orf80-Flag and Picogreen in G, C17orf80-Flag and mtDNA in H, and D11Wsu47e-HA and Picogreen in I, respectively. Source data are available for this figure: SourceData F1.