The N-terminal domain of SAMM50 is dispensable for its activity and is oriented to the cytoplasm. (A and B) Whole-cell lysates of WT, SAMM50 KD, and SAMM50 KD HeLa cells reconstituted with Myc-tagged SAMM50 WT and SAMM50 Δ1–125 mutant analyzed for mitochondrial protein expression by immunoblotting (A), and relative expression levels were quantified (B). Values are mean ± SD from three independent experiments. **, P < 0.005; one-way ANOVA. (C) SIM of WT, SAMM50 KD, and SAMM50 KD cells reconstituted with Myc-SAMM50 and stained with antibodies to SAMM50 and TIMM23. Scale bars, 10 µm. (D) TEM of mitochondrial ultrastructure of WT, SAMM50 KD, and SAMM50 KD cells reconstituted with either WT Myc-SAMM50 or Myc-SAMM50 Δ1–125. Scale bars, 0.2 µm. (E) Cristae morphology shown in D was scored based on 150 mitochondria from 9–10 micrographs per sample (see Materials and methods). Values are mean ± SD. **, P < 0.005; one-way ANOVA. (F) Mitochondria from SAMM50 KD cells reconstituted with Myc-SAMM50 subjected to increasing concentrations of proteinase K analyzed by immunoblotting. (G) SAMM50 and p62 were N-terminally tagged, while MIC19 was C-terminally tagged, with the 11th β-sheet of split fluorescent modified mNG₁₁ and stably coexpressed, respectively, in HeLa cells stably expressing the first 10 β-sheets of modified mNG_1-10 in a Tet-off/on system. Fluorescence complementation was induced with 1 µg/ml Tet overnight and analyzed by live-cell imaging. Mitochondria were imaged with MitoTracker Deep Red. Scale bars, 20 µm (main), 5 µm (inset). (H) Mitochondria from SAMM50 KD cells reconstituted with Myc-SAMM50 Δ1–40 treated with increasing concentrations of proteinase K and immunoblotted with the indicated antibodies.