The SAMM50 N-terminal region with the NTS and POTRA domain is not required for mitochondrial protein biogenesis. (A and B) Whole cell lysates from WT cells and two clones of SAMM50 KD cells were immunoblotted to reveal PINK1, p62 and LC3B protein levels (A) and quantified (B). Values are mean ± SD from three different experiments. **, P < 0.005; *, P < 0.01; †, NS; one-way ANOVA. (C and D) HeLa cells were either treated with CTL siRNA or two different siRNA to TOMM40 and analyzed for the levels of indicated proteins by immunoblotting (C) and quantified (D). Values are mean ± SD from three different experiments. **, P < 0.005; *, P < 0.01; one-way ANOVA. (E and F) WT and SAMM50 KD cells were treated with a combination of OA for 3h and protein levels of PINK1 were analyzed by immunoblotting (E) and quantified (F). Values are mean ± SD from three different experiments. ***, P < 0.001; **, P < 0.005; one-way ANOVA. (G) Domain architecture of SAMM50 showing WT and various deletion constructs used in reconstituting SAMM50 KD cells. (H) Expression of the indicated mitochondrial proteins in WT, SAMM50 KD, AND SAMM50 KD cells reconstituted with WT SAMM50 or SAMM50 Δ1-125 mutant was analyzed by immunoblotting. (I and J) WT, SAMM50 KD, and SAMM50 KD cells reconstituted with Myc-SAMM50 and indicated deletion mutants were analyzed for expression of the indicated mitochondrial proteins by immunoblotting (I) and quantified (J). Values are mean ± SD from three different experiments. ***, P < 0.001; **, P < 0.005; one-way ANOVA. (K) Isolated mitochondria from SAMM50 KD cells reconstituted with Myc-SAMM50 were subjected to digestion with different concentration of proteinase K. Mitochondria protein levels were analyzed by immunoblotting with indicated antibodies.