Figure 4.

FAM210B is transported into and is localized in the mitochondria. (a) Schematic representation of FAM210B domains based on the primary structure of FAM210B. (b) Confocal microscopy of HeLa cells transfected with GFP-tagged human FAM210B and RFP-tagged mitochondria. FAM210B-GFP, GFP sequence introduced at the C terminus of FAM210B; FAM210B (MTS)-GFP, the MTS (aa 1–47) of FAM210B was added to the GFP N terminus; FAM210B (ΔMTS)-GFP, GFP was added to the C terminus of MTS (1–47)-deleted CRIF1. Scale bar, 20 mm. (c) Confocal microscopy of HeLa cells transfected with GFP-tagged human FAM210B and RFP-tagged endoplasmic reticulum. (d) Western blotting analysis following subcellular fractionation of GFP-tagged human FAM210B HeLa cells. (e) Western blotting analysis following subcellular fractionation of endogenous in HeLa cells. (f) The mitochondria of HeLa cells were swollen and sonicated to disrupt membranes, washed with alkali buffer (pH 11.5) to detach loosely associated proteins from membranes, and then re-isolated by ultracentrifugation. The supernatant (Supe) and membrane fractions (Pellet) were subjected to western blotting for FAM210B, TOM20, or MnSOD. (g) Mitochondria isolated from HeLa cells were subjected to proteinase K (PK) proteolysis to digest exposed proteins, and detergent (SDS) was used to disrupt both IMMs (inner membrane of mitochondria) and OMMs (outer membrane of mitochondria). The lysates were resolved and subjected to immunoblot analyses. The submitochondrial markers used are Tom20 (OMM), Cyt C (Cytochrome c, intermembrane space), NDUFS1 (IMM), and MnSOD (mitochondrial matrix)