FIGURE 1.

hCOA3 is a transmembrane protein of the inner mitochondrial membrane. A, immunocytochemistry of HeLa cells overexpressing hCOA3-FLAG (hCOA3-FLAG). hCOA3-FLAG was visualized using anti-FLAG M2 antibodies. Mitochondria were stained using Mitotracker Red. Images were obtained in a Leica SP5 confocal microscope with a Leica plan apochromat 63×/1.4 numerical aperture objective lens. Scale bars represent 10 μm. B, purified mitochondria from hCOA3-FLAG-overexpressing cells (hCOA3-FLAG) were sonically irradiated and centrifuged to separate the soluble (S) and membrane (M) proteins. The membrane pellet was treated with 0.1 m Na₂CO₃ and centrifuged to separate the soluble membrane extrinsic (CS) from the insoluble intrinsic membrane proteins (P). Equivalent volumes of each fraction were analyzed by Western blotting. C, mitochondria (Mc) and mitoplasts (Mp) from hCOA3-FLAG-overexpressing cells were treated with proteinase K where indicated. After proteinase K treatment, mitochondria and mitoplasts were recovered by centrifugation and analyzed by Western blotting. D, purified mitochondria from hCOA3-FLAG overexpressing cells were treated with 1% digitonin to selectively solubilize the mitochondrial outer membrane. Membranes (P) and the soluble fraction (S) were separated by centrifugation and analyzed by Western blotting. 0.5, 1, 2, and 3 times of equivalent soluble fraction were loaded on the gel.