HCCR-1 is localized on the mitochondria outer membrane. (A) COS-7 (top and second panels), MCF-7 (third and forth panels) and HEK293/HCCR-1-V5 (bottom panel) cells were cultured on the pre-coated coverslip for overnight. COS-7 and MCF-7 cells were transiently transfected with the pEGFP-N1 vector (GFP) or the vector carrying the human HCCR-1 gene tagged with GFP at its COOH-terminus (HCCR-1/GFP). The cells were incubated with MitoTracker (Red), fixed with 4% paraformaldyhyde. HEK293/HCCR-1-V5 cells were stained with MitoTracker (Red) and fixed with 100% methanol for 1 min at -20°C. Mouse anti-V5 antibody (1:300 dilution) and goat anti-mouse IgG conjugated with HRP (5 μg/ml) were applied to the cells for 1 h at room temperature (HCCR-1-V5). The cells were mounted and examined using a confocal microscope. (B) HCCR-1 is expressed at the mitochondrial membrane in the HEK293 cell line. Subcellular fraction of HEK293/HCCR-1-V5 cell line. HEK293/HCCR-1-V5 cells were fractionated into the nuclei (lane 1), post-mitochondria (lane 2), and mitochondria (lane3) fractions by differential centrifugation. Isolated mitochondria were treated with 0.1 M Na2CO3 and fractionated by centrifugation at 100,000 g into pellets (lane 4) and supernatant (lane 5). Proteins were analyzed by immunoblotting using antibodies to the V5 epitope (top panel), the mitochondrial membrane protein VDAC-1 (middle panel) and the mitochondrial matrix protein SOD-2 (bottom panel). (C) HCCR-1 topology on the mitochondrial outer membrane. Intact mitochondria were incubated for 30 min at room temperature without (lane 1) and with 50 μg/ml proteinase K (lane 2) or with 50 μg/ml proteinase K plus 1% Triton X-100 (lane 3). Each sample was precipitated with TCA and separated by SDS-PAGE and analyzed by immunoblotting with antibodies to V5 (top panel), to the outer membrane protein TOM40 (second panel) and to the inner membrane protein TIM23 (third panel), and to the matrix protein SOD-2 (bottom panel). (D) Schematic diagram shows the HCCR-1 topology at the outer mitochondrial membrane.