Targeting of mitoChR2(SSFO)-YFP to the inner mitochondrial membrane. Representative YFP fluorescence traces of the indicated constructs and experimental procedures. (A) YFP fluorescence monitored in HeLa cells expressing mitoChR2(SSFO)-YFP, the outer mitochondrial membrane marker N33D3cpv, or the matrix-localized 4mtD3cpv. Where indicated, cells were treated with protease (proteinase K; 20 μg/μL), digitonin (100 μM), and trypan blue (0.5 mg/mL). (B) Cells expressing mitoChR2(SSFO)-YFP or 4mtD3cpv were permeabilized with digitonin. Where indicated, alamethicin (20 μg/μL) was added. (C) To verify that mitoChR2(SSFO)-YFP was correctly folded and the YFP faced the matrix, after permeabilization and alamethicin treatment trypan blue was used to quench YFP fluorescence. a.u., arbitrary units.