Revised transmembrane orientation of the NADH:quinone oxidoreductase subunit NuoA

► The orientation of NuoA was investigated using a novel cytochrome c fusion protein technique. ► Conventional PhoA-fusions of NuoA and control proteins were analyzed in parallel. ► Theoretical predictions of the transmembrane topology were improved. ► The NuoA polypeptide was demonstrated to have the C-terminal end exposed in the bacterial cytoplasm.