External alternative NADH : ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency in Yarrowia lipolytica

Alternative NADH:ubiquinone oxidoreductases are single subunit enzymes capa ble of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aero bic yeast Yarrowia lipolytica has only one such enzyme, encoded by the NDH2 gene and located on the external face of the mitochondrial inner membrane. In sharp contrast to ndh2 deletions, deficiencies in nuclear genes for cen tral subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondr ial inner membrane by N-terminally attaching the mitochondrial targeting se quence of NUAM, the largest subunit of complex I. Lethality of complex I mu tations was rescued by the internal, but not the external version of altern ative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth i n the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial in ner membrane indicates that alternative NADH:ubiquinone oxidoreductase requ ires no additional components for catalytic activity. Our findings also dem onstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are i nsufficient in Y lipolytica.