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 capable of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aerobic 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 central subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondrial inner membrane by N-terminally attaching the mitochondrial targeting sequence of NUAM, the largest subunit of complex I. Lethality of complex I mutations was rescued by the internal, but not the external version of alternative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth in the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial inner membrane indicates that alternative NADH:ubiquinone oxidoreductase requires no additional components for catalytic activity. Our findings also demonstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are insufficient in Y. lipolytica.
Kerscher
S J
SJ
Universitätsklinikum Frankfurt, Institut für Biochemie I, D-60590 Frankfurt am Main, Federal Republic of Germany. kerscher@zbc.klinik.uni-frankfurt.de
Eschemann
A
A
Okun
P M
PM
Brandt
U
U
D001692:Biological Transport; D042967:Electron Transport Complex I; D015870:Gene Expression; D007425:Intracellular Membranes / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I