In Saccharomyces cerevisiae, the NDI1 gene encodes a mitochondrial NADH dehydrogenase, the catalytic side of which projects to the matrix side of the inner mitochondrial membrane. In addition to this NADH dehydrogenase, S. cerevisiae exhibits another mitochondrial NADH-dehydrogenase activity, which oxidizes NADH at the cytosolic side of the inner membrane. To investigate whether open reading frames YMR145c/NDE1 and YDL 085w/NDE2, which exhibit sequence similarity with NDI1, encode the latter

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... yme, NADH-dependent mitochondrial respiration was assayed in wild-type S. cerevisiae and nde deletion mutants. Mitochondria were isolated from aerobic, glucose-limited chemostat cultures grown at a dilution rate (D) of 0.10 h ؊1 , in which reoxidation of cytosolic NADH by wild-type cells occurred exclusively by respiration. Compared with the wild type, rates of mitochondrial NADH oxidation were about 3-fold reduced in an nde1⌬ mutant and unaffected in an nde2⌬ mutant. NADH-dependent mitochondrial respiration was completely abolished in an nde1⌬ nde2⌬ double mutant. Mitochondrial respiration of substrates other than NADH was not affected in nde mutants. In shake flasks, an nde1⌬ nde2⌬ mutant exhibited reduced specific growth rates on ethanol and galactose but not on glucose. Glucose metabolism in aerobic, glucose-limited chemostat cultures (D ‫؍‬ 0.10 h ؊1 ) of an nde1⌬ nde2⌬ mutant was essentially respiratory. Apparently, under these conditions alternative systems for reoxidation of cytosolic NADH could replace the role of Nde1p and Nde2p in S. cerevisiae. During dissimilation of sugars via respiration by eukaryotic cells, glycolysis leads to NAD ϩ reduction in the cytosol, whereas mitochondrial oxidation of pyruvate via the pyruvatedehydrogenase complex and the trichloroacetic acid cycle yields NADH in the mitochondrial matrix. As the mitochondrial inner membrane is impermeable to NADH (1, 2), respiratory growth requires continuous reoxidation of this cofactor in the cytosol as well as in the mitochondrial matrix. The mitochondrial inner membrane of the yeast Saccharo-myces cerevisiae contains at least two NADH:ubiquinone-6 oxidoreductases ('NADH dehydrogenases') that may couple the oxidation of NADH to the mitochondrial respiratory chain (2 -5). The catalytic site of one of these, commonly referred to as the 'internal' NADH dehydrogenase, faces the mitochondrial matrix. Thus, it can oxidize the intramitochondrial NADH generated by the pyruvate-dehydrogenase complex and the TCA cycle (2). In contrast to the classical 'complex I' NADH dehydrogenases of higher eukaryotes, the 'internal' NADH dehydrogenase of growing S. cerevisiae cells is not proton translocating (5, 6). The enzyme consists of a single subunit encoded by the nuclear NDI1 gene (7) . Mutants in which NDI1 is inactivated do not oxidize intramitochondrial NADH (8). In addition to the NDI1-encoded 'internal' NADH dehydrogenase, S. cerevisiae is able to synthesize another inner membrane NADH dehydrogenase, commonly referred to as external NADH dehydrogenase, the catalytic site of which faces the intermembrane space (Refs. 2, 3, and 5; Fig. 1 ). In contrast to the mitochondria of fungi and plants (9, 10), mammalian mitochondria do not harbor external NADH dehydrogenases and therefore depend on redox shuttle mechanisms to couple the oxidation of cytosolic NADH to internal NADH dehydrogenases (11). The presence of an external NADH dehydrogenase in yeast mitochondria correlates with the absence of a functional malate-aspartate shuttle (5, 12), one of the major redox shuttles in mammalian mitochondria (11). However, the key enzymes for two alternative systems, the glycerol-3-phosphate dehydrogenase system and the ethanol-acetaldehyde shuttle (Fig. 1) , have both been demonstrated in S. cerevisiae (2, 13, 14) . A recent study indicated that the glycerol-3-phosphate dehydrogenase system contributes to the oxidation of cytosolic NADH under certain conditions but that it is not essential for respiratory growth of S. cerevisiae (15). The relative importance of the various proposed systems for respiratory oxidation of cytosolic NADH by S. cerevisiae mitochondria is at present unclear. Even under aerobic conditions, alcoholic fermentation rather than respiration is the predominant mode of sugar metabolism in S. cerevisiae (16). Fully respiratory growth on sugars is only possible during sugar-limited cultivation below the so called critical specific growth rate ( crit ). Above crit , respiration and aerobic alcoholic fermentation occur simultaneously, even in sugar-limited cultures (17) (18) (19) . Aerobic fermentation negatively affects the biomass yield on sugars (20). Therefore, biomass-directed industrial applications of S. cerevisiae, such as the production of bakers' yeast and heterologous proteins, have to be performed at submaximal growth rates in aerobic, sugarlimited fed-batch cultures (21, 22) . Competition between mitochondria and alcohol dehydrogenase for cytosolic NADH