The gene encoding the yeast mitochondrial rotenone-insensitive internal NADH : ubiquinone-6 oxidoreductase has been sequenced. The DNA sequence indicates the presence of an open reading frame of 1539 bp predicted to encode a protein of 513 amino acid residues (57.2 kDa). The NADH dehydrogenase is synthesized as a precursor protein containing a signal sequence of 26 residues. In vitro import experiments show that the precursor NADH dehydrogenase is cleaved to the mature size by the matrix processing peptidase. Both cleavage and translocation across the mitochondrial membrane(s) are dependent on the membrane potential component of the proton-motive force.Comparison ofthe protein sequence of the yeast NADH dehydrogenase with the data bank indicates that the enzyme from yeast is homologous to the NADH dehydrogenase of Escherichia coli (22.2% identical residues). Both NADH dehydrogenases contain in the central part of the protein a sequence predicted to fold into a Pap structure involved in the binding of NADH or FAD(H2). Various aspects of the protein structure are discussed.The mitochondria of the yeast Saccharomyces cerevisiae contain at least two different NADH : ubiquinone-6 oxidoreductases, cach bound to the mitochondrial inner membrane, both being rotenone-insensitive and not coupled to site-1 phosphorylation [I -51. The enzyme facing the intermembrane space (external NADH dehydrogenase), is involved in the oxidation of NADH produced in the cytosol and may thus considered to be the physiological equivalent of the malate/ aspartate shuttle present in higher eukaryotes, but apparently absent in S. cercrisiae [4,6]. The external NADH dehydrogenase is also present in plant mitochondria, which, like yeast, apparently lack a functional malate/aspartate shuttle system The other type of dehydrogenase faces the matrix site (internal NADH dehydrogenase), oxidizes NADH produced by the Krebs cycle and the mitochondrial alcohol dehydrogenase and is the physiological counterpart of complex I [4], not present in S. cerevisiue. Neither of these rotenone-insensitive NADH dehydrogenases are present in mammalian mitochondria.[71.-___ Correspondence lo S. de Vrics, Section for Molecular Biology, Department of Molecular Ccll Biology, University of Amsterdam, Kruislaan 318, NL-1098 SM Amsterdam, Thc Nethcrlands.Abbreviation. DecylQ, 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinonc.l k z y n w . NADH : ubiquinone-6 oxidoreductase, NADH dehydrogenase (EC 3.6.5.3).N o f e . The novel nucleotide sequence data published here have bcen submitted to the EMBL sequencc data bank and are available under accession number X 61590.We have described the purification of a NADH :ubiquinone oxidoreductase from yeast mitochondria [3] and have subsequently established its identity as the internal NADH dehydrogenase by analysis of the properties of the mitochondria of a null mutant [5].Rotenone-insensitive NADH : ubiquinone oxidoreductases are not unique to yeast. For example, an enzyme similar to the single subunit, FAD-containing internal NADH dehydr...