Electron transport is a general cellular process in which electrons generated from the oxidation of molecules such as NADH and FADH.sub.2 are transferred, through the action of various enzymes, to a series of electron carriers. These electron carriers may act as electron donors for various reductive reactions in the cell or may transport their electrons to other electron carriers along an electron transport chain. The change in oxidation potential as electrons are passed along such a chain generates energy which may be used by the cell.
The mitochondrial electron transport (or respiratory) chain is comprised of a series of enzyme complexes in the mitochondrial membrane. This transportation chain is responsible for the transfer of electrons from NADH through a series of redox centers (electron carriers) within these complexes to oxygen, as well as for the coupling of this oxidation to the synthesis of ATP (oxidative phosphorylation). ATP provides the primary source of energy for driving a cell's many energy-requiring reactions. The key enzyme complexes in the respiratory chain are NADH:ubiquinone oxidoreductase (NADH-D), succinate:ubiquinone oxidoreductase, cytochrome c.sub.1 -b oxidoreductase, cytochrome c oxidase (COX), and ATP synthase. These complexes are located on the inner matrix side of the mitochondrial membrane with the exception of succinate:ubiquinone oxidoreductase, which is located on the cytosolic side. NADH-D initiates the first step in the respiratory chain by accepting electrons from NADH and passing them through a flavin molecule and several iron-sulfur centers to ubiquinone. Succinate:ubiquinone oxidoreductase also transports electrons generated by oxidation of succinate to fumarate in the citric acid cycle through electron carriers (FAD and iron-sulfur centers) to the membrane bound ubiquinone. Cytochrome c.sub.1 -b oxidoreductase accepts electrons from ubiquinone and passes them on to cytochrome c. COX accepts electrons from cytochrome c and catalyzes the last, and most important, transfer of electrons to oxygen. Energy released in the course of each of these electron transfers is harnessed by ATP synthase to form ATP (oxidative phosphorylation).
NADH-D, the largest of these complexes with an estimated mass of 800 kDa, contains approximately 40 polypeptide subunits of widely varying size and composition. NADH-D is highly conserved in a variety of mammalian species including rat, rabbit, cow, and human (Cleeter, M. W. J. and Ragan, C. I. (1985) Biochem. J. 230: 739-46). The best characterized NADH-D is from bovine heart mitochondria and is composed of 41 polypeptides (Walker, J. E. et al. (1992) J. Mol. Biol. 226: 1051-72). Seven of these polypeptides are encoded by mitochondrial DNA, while the remaining 34 are nuclear gene products that are imported into the mitochondria. Six of these imported polypeptides are characterized by N-terminal signal peptide sequences which target these polypeptides to the mitochondria and are then cleaved from the mature proteins. A second group of polypeptides lack N-terminal targeting sequences and appear to contain import signals which lie within the mature protein (Walker et al., supra). The measured molecular masses of several of the smaller polypeptides, B8, B13, B14, B15, and B22, are consistent with post-translational removal of the terminal methionine residue and N-acetylation of the adjacent amino acid.
The functions of many of the individual subunits in NADH-D are largely unknown. The 24-, 51-, and 75-kDa subunits have been identified as being catalytically important in electron transport, with the 51-kDa subunit forming part of the NADH binding site and containing the flavin moiety that is the initial electron acceptor (Ali, S. T. et al. (1993) Genomics 18:435-39). The location of other functionally important groups, such as the electron-carrying iron-sulfate centers, remains to be determined. Many of the smaller subunits (&lt;30 kDa) contain hydrophobic sequences that may be folded into membrane spanning .alpha.-helices. These subunits presumably are anchored into the inner membrane of the mitochondria and interact via more hydrophilic parts of their sequence with globular proteins in the large extrinsic domain of NADH-D. The remaining proteins are likely to be globular and form part of a domain outside the lipid bilayer.
The PDSW subunit of bovine mitochondrial NADH-D is one of these smaller subunits (22 kDa) that is mostly hydrophilic in nature and contains no apparent hydrophobic anchor. It is nuclear encoded but does not contain an N-terminal signal sequence nor a modified N-terminal residue. The function of the PDSW subunit is unknown, but it is probably one of the globular subunits located outside of the lipid bilayer (Walker et al., supra) Defects and altered expression of NADH-D are associated with a variety of human diseases, including neurodegenerative diseases, myopathies, and cancer (Singer, T. P. et al. (1995) Biochim. Biophys. Acta 1271:211-19; Selvanayagam, P. and Rajaraman, S. (1996) Lab. Invest. 74:592-99). In addition, NADH-D reduction of the quinone moiety in chemotherapeutic agents such as doxorubicin is believed to contribute to the antitumor activity and/or mutagenicity of these drugs (Akman, S. A. et al. (1992) Biochemistry 31:3500-6).
The discovery of a new NADH dehydrogenase subunit and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cancer, immune disorders, and myopathies.