THE NADH-BINDING SUBUNIT OF RESPIRATORY-CHAIN COMPLEX-I IS NUCLEAR-ENCODED IN PLANTS AND IDENTIFIED ONLY IN MITOCHONDRIA

In higher plants, genes for subunits of respiratory chain complex I (N ADH:ubiquinone oxidoreductase) have so far been identified solely in o rganellar genomes. At least nine subunits are encoded by the mitochond rial DNA and 11 homologues by the plastid DNA. One of the 'key' compon ents of complex I is the subunit binding the substrate NADH. The corre sponding gene for the mitochondrial subunit has now been cloned and id entified in the nuclear genome from potato (Solanum tuberosum). The ma ture protein consists of 457 amino acids and is preceded by a mitochon drial targeting sequence of 30 amino acids. The protein is evolutionar ily related to the NADH-binding subunits of complex I from other eukar yotes and is well conserved in the structural domains predicted for bi nding the substrate NADH, the FMN and one iron-sulphur cluster. Expres sion examined in different potato tissues by Northern blot analysis sh ows the highest steady-state mRNA levels in flowers. Precursor protein s translated in vitro from the cDNA are imported into isolated potato mitochondria in a Delta psi-dependent manner. The processed translatio n product has an apparent molecular mass of 55 kDa, identical to the m ature protein present in the purified plant mitochondrial complex I. H owever, the in-vitro translated protein is not imported into isolated chloroplasts. To further investigate whether the complex I-like enzyme in chloroplasts contains an analogous subunit for binding of NAD(P)H, different plastid protein fractions were tested with a polyclonal ant iserum directed against the bovine 51 kDa NADH-binding subunit. In non e of the different thylakoid or stroma protein fractions analysed were specific crossreactive polypeptides detected. These results are discu ssed particularly with respect to the structure of a potential complex I in chloroplasts and the nature of its acceptor site.