Homologues of yeast and bacterial rotenone-insensitive NADH dehydrogenasesin higher eukaryotes: two enzymes are present in potato mitochondria

Two different cDNAs, homologous to genes for rotenone-insensitive NADH dehy drogenases of bacteria and yeast, were isolated from potato. The encoded pr oteins, called NDA and NDB, have calculated molecular masses of 55 and 65 k Da, respectively. The N-terminal parts show similarity to mitochondrial tar geting peptides and the polypeptides are in vitro imported into potato mito chondria. Import processing to a smaller polypeptide is seen for the NDA bu t not the NDB protein. After import, NDA is intramitochondrially sorted to the matrix side of the inner membrane, whereas NDB becomes exposed to the i ntermembrane space. Imported proteins are associated to membranes upon digi tonin permeabilization. On expression in Escherichia coil, NDB is released from the bacterial membrane in the absence of divalent cations whereas dete rgents are necessary for solubilization of NDA. Both deduced amino-acid seq uences contain the dual motifs for nucleotide binding with the characterist ics of the core criteria, similar to the bacterial homologues. Unique among NADH dehydrogenases, the NDB amino-acid sequence contains a non-conserved insert, which is similar to EF-hand motifs for calcium binding. Phylogeneti c analyses group the rotenone-insensitive NADH dehydrogenases largely by sp ecies, but suggest ancient gene duplications.