NUCLEAR AND MITOCHONDRIAL FORMS OF HUMAN URACIL-DNA GLYCOSYLASE ARE ENCODED BY THE SAME GENE

Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of M(r) = 33,800 contains an N -terminal sequence of 77 amino acids not present in the presumed matur e form of M(r) = 25,800. This led to the hypothesis that the N-termina l sequence might be involved in intracellular targeting. To examine th is hypothesis, we analysed UDG from nuclei, mitochondria and cytosol b y western blotting and high resolution gel filtration. An antibody tha t recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they are products of the same gene. The nuclear and mitochondrial form had an apparent M(r) = 27,500 and t he cytosolic form an apparent M(r) = 38,000 by western blotting. Gel f iltration gave essentially similar estimates. An antibody with specifi city towards the presequence recognised the cytosolic form of M(r) = 3 8,000 only, indicating that the difference in size is due to the prese quence. Immunofluorescence studies of HeLa cells clearly demonstrated that the major part of the UDG activity was localised in the nuclei. T ransfection experiments with plasmids carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochon dria, whereas UNG lacking the presequence was translocated to the nucl ei. We conclude that the same gene encodes nuclear and mitochondrial u racil-DNA glycosylase and that the signals for mitochondrial transloca tion resides in the presequence, whereas signals for nuclear import ar e within the mature protein.