Human mitochondrial 5'-deoxyribonucleotidase. Overproduction in cultured cells and functional aspects.
Deoxynucleoside triphosphates (dNTPs) used for mitochondrial DNA replication are mainly formed by phosphorylation of deoxynucleosides imported into mitochondria from the cytosol. We earlier obtained evidence for a mitochondrial 5'-nucleotidase (dNT2) with a pronounced specificity for dUMP and dTMP and suggested that the enzyme protects mitochondrial DNA replication from excess dTTP. In humans, accumulation of dTTP causes a mitochondrial genetic disease. We now establish that dNT2 in vivo indeed is located in mitochondria. The native enzyme shows the same substrate specificity and affinity for inhibitors as the recombinant dNT2. We constructed ponasterone-inducible cell lines overproducing dNT2 with and without the green fluorescent protein (GFP) linked to its C terminus. The fusion protein occurred in mitochondria mostly in an inactive truncated form, with only a short C-terminal fragment of dNT2 linked to GFP. No truncation occurred when dNT2 and GFP were not linked. The cell mitochondria then contained a large excess of active dNT2 with or without the mitochondrial presequence. After removal of ponasterone overproduced dNT2 disappeared only slowly from the cells, whereas dNT2-mRNA was lost rapidly. Overproduction of dNT2 did not lead to an increased excretion of pyrimidine deoxyribonucleosides, in contrast to overproduction of the corresponding cytosolic deoxynucleotidase, suggesting that the mitochondrial enzyme does not affect overall cellular deoxynucleotide turnover.
Gallinaro
Lisa
L
Department of Biology, University of Padova, I-35131 Padova, Italy.
Crovatto
Katia
K
Rampazzo
Chiara
C
Pontarin
Giovanna
G
Ferraro
Paola
P
Milanesi
Eva
E
Reichard
Peter
P
Bianchi
Vera
V
D015720:5'-Nucleotidase / Q000096:biosynthesis / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004790:Enzyme Induction; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D009705:Nucleosides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D017931:DNA Primers; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D009705:Nucleosides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C063766:5'(3')-nucleotidase; D015720:5'-Nucleotidase