Human Mitochondrial 5′-Deoxyribonucleotidase

Abstract

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. 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. Mammalian 5′-nucleotidases form a heterogeneous group of enzymes differing in primary structure, cell localization, and tissue and substrate specificity. They dephosphorylate various nucleotides to nucleosides and inorganic phosphate. Originally they were distinguished from their enzyme activity, more recently via cloning of their cDNAs resulting in the clear definition of 7 different species (1Resta R. Yamashita Y. Thompson L.F. Immunol. Rev. 1998; 161: 95-109Crossref PubMed Scopus (265) Google Scholar, 2Hunsucker S.A. Spychala J. Mitchell B.S. J. Biol. Chem. 2001; 276: 10498-10504Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 3Sala-Newby G.B. Newby A.C. Biochim. Biophys. Acta. 2001; 1521: 12-18Crossref PubMed Scopus (27) Google Scholar, 4Itoh R. Comp Biochem. Physiol. B. 1993; 105: 13-19Crossref PubMed Scopus (56) Google Scholar, 5Amici A. Emanuelli M. Raffaelli N. Ruggieri S. Saccucci F. Magni G. Blood. 2000; 96: 1596-1598Crossref PubMed Google Scholar, 6Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). One enzyme is a ubiquitous ectonucleotidase anchored to the surface of the plasma membrane (1Resta R. Yamashita Y. Thompson L.F. Immunol. Rev. 1998; 161: 95-109Crossref PubMed Scopus (265) Google Scholar). Five enzymes occur in the cytosol (2Hunsucker S.A. Spychala J. Mitchell B.S. J. Biol. Chem. 2001; 276: 10498-10504Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 3Sala-Newby G.B. Newby A.C. Biochim. Biophys. Acta. 2001; 1521: 12-18Crossref PubMed Scopus (27) Google Scholar, 4Itoh R. Comp Biochem. Physiol. B. 1993; 105: 13-19Crossref PubMed Scopus (56) Google Scholar, 5Amici A. Emanuelli M. Raffaelli N. Ruggieri S. Saccucci F. Magni G. Blood. 2000; 96: 1596-1598Crossref PubMed Google Scholar, 6Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and one in mitochondria (7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). The mitochondrial nucleotidase and one of the cytosolic enzymes are structurally related and show a preference for the hydrolysis of deoxyribonucleotides and were for this reason named deoxyribonucleotidases (dNTs), 1The abbreviations used are: dNTs, deoxyribonucleotidases; dNTPs, deoxyribonucleoside triphosphates; dNT2, mitochondrial 5′(3′) deoxyribonucleotidase; GFP, green fluorescent protein; dNT1, cytosolic 5′(3′) deoxyribonucleotidase; TK1, cytosolic thymidine kinase; TK2, mitochondrial thymidine kinase; BPE-T, (1-[2-deoxy-3,5,-O-(2-bromo-1-phosphono)ethylidene-β-d-threo-pentofuranosyl]thymine); PMcH-U, ((+/-)-1-trans-(2-phosphonomethoxycyclohexyl)uracil); TBS, Tris-buffered saline; DLU, digital light units; AP, alkaline phosphatase. 1The abbreviations used are: dNTs, deoxyribonucleotidases; dNTPs, deoxyribonucleoside triphosphates; dNT2, mitochondrial 5′(3′) deoxyribonucleotidase; GFP, green fluorescent protein; dNT1, cytosolic 5′(3′) deoxyribonucleotidase; TK1, cytosolic thymidine kinase; TK2, mitochondrial thymidine kinase; BPE-T, (1-[2-deoxy-3,5,-O-(2-bromo-1-phosphono)ethylidene-β-d-threo-pentofuranosyl]thymine); PMcH-U, ((+/-)-1-trans-(2-phosphonomethoxycyclohexyl)uracil); TBS, Tris-buffered saline; DLU, digital light units; AP, alkaline phosphatase. with dNT1 in the cytosol (6Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and dNT2 in mitochondria (7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). We are investigating the regulation of deoxyribonucleoside triphosphate (dNTP) pools in relation to DNA synthesis and became first interested in the physiological function of dNT1 and cloned its cDNA (6Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We obtained evidence that dNT1 participates in the regulation of pyrimidine but not purine dNTP pools via cytosolic substrate cycles (8Gazziola C. Ferraro P. Moras M. Reichard P. Bianchi V. J. Biol. Chem. 2001; 276: 6185-6190Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Subsequently the related dNT2, in its cDNA a mitochondrial and obtained evidence that the enzyme was in mitochondria (7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). We overproduced dNT2 in the recombinant protein to and that its substrate specificity was that of dNT1 and to of dTMP and specificity suggested that dNT2 participates in the regulation of thymidine in mitochondria. The of a mitochondrial thymidine M. Karlsson A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. B. A. Hellman U. S. PubMed Scopus Google Scholar), from the cytosolic thymidine Biol. PubMed Scopus Google Scholar) is The enzyme in the synthesis of mitochondrial via of thymidine from the The of this was recently by the that of a genetic by of mitochondrial DNA A. A. Y. S. 2001; PubMed Scopus Google Scholar). In one or for triphosphates to of thymidine from the cytosol. We dNT2 to accumulation of dTTP. The for a from an excess of dTTP is by a mitochondrial genetic by the of the cytosolic thymidine A. M. PubMed Scopus Google Scholar). in this enzyme to accumulation of thymidine and dTTP. show that and overproduction of thymidine lead to of mitochondrial DNA synthesis suggesting that a of the mitochondrial thymidine is of The mitochondrial and the substrate specificity of dNT2 that the enzyme a in this We that dNT2 is the of an substrate with the dNT2 in a to the accumulation of dTTP. In earlier the mitochondrial and of dNT2 was from the of the cloned cDNA and the recombinant protein (7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). We now establish the of dNT2 in mitochondria and the of of the native enzyme with of the recombinant We the of various cell lines with cDNA of dNT2 and the overproduced protein the of overproduction of dNT2 the of pyrimidine The inhibitors M. M. M. 1998; Scopus Google Scholar) and R. M. Chem. Scopus Google Scholar) were from dUMP and were obtained from and were from from was not the the for with and the by The for were from and and were from from from and and from recombinant dNT1 and dNT2 were as E. was used for and to J. Scholar). E. was the for the of recombinant from in of the were with or was in C. Moras M. Ferraro P. Gallinaro L. R. C. Reichard P. Bianchi V. PubMed Scopus Google Scholar). The cytosolic thymidine cell was from of were in with and in a with of was by an We for of dNT2 in One from the and contained the dNT2 by were of from and contained the dNT2 or to the GFP The dNT2 and GFP as from the same The for the were obtained from the cDNA (7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar) by with and or The for the fusion protein obtained from (7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar) was as a fragment into to cell were by and or with from After were and by of and by fluorescent in the of the overproducing dNT2 the were obtained by with (7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar) and with After a dNT2 of and with the the overproduction of dNT2 in used an in as C. Moras M. Ferraro P. Gallinaro L. R. C. Reichard P. Bianchi V. PubMed Scopus Google Scholar). of that the C. Moras M. Ferraro P. Gallinaro L. R. C. Reichard P. Bianchi V. PubMed Scopus Google Scholar) were the of the of this were used for the with and with were and with ponasterone was different of were used for cell and of cell for enzyme In the was when was and the were for to to the of dNT2 We dNT1 and dNT2 from by of of protein into for were from and were with S. J. Immunol. 1993; PubMed Scopus Google Scholar) and by the first the of the The were by affinity dNT1 or dNT2 was a the protein was to a first with Tris-buffered by and with The were into and with of in The were first with TBS, and the was then with of was with of The of the protein not They with the related nucleotidase with a We used by of with that form of dNT2. The were to C to C and to a for dNT2 the but did not with with the was and and were We for their We used and for this and to with earlier for of mitochondria from C. A. PubMed Google Scholar). We the with and then the of and the cell for We the with of of by the a was by for and with of The from the first was to an and a mitochondrial was for The from the is to as cytosol and was used for enzyme and the same the mitochondrial and the were as for the of the from overproducing dNT2 were by and enzyme for the of enzymes the and mitochondrial were only in the mitochondrial and contained mitochondrial enzymes and and the were more in the a with mitochondria. The cytosol contained and a with mitochondrial of in mitochondria and the of cytosol in cell its the of enzymes and dNT2. and the mitochondria and cytosol. of increased the of the mitochondrial and the of the by mitochondria. the with or a the of mitochondrial enzymes and dNT2 in this but did not We did not but in in were with and They were the in and After of to a of and the was for were to and without of of dNT1 and dNT2 of the were with as earlier (6Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). The inhibitors and were used to dNT1 and dNT2 C. C. Reichard P. Bianchi V. Biochem. Biophys. PubMed Scopus Google Scholar). In substrate was the of substrate specificity nucleotides were used and enzyme was from the of formed of Biochem. PubMed Scopus Google Scholar). were used for P. Scopus Google Scholar) and E. Scholar). one of is as the of of is of was M. Biochem. PubMed Scopus Google Scholar) with as was with the as by the and by of the were by J. Scholar). fragment of dNT2 cDNA was from by with and and used as a The dNT2 was by with and was in were and by with the was used to the of in the different The of dNT2 were by the of the dNT2 with that of the corresponding The for were in earlier (8Gazziola C. Ferraro P. Moras M. Reichard P. Bianchi V. J. Biol. Chem. 2001; 276: 6185-6190Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and only We of ponasterone and for with We cell for an in and and of In the cells, into DNA and the of the and dTTP in the into and the of dTTP and then the of and into DNA as as the of pyrimidine deoxyribonucleoside by the from were by M. P. 1993; Scholar), and a of The mitochondria were then by in of of a of inhibitors The for contained of protein and of 5′-nucleotidase with dUMP as was and of the was in a of with of of protein and of 5′-nucleotidase was to a of were with a of in was in one the The C. C. Reichard P. Bianchi V. Biochem. Biophys. PubMed Scopus Google Scholar) the in the first but not the The in the first of of nucleotidase was used for the earlier that recombinant dNT2 obtained overproduction dUMP and dTMP with the and of was to that a native dNT2 a substrate specificity. used a of dNT2 from mitochondria for the The in show that the substrate specificity of the enzymes indeed was the the with nucleotides or as the and to as the We a of and show the earlier substrate of recombinant and native enzymes deoxyribonucleotides but are not for specificity of recombinant and native (7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google L. Reichard P. J. Biol. Chem. Full Text PDF PubMed Google (6Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google of the different enzyme were as recombinant dNT2, native dNT2, recombinant dNT1, native dNT1, The for recombinant dNT2 and the of dNT1 are from earlier (6Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar, L. Reichard P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). in a The of the different enzyme were as recombinant dNT2, native dNT2, recombinant dNT1, native dNT1, The for recombinant dNT2 and the of dNT1 are from earlier (6Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar, L. Reichard P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We the of the inhibitors and the of dNT2 and recombinant earlier C. C. Reichard P. Bianchi V. Biochem. Biophys. PubMed Scopus Google Scholar), recombinant dNT1 and dNT2 whereas mainly dNT2 with a of dNT2 only in the of the inhibitors recombinant and native dNT2 were by the dNT1, activity, whereas of enzyme with of inhibitors recombinant and native dNT2 and recombinant of in different of the enzyme were with in the and of or in a different of the enzyme were with in the and of or After of with the cDNA of dNT2 linked by its to the cDNA of GFP the cell mitochondria a green suggesting a of the fusion protein in mitochondria. The mitochondrial of the the mitochondrial of dNT2 (7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). as the mitochondria contained mostly a truncated form of dNT2 and not the enzyme linked to GFP. became to the mitochondrial of dNT2. We the of dUMP in from as in or in the of one of the inhibitors or dUMP is a substrate for dNT1 and dNT2. the in that the in enzyme in the and of the of dNT1 and the the without and with the of dNT2 of dNT1 and dNT2 in cell from of and the of dUMP by from cell were used to the of dNT1 and dNT2 dNT1 and The of dNT1 and dNT2 is from the in of dUMP in the of this and in its only dNT2 is from the of dUMP in and in the of BPE-T, dNT1 is obtained from the of the in the of and in a The of and the of dUMP by from cell were used to the of dNT1 and dNT2 dNT1 and The of dNT1 and dNT2 is from the in of dUMP in the of this and in its only dNT2 is from the of dUMP in and in the of BPE-T, dNT1 is obtained from the of the in the of and in this of the in the mitochondrial was dNT2 whereas the cytosol contained dNT1 The contained mostly dNT2, the by mitochondria earlier in In that was The inhibitors only of the is not for dNT2 but dNT1 not the that from dNT1 is in the dNT2 in mitochondria. the that of the of from The for the fusion protein and was used earlier with to the mitochondrial of dNT2 (7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). in the in and In to the mitochondrial of dNT2 in cell the from into the with from of and with the now an In the overproduced dNT2 and a of mitochondria. of the in a of in to a the for a fusion protein the dNT2 and GFP The was with an GFP and the but not with and is the C the and the of dNT2. specificity and the of the that the from a truncated form of dNT2, linked by its C to GFP. GFP a The same were obtained with with truncation was by the overproduction of dNT2 or by the of dNT2 to GFP with a different in a the cDNAs for dNT2 and GFP. In dNT2 and GFP were with GFP a mitochondrial dNT2 increased more and the a green in the with dNT2 of and are for dNT2, corresponding to the recombinant enzyme with or without the from an E. overproducing dNT2 with the the for the The of the from the to of the the of dNT2 with and without the in the the the with the but not with the to the We that this is by the of the of the same a was with an GFP One was with to show the of the from A. The GFP did not but a in the of GFP. for is a of GFP linked to the truncated dNT2 species of is located GFP, its that of with in the overproduction of dNT2 and GFP, with GFP located in the and dNT2 in mitochondria of the dNT2 lost the mitochondrial presequence. The that the truncation of dNT2 in was not by overproduction of the enzyme but its to GFP in the fusion The enzyme in with in with for the fusion protein to enzyme by truncation in the In used an with a for dNT2 but not for GFP. of ponasterone dNT2 was in from this in corresponding from of overproduction the of the of the overproduction in the cell lines with or ponasterone are in The was a of and then with cell for a or various cell by enzyme and shows the for of enzyme more pronounced ponasterone with After removal of the enzyme for the fusion protein and its truncated form increased and in with enzyme shows the corresponding with were resulting in a of the of of protein ponasterone A. of in of show for dNT2 corresponding to an enzyme with and without increased and are of but the was more pronounced After removal of the the of with the more rapidly. After of for with ponasterone the mitochondrial and the of dNT2, but the cytosol contained In dNT2 was the cytosol and mitochondria the of in the of dNT2 disappeared from the cytosol but slowly from mitochondria. After the of in the cytosol to only of the in mitochondria a with overproduced earlier from the cytosol. of from cell of overproducing the fusion protein a with to of not the and truncated fusion were mainly in mitochondria but a was in the cytosol. After removal of ponasterone enzyme and the dNT2 the form of the disappeared this a of the or was the only slowly with synthesis of and the large in dNT2-mRNA but disappeared removal of ponasterone A. this dNT2 by only the of enzyme is to a of the protein and not to synthesis of that the enzyme and of and ponasterone A. We earlier excretion is a of substrate cycles and (8Gazziola C. Ferraro P. Moras M. Reichard P. Bianchi V. J. Biol. Chem. 2001; 276: 6185-6190Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). the of dNT2 overproduction pyrimidine excretion in an in We now only in and overproducing the excretion of and as the of synthesis of show in corresponding from an earlier with overproducing dNT1 (8Gazziola C. Ferraro P. Moras M. Reichard P. Bianchi V. J. Biol. Chem. 2001; 276: 6185-6190Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In that excretion increased in thymidine and were but with of or In the overproduction of the mitochondrial dNT2 did not the excretion of thymidine and the excretion of excretion was to in the were obtained in with the for overproduction of the mitochondrial dNT2 did not the excretion of pyrimidine in the earlier that by ponasterone increased the excretion of the pyrimidine and not in a with ponasterone that are with a for the ponasterone but not for dNT1 or dNT2 overproduction of and is not linked to overproduction of but from the of ponasterone with recombinant dNT2 evidence for the mitochondrial of the enzyme (7Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). We now establish that the recombinant and the native enzyme They show the same substrate specificity and the same by After of in the of dNT2 was in mitochondria and in a that was by mitochondria. In the related dNT1 was located in the cytosol show that dNT2 is a mitochondrial enzyme and that obtained with the recombinant protein the of the native The of a in mitochondria a of evidence from the of M. Karlsson A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. B. A. Hellman U. S. PubMed Scopus Google Scholar) and a mitochondrial J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In earlier with 5′-nucleotidases overproducing one nucleotidase a to the of enzyme overproduction (8Gazziola C. Ferraro P. Moras M. Reichard P. Bianchi V. J. Biol. Chem. 2001; 276: 6185-6190Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). suggested the of cytosolic enzymes via substrate cytosolic nucleotidase participates in the regulation of and dNT1 in the regulation of dUMP and dTMP The overproducing cell lines used in were with nucleotidase the of an and DNA synthesis and a with dNT2 now constructed cell lines that to this an fluorescent fusion protein in dNT2 was linked via its C to GFP. with ponasterone this cell overproduced dNT2 but in to the fusion a truncated form in a short C-terminal fragment of dNT2 was linked to GFP form was more the fusion enzyme as the of dNT2 is located in the (6Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, S. A. L. R. P. F. M. S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The same occurred in of and that overproduced the fusion protein of to GFP is a for their and a an of this did not in that overproduced dNT2 from GFP or the cDNA The of dNT2 in increased with the dNT2 to of the protein in cellular The contained of dNT2 with and without the mitochondrial After cell of dNT2 in the mitochondrial and as as the cytosol The of dNT2 in the is in an by mitochondria. in the contained large of mitochondrial of the enzymes and dNT2 in but were to mitochondria We reason to the as this and to the of dNT2 mitochondria and cytosol In that dNT2 of the mitochondrial The of a large of dNT2 in the cytosol of overproducing is more to the of overproduction only of the enzyme is in the is in the cytosol dNT2 is a mitochondrial and the more overproduction of the enzyme the the mitochondrial resulting in accumulation of the enzyme in the cytosol. In with this that removal of the dNT2 disappeared more from the cytosol from the mitochondria not the as the cytosol contained dNT2 without the is of the protein into mitochondria N. Rev. Biochem. PubMed Scopus Google E. C. G. Biol. PubMed Scopus Google Scholar). The large overproduction of dNT2 affect the of the mitochondrial and of mitochondrial into the cytosol in the or the the of activity, and in the cytosol of the mitochondrial a of the of and dNT2 shows a accumulation of the in the enzymes are located in the mitochondrial We that to of dNT2 in the cytosol of overproducing In this by of the when of the cytosolic disappeared After removal of the the dNT2-mRNA but disappeared from the a in contrast to the of the that removal of the dNT2 and overproduction of dNT2 of dNT2 is by protein but only The of in in the of enzyme overproduction the mitochondrial are We one in the of a overproduction of dNT2 the pyrimidine by the used earlier to the of the cytosolic dNT1 for the of pyrimidine pools (8Gazziola C. Ferraro P. Moras M. Reichard P. Bianchi V. J. Biol. Chem. 2001; 276: 6185-6190Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). pools only a of the pools and the the overproduction the of cytosolic We of dNT2 overproduction a of the overproduced enzyme was in the cytosol. The of this from an of 7 that overproduction of mitochondrial dNT2 or cytosolic dNT1, with a specificity of the for the cytosolic In to thymidine nucleotides in the mitochondrial We for the and and to for the inhibitors and