Cloning and characterization of full-length mouse thymidine kinase 2: the N-terminal sequence directs import of the precursor protein into mitochondria.
The subcellular localization of mitochondrial thymidine kinase (TK2) has been questioned, since no mitochondrial targeting sequences have been found in cloned human TK2 cDNAs. Here we report the cloning of mouse TK2 cDNA from a mouse full-length enriched cDNA library. The mouse TK2 cDNA codes for a protein of 270 amino acids, with a 40-amino-acid presumed N-terminal mitochondrial targeting signal. In vitro translation and translocation experiments with purified rat mitochondria confirmed that the N-terminal sequence directed import of the precursor TK2 into the mitochondrial matrix. A single 2.4 kb mRNA transcript was detected in most tissues examined, except in liver, where an additional shorter (1.0 kb) transcript was also observed. There was no correlation between the tissue distribution of TK2 activity and the expression of TK2 mRNA. Full-length mouse TK2 protein and two N-terminally truncated forms, one of which corresponds to the mitochondrial form of TK2 and a shorter form corresponding to the previously characterized recombinant human TK2, were expressed in Escherichia coli and affinity purified. All three forms of TK2 phosphorylated thymidine, deoxycytidine and 2'-deoxyuridine, but with different kinetic efficiencies. A number of cytostatic pyrimidine nucleoside analogues were also tested and shown to be good substrates for the various forms of TK2. The active form of full-length mouse TK2 was a dimer, as judged by Superdex 200 chromatography. These results enhance our understanding of the structure and function of TK2, and may help to explain the mitochondrial disorder, mitochondrial neurogastrointestinal encephalomyopathy.
Wang
L
L
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, P.O. Box 575, SE-751 23 Uppsala, Sweden. liya.wang@vmk.slu.se
Eriksson
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D003841:Deoxycytidine / Q000378:metabolism; D003857:Deoxyuridine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000737:chemistry* / Q000235:genetics*; D014018:Tissue Distribution
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003841:Deoxycytidine; C105339:thymidine kinase 2; D013937:Thymidine Kinase; D013936:Thymidine; D003857:Deoxyuridine