Abstract
In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level.
Highlights
In the former pool dNTPs are produced from ribonucleotides by the de novo pathway, where ribonucleotide reductase and thymidylate synthase are key enzymes, and by phosphorylation of deoxyribonucleosides via the salvage pathway. mt dNTPs derive from the salvage of deoxyribonucleosides catalyzed by mt kinases and from import of deoxyribonucleotides preformed in the cytosol
The mt inner membrane is impermeable to nucleotides, cytosolic and mt dNTP pools communicate via specific transporters. mt carriers for deoxyribonucleotides have been cloned in yeasts [1, 2] and biochemically characterized in mammalian mitochondria [3, 4]
The intramitochondrial salvage pathway for dNTP synthesis starts with the phosphorylation of imported deoxyribonucleosides by the two mt deoxyribonucleoside kinases, thymidine kinase 2 (TK2) and deoxyguanosine kinase, two constitutively expressed enzymes encoded by two nuclear genes [16, 17]
Summary
Cell Lines and Cell Growth—The established human tumor cell lines HEK 293, Ost TK1Ϫ, and HOS TK1ϩ and the human skin fibroblast lines C63 and C72 were grown in Dulbecco’s modified Eagle’s medium with 7–10% heat-inactivated fetal calf serum (FCS) and antibiotics. To obtain inducible expression of the shRNAs we prepared a stable clone of Ost TK1Ϫ cells transfected with plasmid pcDNA6/TR (Invitrogen), coding for a tetracycline repressor that is inactivated by tetracycline. The same cultures underwent a second transfection with 25 nM siRNAs in Dulbecco’s modified Eagle’s medium with 0.1% dialyzed FCS, and the isotope experiments were performed 3 days later By this protocol the cells remained in the presence of the siRNAs for a total of 10 days without replating. We prepared protein extracts as described previously [24] by adding protease inhibitor mixture (Roche Applied Science) to the lysis buffer. We centrifuged the lysate at 19,000 ϫ g for 20 min at 4 °C to precipitate membrane debris and used the supernatant for the enzymatic assays
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