Mitochondrial deoxynucleotide pool sizes in mouse liver and evidence for a transport mechanism for thymidine monophosphate

Abstract

Dividing cultured cells contain much larger pools of the four dNTPs than resting cells. In both cases the sizes of the individual pools are only moderately different. The same applies to mitochondrial (mt) pools of cultured cells. Song et al. [Song S, Pursell ZF, Copeland WC, Longley MJ, Kunkel TA, Mathews CK (2005) Proc Natl Acad Sci USA 102:4990-4995] reported that mt pools of rat tissues instead are highly asymmetric, with the dGTP pool in some cases being several-hundred-fold larger than the dTTP pool, and suggested that the asymmetry contributes to increased mutagenesis during mt DNA replication. We have now investigated this discrepancy and determined the size of each dNTP pool in mouse liver mitochondria. We found large variations in pool sizes that closely followed variations in the ATP pool and depended on the length of time spent in the preparation of mitochondria. The proportion between dNTPs was in all cases without major asymmetries and similar to those found earlier in cultured resting cells. We also investigated the import and export of thymidine phosphates in mouse liver mitochondria and provide evidence for a rapid, highly selective, and saturable import of dTMP, not depending on a functional respiratory chain. At nM external dTMP the nucleotide is concentrated 100-fold inside the mt matrix. Export of thymidine phosphates was much slower and possibly occurred at the level of dTDP.

Fig. 1.

The measured size of the mt dNTP and rNTP pools depends on the measured size of the ATP pool. (A) Size of dNTP pools and ATP pool in consecutive preparations of liver mitochondria. The ordinate is in pmol/mg mitochondria for dNTPs and in nmol/mg mitochondria for ATP. Exp. 1 is the earliest experiment, and Exp. 9 is the most recent one. Buffer A contained 25 μg/ml oligomycin in Exp. 5, 25 μM rotenone in Exp. 6, and 50 μM atractyloside in Exp. 8. (B) Size of rNTP pools. rNTPs are given as pmol, and ATP is given as nmol. dNTP and rNTP pools were determined by the polymerase assay and by HPLC, respectively.

Fig. 2.

Import of dTMP into liver mitochondria. We incubated isolated mitochondria with 5 nM [³H]dTMP (45,000 cpm/pmol) with and without added 0.1 mM ADP (transformed to ATP by the mitochondria) as detailed for transport experiments in Materials and Methods. After either 2 or 10 min, mitochondria and external medium were separated by centrifugation and analyzed for the presence of labeled thymidine, dTMP, and dTDP+dTTP. (A) Import into mitochondria. (B) Remainder in external medium. Note the break in the ordinate in both panels. ■, dTDP+dTTP; ▴, dTMP; ●, thymidine. Solid lines are data from incubations without added ADP, and broken lines are data from incubations with ADP.

Fig. 3.

Incubation of mitochondria with labeled dTTP. We incubated mitochondria for 2 and 10 min with [³H]dTTP (35,000 cpm/pmol) with and without added ADP and determined labeled thymidine compounds as described for Fig. 2. (A) Data from mitochondria. (B) Data from the external medium. No dTTP was found inside mitochondria, but dTMP derived from the breakdown of dTTP in the external medium had been imported. Note that B contains a break in the ordinate. ■,dTTP+dTTP; ▴, dTMP; ●, thymidine. Broken lines show results from the experiment with ADP.

Fig. 4.

Dependence of dTMP import on concentration of dTMP. We incubated mitochondria with increasing concentrations of [³H]dTMP (430 cpm/pmol) for 2 min and determined the amount of intramitochondrial dTMP (▴), thymidine (●), and dTTP+dTDP (■) from their radioactivity.

Fig. 5.

Export of radioactive thymidine and thymidine phosphates from mitochondria labeled in vivo from [³H]thymidine (20,000 cpm/pmol). We incubated the labeled mitochondria under standard conditions and determined the amount of radioactive dTTP (□), dTDP (■), dTMP (▴), and thymidine (●) remaining in mitochondria (A) or exported into the medium (B) between 1 and 20 min. The specific radioactivity of the thymidine phosphates was 2,200 cpm/pmol.

Fig. 6.

Export of thymidine phosphates does not involve an exchange process. We incubated labeled mitochondria under standard conditions for 2 min (open staples) or 10 min (filled staples). First column, controls without further addition; second column, averaged data from two experiments with 0.5 and 5 μM external dTMP; third column, 0.5 and 5 μM external dTTP; fourth column, 0.5 and 5 μM dTTP plus 0.1 mM ADP. The data show the export in pmol of dTTP (first row), dTDP (second row), dTMP (third row), and thymidine (fourth row).