Ribonucleotide reductase association with mammalian liver mitochondria

Abstract

Deoxyribonucleoside triphosphate pools in mammalian mitochondria are highly asymmetric, and this asymmetry probably contributes to the elevated mutation rate for the mitochondrial genome as compared with the nuclear genome. To understand this asymmetry, we must identify pathways for synthesis and accumulation of dNTPs within mitochondria. We have identified ribonucleotide reductase activity specifically associated with mammalian tissue mitochondria. Examination of immunoprecipitated proteins by mass spectrometry revealed R1, the large ribonucleotide reductase subunit, in purified mitochondria. Significant enzymatic and immunological activity was seen in rat liver mitochondrial nucleoids, isolated as described by Wang and Bogenhagen (Wang, Y., and Bogenhagen, D. F. (2006) J. Biol. Chem. 281, 25791-25802). Moreover, incubation of respiring rat liver mitochondria with [(14)C]cytidine diphosphate leads to accumulation of radiolabeled deoxycytidine and thymidine nucleotides within the mitochondria. Comparable results were seen with [(14)C]guanosine diphosphate. Ribonucleotide reduction within the mitochondrion, as well as outside the organelle, needs to be considered as a possibly significant contributor to mitochondrial dNTP pools.

Keywords: DNA Precursors; Mitochondria; Mitochondrial DNA; Nucleoside Nucleotide Metabolism; Nucleotide; Ribonucleotide Reductase; dNTP Synthesis.

FIGURE 1.

Quantitation of dGTP in rat heart mitochondria by HPLC. An extract of rat heart mitochondria was analyzed by HPLC before (left panel) and after (right panel) passage through a boronate column to remove ribonucleotides. Chromatography was carried out on a Whatman Partisil 10 SAX column as previously described for separation of ribonucleoside triphosphates (6). Nucleotides were identified by retention time and quantitated by peak area with reference to standards. dGTP content in the extracts before and after boronate chromatography was determined also by the DNA polymerase-based assay, and all of the determined values for dGTP are tabulated.

FIGURE 2.

Ribonucleotide reductase activity in mitochondria and cytosol of mammalian livers and of HeLa cells. Lactate dehydrogenase was assayed as a marker for cytosolic contamination. The data for rat liver represent average values for duplicate assays on three different mitochondrial preparations ± standard deviation. The values for the remaining assays represent averages of duplicate assays on single preparations. cyto, cytosol; mt, mitochondria.

FIGURE 3.

Sucrose gradient centrifugal analysis of ribonucleotide reductase activity in pig liver cytosol and mitochondrial extracts. The details are given under “Experimental Procedures.” Human hemoglobin was centrifuged in a separate tube as a sedimentation rate marker. The direction of sedimentation is from right to left. Each data point in the analyses of RNR activity represents the average ± standard deviation of triplicate assays. A, pig liver cytosol; B, pig liver mitochondria; C, hemoglobin standard.

FIGURE 4.

Association of ribonucleotide reductase activity with mitochondrial nucleoids from rat liver. Nucleoids were prepared and subjected to velocity gradient centrifugation as described by Wang and Bogenhagen (24). DNA and protein in the gradient fractions were analyzed by the Bradford and Pico-Green assays, as specified by the manufacturer. Selected gradient fractions were analyzed by immunoblotting for R1, R2, and selected nucleoid markers as shown. HEK293 depicts immunoblot analysis of an extract of cultured human kidney cells as a positive control. a, immunoblotting; b, DNA and protein concentrations; c, RNR activity.

FIGURE 5.

Association of ribonucleotide reductase R1 and R2 proteins with nucleoids from pig liver mitochondria. Nucleoids were isolated from pig liver and analyzed by velocity sedimentation as described by Wang and Bogenhagen (24). Selected fractions were analyzed by immunoblotting for R1, R2, p53R2, and selected nucleoid marker proteins. a, immunoblotting; b, DNA and protein concentrations.

FIGURE 6.

Conversion of [¹⁴C]CDP to deoxyribonucleotides isolated rat liver mitochondria. For details, see “Experimental Procedures.” Each data point is the average of five analyses of each sample ± standard deviation from one experiment of three that were performed.

FIGURE 7.

Conversion of [¹⁴C]GDP to deoxyribonucleotides in isolated rat liver mitochondria. Each data point is the average of four analyses of each sample ± standard deviation from an experiment that was done twice.