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Home , Deoxyribonucleotide, Guanosine diphosphate, Ribonucleotide, Ribonucleotide reductase

Ribonucleotide Association with Mammalian Liver Mitochondria

Korakod Chimploy1, Shiwei Song2, Linda J. Wheeler, and Christopher K. Mathews3

From the Department of and Biophysics, Oregon State University, Corvallis, OR 97331-7305

Running Title: Reductase in Mammalian Mitochondria

#Address correspondence to: Christopher K. Mathews, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-7305; Tel. (541)737-1865; Fax (541)737-0481; E-mail: [email protected]

Key Words: Mitochondria, , , pool asymmetry Background: Mitochondrial DNA replication is supplied by precursor pools distinct from pools supplying nuclear DNA replication. Results: Liver mitochondrial extracts contain significant ribonucleotide reductase activity, associated with nucleoids. Conclusion: Intramitochondrial ribonucleotide reduction may be a significant source of mitochondrial dNTPs. Significance: Knowing sources of mitochondrial dNTP pools may lead toward understanding mitochondrial genomic stability.

SUMMARY

Deoxyribonucleoside triphosphate pools in mammalian mitochondria are highly asymmetric, and this asymmetry probably contributes toward 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 RNR subunit, in purified mitochondria. Significant enzymatic and immunological activity was seen in rat liver mitochondrial nucleoids, isolated as described by Wang, Y., and Bogenhagen, D. F. (2006) J. Biol. Chem. 281, 25791–25802. Moreover, incubation of respiring rat liver mitochondria with [ 14C] diphosphate leads to acccumulation of radiolabeled and within the mitochondria. Comparable results were seen with [ 14C] 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.

Control of DNA precursor pool sizes is underrepresentation does not significantly closely linked to genomic stability (1,2). affect replication fidelity (4). On the other Most analyses of dNTP4 pools in eukaryotic hand, our analyses of dNTP pool sizes in or bacterial cell extracts show a pronounced mammalian tissue mitochondria have pool asymmetry, with dGTP strongly yielded a different picture. In mitochondrial underrepresented (3). However, as shown by extracts dGTP is greatly overrepresented, in vitro measurements of replication errors amounting to as much as 90 per cent of total during DNA synthesis, this dNTP in rat heart mitochondria (5,6). In

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vitro analyses of replication fidelity suggest the family of mitochondrial DNA depletion that this dNTP pool asymmetry contributes diseases results from a deficiency of p53R2, toward the mitochondrial mutation rate, the DNA damage-inducible form of which is one to two orders of magnitude ribonucleotide reductase small subunit. higher than that for the nuclear genome Thus, with respect to the effects of genetic (7,8). deficiency on human health, both the mitochondrial salvage pathways and the To assess the biological significance of RNR-related pathway that begins in the mitochondrial dNTP pool asymmetry, it is cytosol play significant metabolic functions. important to understand the metabolic sources of dNTPs within the mitochondrion. In 1994 our laboratory reported evidence for Most attention has focused upon salvage RNR activity in mitochondria isolated from synthesis within the mitochondrion (9) and HeLa cells (16). However, because of the cytosolic , involving high RNR activity in the cytosol of these ribonucleotide reductase, followed by rapidly proliferating cells, we could not deoxyribonucleotide transport into the definitively rule out the possibility that the organelle (10,11). Data of Rampazzo et al activity we were measuring resulted from (11) and Pontarin et al (10) support the cytosolic contamination of our premise that in quiescent cells the salvage mitochondrial preparations. pathway predominates, whereas proliferating cells depend upon the de novo Our preliminary results (16) suggested the pathway beginning with ribonucleotide possibility of a third pathway to reductase (RNR) in the cytosol. mitochondrial dNTPs, a pathway beginning with reduction of within the RNR is an oligomeric consisting of mitochondrion. The issue is timely for a homodimeric large subunit, R1, and a several reasons. First, Pontarin et al (10) homodimeric small subunit, R2. The level of briefly mentioned their inability to detect a R2 is cell cycle-regulated, rising during S mitochondrial RNR activity, and the phase to meet the increased demand for apparent disagreement between our results DNA precursors. More recently discovered and theirs needs to be resolved. Second, the is p53R2, an alternative small subunit that is possibility that RNR acts both in cytosol and not cell cycle-regulated and is expressed in mitochondria to produce mitochondrial response to DNA damage. Pontarin et al dNTPs suggests a possibly wasteful (12,13) have presented evidence that RNR duplication of function. Third, Anderson et containing the p53R2 subunit plays an al (17) demonstrated a de novo pathway in essential function in providing mammalian mitochondria for dTMP mitochondrial DNA precursors in quiescent from dUMP, involving cells. intramitochondrial , , and serine The metabolic significance of both pathways hydroxymethyltransferase. Might other de is unquestioned. With respect to the salvage novo pathways to pathways, two of the four exist within mitochondria? in human cells— and For the present study we first confirmed by thymidine kinase 2—are localized within an independent approach our previous mitochondria (9), and genetic deficiency of finding of asymmetric dNTP pools in either one has serious clinical consequences mitochondria. Next we examined (14), resulting from depletion of ribonucleotide reductase activity in mitochondrial DNA. With respect to the de mitochondria and submitochondrial novo pathway operating in the cytosol, fractions from tissues in the adult rat, where Bourdon et al (15) found that one member of we expected cytosolic RNR activity to be

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relatively low in nonproliferating tissues. [3H]dCDP as previously described (19). Next, we used immunoprecipitation and Mitochondrial and subcellular marker mass spectrometry to search for RNR enzyme activities were determined by subunits in purified rat liver mitochondria. standard procedures: lactate dehydrogenase We also prepared nucleoids from rat liver (20), citrate synthase (21), adenylate kinase mitochondria and analyzed these (22), and cytochrome c oxidase (23). preparations for RNR. Finally, we asked whether isolated respiring rat liver Immunoprecipitation. All operations were mitochondria could take up radiolabeled carried out at 4oC. Three aliquots of rat liver diphosphates and convert mitochondria, each representing about 0.75 them in situ to deoxyribonucleotides. g of tissue, were washed by two cycles of resuspension in isolation buffer, followed by EXPERIMENTAL PROCEDURES centrifugation. Each pellet was resuspended in 0.75 ml of RIPA buffer (Santa Cruz Materials. [14C]Deoxycytidine diphosphate Biotechnology, Inc.) and disrupted by sonic and [14C]deoxyguanosine diphosphate, both oscillation. After centrifugation for 30 uniformly labeled, were obtained from minutes at 14,000 rpm in a microcentrifuge, Moravek. Antibodies to RNR proteins R1, 0.5 µg of donkey anti-goat IgG was added to R2, and p53R2 were obtained from Santa each supernatant along with 20 µl of protein Cruz Biotechnologies, Inc. The antisera A/G PLUS agarose bead suspension. After were raised against portions of the 30 minutes’ standing, each mixture was respective human proteins in goats, and the centrifuged briefly (one minute at 5000 rpm) supplier certified that the antibodies cross- and to each supernatant was added 1.0 µg of reacted with the corresponding mouse and primary antibody (R1, R2, or p53R2). Each rat proteins. Anti-goat IgG and protein A/G mixture was allowed to stand for two hours, PLUS-agarose beads were also obtained following which 20 µl of protein A/G PLUS from Santa Cruz Biotechnologies. bead suspension was added to each mixture, and the three mixtures were slowly rotated Methods. Mitochondria were isolated from overnight. Each mixture was then livers of freshly sacrificed rats by rapid centrifuged for one minute at 5000 rpm, and chilling of the organ, followed by the supernatants were discarded. Each pellet homogenization in isolation buffer and was washed by suspension followed by differential centrifugation, as previously recentrifugation three times with RIPA described (5,6). Isolation buffer is 220 mM buffer and twice with 0.1M Tris-HCl buffer, mannitol, 70 mM sucrose, 5 mM MOPS (pH pH 8.0. 7.4), 2 mM EGTA, and 0.2 mg/ml BSA. The same procedure was used for isolating Digestion and LC/MS/MS analysis of mitochondria from mouse liver. For some immunoprecipitates. Proteomic analyses experiments mitochondria were isolated were performed in the OSU Environmental from pig liver or pig heart. In these cases the Health Sciences Center mass spectrometry isolation procedures were essentially the facility and core. In-solution digestion was same, but organs were obtained from freshly performed using Promega protease enhancer killed animals at a local slaughterhouse. and trypsin (modified, gold) following the Mitochondria from HeLa cells were isolated manufacturer’s protocol (Promega, as previously described (18). Madison, WI). Tryptic were trapped on a Michrom CapTrap Enzyme assays. Enzyme assays were carried column and a C18 column (Agilent Zorbax out on mitochondrial extracts or 300SB-C18, 250 x 0.3 mm, 5 µm). A binary subfractions, as described in Results. solvent system consisting of solvent A (2% Ribonucleotide reductase activity was aqueous acetonitrile with 0.1% formic acid) determined by conversion of [3H]CDP to and solvent B (acetonitrile with 0.1 %

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formic acid) was used. Peptides were hours at 26,000 rpm. The cytosol, analyzed trapped and washed with 1% solvent B for 3 in a separate gradient, was simply the post- min. Peptide separation was achieved using mitochondrial supernatant from the original a linear gradient from 3% B to 30% B at a fractionation of a liver homogenate. flow rate of 4 µl/min over 35 minutes. LC- MS/MS analysis was conducted on a Nucleoids. Nucleoids were isolated from rat Thermo LTQ-FT MS instrument coupled to liver and pig liver mitochondria essentially a Waters nanoAcquity UPLC system. The as described by Wang and Bogenhagen (24) LTQ-FT mass spectrometer was operated for HeLa cell nucleoids. Briefly, using data-dependent MS/MS acquisition mitochondria were isolated by centrifugation with an MS precursor ion scan, performed in of a post-nuclear supernatant through a the ICR cell, from 350-2000 m/z with the Percoll step-gradient, then purified by resolving power set to 100,000 at m/z 400. nuclease treatment and recentrifugation MS/MS scans were performed by the linear through sucrose. The purified mitochondria ion trap on the five most abundant doubly or were next treated with digitonin to create triply charged precursor ions detected in the mitoplasts. After washing and re-pelleting, MS scan. the mitoplasts were lysed with Triton X-100 and subjected to brief centrifugation. The Thermo RAW data files were processed supernatant, containing lysed mitoplasts, with Proteome Discoverer v1.3.0. For was layered onto a 17-to-45% glycerol database searching Mascot (v2.3) and gradient over a pad of 30% Nycodenz and X!Tandem (embedded in Scaffold proteome 30% glycerol, and then centrifuged for 1.5 analysis software) were used to search the hours at 186,000xg in a Beckman SW41 Rattus norvegicus database, using the rotor. following parameters: the digestion enzyme was set to Trypsin/P and two missed Nucleotide incorporation and metabolism in cleavage sites were allowed. The precursor intact mitochondria. For the experiments ion mass tolerance was set to 50 ppm, while involving radiolabeled nucleotide uptake fragment ion tolerance of 1.0 Da was used. and reduction in situ, rat liver mitochondria, Dynamic modifications that were after isolation and washing, were aliquoted considered: carbamidomethylation (+57.02 as a series of centrifugal pellets in Da) for ; oxidation (+15.99 Da) for microcentrifuge tubes, with each pellet ; phosphorylation (+97.98 Da) representing about one g wet weight of the for serine, threonine and ; original liver. Mitochondria were used on deamidination (-0.98 Da) for and their day of isolation. Each pellet was , and acetylation (42.02 Da) for suspended in 1.0 ml of an incubation lysine and the N-terminus. Scaffold_3.3.1 mixture containing 70 mM sucrose, 220 mM (Proteome Software, Portland, OR) was mannitol, 5 mM MOPS (pH 7.4), 5 mM used for search data compilation and data KH2PO4, 5 mM MgCl2, 1 mM EGTA, 10 evaluation. mM potassium glutamate, 2.5 mM sodium malate, 1.0 mM ADP, and 1.0 mg/ml bovine Sucrose gradient centrifugation. serum albumin. Each mixture was incubated Mitochondria were suspended in isolation at 37 oC for ten minutes with occasional buffer plus 2 mM DTT, 0.2 mM PMSF, 2 shaking. At that point 1.0 µCi of isotopic mM pepstatin A, 5 mg/ml leupeptin, 2 precursor ([14C]CDP or [14C]GDP as mg/ml E-64, and 1% Triton X-100. The specified, uniformly labeled, without suspension was incubated for ten minutes at dilution) was added to each mixture. At each 4 oC, then centrifuged at 18,000 x g for ten indicated time (5, 10, 20, and 30 minutes) minutes at 4 oC. The supernatant was layered one tube was removed and rapidly chilled to onto a 5-20% sucrose gradient in a Beckman prevent further incorporation. After the final SW41 rotor and centrifuged at 4 oC for 24 sample had been taken, all samples were

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centrifuged and the mitochondrial pellets An obvious approach is HPLC, monitored washed by resuspension in incubation buffer by UV absorbance. In general, followed by recentrifugation. Nucleotide mitochondrial nucleotide pools are so low in extraction was carried out with 60% aqueous relation to the amount of material available methanol followed by heating, as previously as to make their quantitation by HPLC described (6). problematical. However, our analyses of rat heart mitochondria by the enzymatic assay Nucleotides were resolved, after addition of indicate that in this organ mitochondrial nonradioactive marker nucleotides, by two- dGTP levels are comparable to those of dimensional thin-layer chromatography on ribo-GTP (6), suggesting that dGTP can be PEI-cellulose sheets. The first dimension quantitated by HPLC. As shown in Figure 1, was 1M lithium chloride saturated with left panel, two peaks in the analysis of a rat sodium borate at pH 7, and the second heart mitochondrial extract were identified dimension was 0.35M ammonium sulfate. as GTP and dGTP, respectively, by Marker nucleotides were located under UV comparison of their elution times with light, and spots on the chromatogram were standards. The extract was then passed cut out and radioactivity measured by liquid through a boronate column, which retains scintillation counting. ribonucleotides but not deoxyribonucleotides (29). Analysis of the RESULTS flowthrough (right profile) showed disappearance of the peak identified as GTP, dNTP pool asymmetry. As stated earlier, with the more abundant ATP not being much of our interest in characterizing quantitatively removed. More to the point, ribonucleotide reductase in mammalian there was no change in the putative dGTP mitochondria was to help illuminate factors peak. The dGTP content of each extract, influencing the pronounced dNTP pool before and after boronate chromatography, asymmetry in these organelles (5,6). These was determined from the respective peak results have been questioned (25,26), based area, with reference to standards. Each partly upon possible nucleotide fraction was also analyzed for dGTP by the redistribution resulting from ATP loss enzymatic assay. Within experimental error during mitochondrial isolation, and partly the dGTP pool size, in pmol per mg of upon technical aspects of the DNA mitochondrial protein, was the same in all polymerase-based assay procedure used to four cases, as shown in the data tabulated in measure dNTP pools. We found (6) that our Figure 1. Hence, the unusually large dGTP ATP pools determined in isolated pool in rat tissue mitochondria has been mitochondria conformed to values published demonstrated by two independent by others, indicating that ATP loss during approaches—the DNA polymerase-based mitochondrial isolation was not a significant assay, as reported previously (6) and by issue. Also, we confirmed the technical HPLC, as shown here. problem with the dNTP assay procedure, but found that it does not apply under our assay Ribonucleotide reductase activity in tissue conditions. Nevertheless, to resolve the mitochondria. As stated earlier, a concern issue, it seemed advisable to reconfirm our about our earlier report (16) was the findings by an independent approach. possibility that the RNR activity we were measuring in mitochondrial extracts We normally assay dNTPs by the DNA represented contamination of our HeLa cell polymerase-based method (27,28). Although mitochondrial preparations with cytosol. For we have dealt with known sources of error this reason we turned to tissue mitochondria (6,26) in this assay, it seemed desirable to from adult animals, where cytosolic activity verify our measurements of dGTP levels in of RNR is expected to be low, and the mitochondria by an independent approach. contamination issue to be minimized. In the

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experiment of Figure 2, we measured RNR In another experiment we prepared activity in mitochondrial and cytosolic mitoplasts from rat liver mitochondria, by extracts from mouse, rat, and pig liver. In all solubilizing the outer membrane with cases the RNR specific activity in digitonin (33). As shown in Table IB, 82 mitochondria was higher than that in percent of total RNR activity was retained in cytosol, by factors of two to four. Also the mitoplasts, comparable to what we saw lactate dehydrogenase, representing a with citrate synthase. Nearly all of the cytosolic marker, showed low activity in the adenylate kinase activity, representing an mitochondrial fractions, indicating minimal intermembrane space marker, was absent cytosolic contamination. On the other hand, from the mitoplasts. Together the combined in HeLa cells the RNR specific activity in data of Table I indicate that in rat liver cytosol was higher than in mitochondria. mitochondria ribonucleotide reductase is This indicates that the RNR activity we largely localized in the matrix. earlier reported in HeLa cell mitochondria could have been a cytosolic contaminant. We hoped to purify the enzyme from pig However, cytosolic contamination is ruled liver mitochondria, because of the larger out in liver, where the mitochondrial amount of starting material available, specific activity is higher than that in compared to rats. As in rodent tissues, pig cytosol. heart and pig liver mitochondria both displayed enzyme activity and contained The enzyme specific activity in material immunoreactive with antibodies to mitochondrial extracts may appear low— human R1. However, much or most of the about 10 pmol/min/mg protein for rat liver pig mitochondrial enzyme was membrane extracts—but the values we observed are bound (data not shown). The enzyme could comparable to those reported for other crude be solubilized with some non-ionic tissue extracts. Earlier our laboratory detergents, but with low recovery of activity. reported about 3 pmol/min/mg protein for By contrast, the enzyme activity in both rat BSC40 kidney cells and 20 pmol/min/mg for liver and mouse liver was soluble. However, the same cells after infection by vaccinia because of the difficulty of obtaining , which induces a new RNR (19). sufficient starting material from rat or mouse Turner et al (30) reported values between 15 liver mitochondria, further purification was and 50 pmol/min/mg protein for mouse L not attempted. cell extracts. Elford et al (31) reported values ranging from below 1 to about 13 RNR immunoreactive material in rat tissue pmol/min/mg protein for extracts from a mitochondria. Immunoblotting experiments series of rat hepatomas. were carried out with mitochondrial extracts and subfractions. Immunoreactive bands Ribonucleotide reductase activity in were seen with antibodies to both R1 and R2 submitochondrial fractions. We next (data not shown). However, the estimated investigated the submitochondrial molecular weights of immunoreactive localization of RNR activity. In the material were lower than those reported for experiment of Table IA we divided rat liver mammalian R1 and R2. Whether this results mitochondria into a soluble fraction and a from degradation of enzyme subunits during membrane fraction, essentially as described mitochondrial isolation or from the by Kang et al (32). The fractionation was possibility that the mitochondrial RNR is a monitored by measurements of citrate distinct protein cannot be ascertained. synthase, a marker for soluble mitochondrial Immunoblot experiments with purified , and cytochrome c oxidase, a mouse R1 and R2 proteins were membrane marker. RNR specific activity inconclusive. So we carried out was enriched nearly fourfold in the soluble immunoprecipitation with extracts of rat fraction. liver mitochondria and analyzed tryptic

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digests of the immunoprecipitates by mass hemoglobin (Mr 65 kDa), centrifuged spectrometry. separately as a marker, while the cytosolic activity sedimented about twice as rapidly as Table II presents results of this experiment. the marker. In this experiment 96 percent of Washed mitochondria were extracted as the mitochondrial activity and 73 percent of described in Experimental Procedures, and the cytosolic activity was recovered from the separate aliquots were treated with gradient. In another experiment (not shown) antibodies to mouse R1, R2, and p53R2. The the activity in solubilized pig heart table presents combined results from three mitochondria behaved similarly to the immunoprecipitations. The R1 behavior shown here for liver mitochondria. immunoprecipitate revealed four unique R1 peptides, covering 77 of the 792 residues in Ribonucleotide reductase in mitochondrial rat R1. The R2 immunoprecipitate also nucleoids. The mitochondrial nucleoid is a revealed four unique R1 peptides , which complex in the matrix containing tightly cover 64 of the 792 R1 residues. One of the folded mitochondrial DNA and proteins, four peptides was found in both most of them connected with mtDNA immunoprecipitates. Neither R2- nor replication and transcription. Mitochondrial p53R2- derived peptides were detected with genome replication evidently occurs at the statistical assurance in any of the three nucleoid level, and much interest is focused immunoprecipitates. We assume that the R2 upon this process as a basis for antibody precipitated a holoenzyme understanding inheritance of mitochondrial containing R1 but that any R2 peptides may characteristics (34). Because of the role of have been present at levels too low to allow ribonucleotide reductase in providing DNA their unambiguous detection. The data precursors, it was of interest to know identify R1 as a mitochondrial protein and whether the enzyme in mitochondria is co- suggest that R2 or an R2-related protein is localized with the mitochondrial replisome. present as well. To investigate this possibility we isolated rat Because the p53R2 amino acid sequence is liver mitochondrial nucleoids by the not in the rat protein database, we broadened procedure of Wang and Bogenhagen (24), in the search, to include sequences from the which mitoplasts are isolated from purified combined rat, mouse, and human amino acid mitochondria, followed by lysis of the sequences. This analysis did not reveal mitoplasts and centrifugation of the lysate p53R2 peptides in any of the three through a velocity gradient. As reported also immunoprecpitates nor did it reveal any R2 by Wang and Bogenhagen, we observed two peptides that might have been missed peaks of DNA and protein in the through analysis of the rat database alone. sedimentation profile (Figure 4b)—although Whether p53R2 might be present within in this experiment some of the heavy mitochondria remains an open question; our material sedimented to the bottom of the analysis did not reveal it. tube. Both the rapidly sedimenting and slowly sedimenting peaks contained material Sucrose gradient centrifugation of immunoreactive with human R2, the RNR mitochondrial RNR. Although it does not small subunit protein, while R1- resolve the question whether the activity in immunoreactive material was found only in mitochondrial extracts represents a novel the slowly sedimenting nucleoids (panel a). form of ribonucleotide reductase, we did The fractions containing both R1- and R2- analyze the RNR activity following sucrose immunoreactive material also displayed gradient centrifugation of pig liver enzyme activity, as shown in panel c. The mitochondrial and cytosolic extracts (Figure physical association of RNR activity with 3). The mitochondrial activity sedimented nucleoids suggests that there may be a slightly more rapidly than human functional relationship as well. The strong

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association of R2 with the rapidly without prior fractionation. As shown in sedimenting nucleoids, in the apparent Figure 6, substantial label from CDP absence of R1, is of interest, but its appeared in dCMP, dCDP, dTMP, and significance is unknown. Note that in our dTDP, with smaller incorporation into dTTP hands the nucleoid markers RNA and negligible radioactivity in dCTP. The polymerase and HSP60 were found only in PEI-cellulose chromatography, which the slowly sedimenting peak (panel a). resolves components by ion exchange, does not separate thymidine nucleotides from the We also investigated RNR association with corresponding nucleotides. nucleoids in pig liver mitochondria, which Therefore, the components migrating with were prepared essentially identically to the marker dTMP, dTDP, and dTTP are labeled procedure used in rat liver. Results were dT/UMP, dT/UDP, and dT/UTP, similar in that nucleoids sedimented in two respectively. forms, with R1 and R2 both in the slowly sedimenting form and R2 alone found in the From the total radioactivity found in rapidly sedimenting material (Figure 5). A deoxyribonucleotides in the first five difference between rat and pig in these minutes of incubation, when incorporation is experiments was that in the pig liver maximal, we can estimate total nucleoids the nucleoid markers that we ribonucleotide reductase activity as being analyzed were found in both rapidly and about five pmol per per minute per mg slowly sedimenting peaks. In this protein, about half the specific activity we experiment we also probed nucleoid observed in mitochondrial lysates (Table 1). fractions for p53R2, the DNA damage- This may reflect a rate limitation at the inducible form of the RNR small subunit. mitochondrial uptake level under our We saw no evidence for existence of this conditions of incubation. Alternatively, protein in nucleoids. degradation of deoxyribonucleotides by the mitochondrial nucleotidase (36) may yield Ribonucleotide reduction in isolated , which would be washed off of mitochondria. In preliminary experiments the TLC plates before chromatography. we found that rat liver mitochondria readily took up radiolabeled With respect to the dGTP overrepresentation (35). It was of obvious interest to determine that we observe in mammalian whether this RNR substrate could undergo mitochondria, this could result from reduction after being taken up into relatively high activity of any biosynthetic mitochondria. reaction or limitation of any reaction that would consume mitochondrial dGTP. To For this study we incubated respiring determine whether intramitochondrial RNR mitochondria with [14C]CDP and followed might be such a rate-controlling step, we its fate by thin-layer chromatographic carried out identical uptake experiments separation of nucleotides on PEI-cellulose, with isolated respiring mitochondria, in followed by determination of radioactivity which [14C]GDP was incubated with in the separated deoxyribonucleotides. In the mitochondria instead of CDP. As seen in earliest experiments we carried out a Figure 7, substantial reduction of the preliminary separation of guanosine ribonucleotide occurred, with deoxyribonucleotides from ribonucleotides most of the deoxyguanosine nucleotides by boronate column chromatography accumulating as dGMP. Total followed by TLC analysis of the ribonucleotide reductase activity was deoxyribonucleotides in the flowthrough. considerably lower than what we observed However, we found that the nucleotides of in the CDP experiments, less than 2 interest could be separated by two- pmol/minute/mg protein total conversion of dimensional thin-layer chromatography GDP to deoxyguanosine nucleotides. Hence,

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this experiment confirms that active phosphorylated to the dNMP. Aside from ribonucleotide reductase exists within the question whether mitochondria contain a mitochondria, but it does not identify significant 1-phosphate pool, ribonucleotide reductase as an essential these alternative pathways would require at element in the processes leading to high least four reactions, with significant dNMP dGTP accumulation in mitochondria. accumulation seen within the first five minutes of incubation. Because we have DISCUSSION shown mitochondria to contain RNR activity, a pathway involving reduction of Several lines of evidence described in this CDP or GDP followed by paper establish the presence of dephosphorylation of the resultant ribonucleotide reductase in mammalian deoxyribonucleoside diphosphate seems mitochondria: (1) enzyme activity in more likely. mitochondrial extracts, at severalfold higher specific activity than seen in corresponding How might ribonucleotide reductase cytosolic extracts; (2) localization of subunits have found their way into enzyme activity to the mitochondrial matrix; mitochondria? We have examined sequences (3) the presence of R1 protein in washed of rat R1, R2, and p53R2 and none contains mitochondria, as determined by mass an N-terminal targeting sequence. However, spectrometric analysis of we note that pathways for mitochondrial immunoprecipitated mitochondrial proteins; protein incorporation have been described (4) association of ribonucleotide reductase other than the classical presequence pathway activity and immunoreactive material with (38,39). Moreover, many of the 1098 liver mitochondrial nucleoids; and (5) the proteins established in the mouse uptake of ribonucleoside diphosphates by mitochondrial proteome in the survey of respiring mitochondria and their conversion Pagliarini et al (40), lack a classical to deoxyribonucleotides. targeting sequence. Finally, we note that mouse and Chinese hamster mitochondria With respect to the isotope uptake have been shown to contain thymidylate experiments, we would have expected to see synthase and a dihydrofolate reductase the respective deoxyribonucleoside isoform, even though both proteins lack diphosphate (dCDP or dGDP) as the first classical targeting sequences (17). Although labeled intermediate to appear. However, in we don’t know the process whereby both the CDP and GDP uptake experiments ribonucleotide reductase subunits are the principal deoxyribonucleotides to transported into mitochondria, it is accumulate were the monophosphates. reasonable to assume that it happens by a Deoxyribonucleotide turnover in process other than the classical preseqence mitochondria is significant (36,37), so it pathway. seems likely that ribonucleoside diphosphate initially taken into mitochondria was What is the molecular form of the reduced to dNDP and rapidly mitochondrial ribonucleotide reductase? The dephosphorylated to dNMP. Might some presence of R1 is clearly established by the other pathway have converted the mass spectrometric analysis of radiolabeled rNDP to dNMP? Any such immunoprecipitated protein. And, as noted pathway would require conversion of the earlier, the presence of four R1 peptides in ribonucleoside diphosphate to the the R2 immunoprecipitate suggests that an corresponding monophosphate and then to R1-R2 oligomer was precipitated by the R2 the , followed by a antibody. We did see one R2 peptide in the phosphorylase reaction using deoxyribose 1- R2 immunoprecipitate, but with lower phosphate as a deoxyribosyl donor. The probability of correct peptide identification. resultant deoxyribonucleoside would then be p53R2 peptides were not seen at all. Our

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failure to detect either R2 or p53R2 with Any discussion of dNTP synthesis for high assurance may mean that these proteins mitochondrial DNA replication needs to were present in our preparations in amounts take into account the findings of Morris et al too small to be detected. Unfortunately, the (41) that in mitochondria from perfused rat small amount of material available in rat hearts dCTP and dTTP are derived entirely liver mitochondria precluded a direct from salvage pathways—a finding that analysis with purified protein, and in an appears to rule out a function for RNR in rat alternate source, namely pig liver heart. Morris et al worked with a different mitochondria, the enzyme was in a organ and a different experimental system membrane-bound form from which it could from that described here and in previous not be removed without inactivation. Our investigations of mitochondrial nucleotide failure to detect p53R2, either by metabolism. Moreover, their analysis immunopreciptation or in the nucleoid involved only the dNTPs. experiments, doesn’t mean that the protein is Whether dNTP synthetic pathways vary absent, just that we could not detect it. among different organs merits systematic investigation. Our detection of ribonucleotide reductase in mitochondria means that at least three One of our reasons for reopening an pathways contribute nucleotides to investigation of rNDP reductase in mitochondrial dNTP pools—first, the well- mitochondria was our hope to better established salvage pathways starting with understand the enzymatic basis for the mitochondrial deoxyribonucleoside kinases; unexpected dNTP pool asymmetry that we second, the equally well-established have described in mammalian mitochondria cytosolic ribonucleotide reductase pathway (6). That hope was not realized. The in situ using proteins R1 and p53R2; and third, as reduction of GDP, observed in the shown in this study, ribonucleotide experiments of Figure 7, was about half the reductase within the mitochondrion using rate of CDP reduction under identical R1 and possibly R2. Because previous conditions. To the extent that freshly studies were carried out with cycling or isolated, respiring rat liver mitochondria quiescent cells in culture and our work was reflect conditions in intact cells, we might done with a differentiated tissue, we are not have expected the in situ activity of in a position to estimate the relative mitochondrial RNR to be higher with GDP contributions of each pathway under than with CDP as the substrate. However, different conditions. However, given the we have not pursued the possibility that the importance of maintaining suitable dNTP GDP reductase activity would be higher in pools within the mitochondrion, perhaps we the presence of proper allosteric modifiers. should not be surprised to learn that there We did find that the activity for CDP are multiple ways to supply these pools. In reduction in our standard assay is sensitive addition the recent demonstration of a to inhibition by dATP to the same extent as thymidylate synthesis cycle within a cytosolic extract (data not shown). mammalian mitochondria (17)—thymidylate Although the mechanism by which synthase, dihydrofolate reductase, and serine mammalian tissue mitochondria maintain a hydroxymethyltransferase—makes it striking dNTP pool asymmetry remains perhaps less surprising that ribonucleotide open, the data presented here indicate that reductase is also present within ribonucleotide reductase activity within the mitochondria. mitochondrion needs to be considered as a possibly significant source of intramitochondrial dNTPs.

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FOOTNOTES

*This work was supported in part by National Institutes of Health Grant R01 GM073744. This work was also supported by Army Research Office Grant 55953-LS. The proteomic analysis was carried out in the OSU Environmental Health Sciences Center mass spectrometry facility supported by NIEHS Center Grant ES000210. We are grateful to Dr. Samanthi Wickremasekara for carrying out this analysis and helping us to interpret the results. We also thank three anonymous reviewers, whose critical comments led to significant improvement of this paper

1Present address: Department of Chemistry, 1253 University of Oregon, Eugene, OR 97403

2Present address: Division of Gerontology and Geriatric Medicine, Baltimore VA Medical Center, 10 N Greene St, Baltimore MD 21201

3To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, 2011 Agricultural and Sciences Bldg., Oregon State University, Corvallis, OR 97331-7305. Tel.: 541-737-1865; Fax: 541-737-0481; E-mail: [email protected].

4The abbreviations used are: dNMP, dNDP, dNTP, deoxyribonucleoside mono-, di-, and triphosphate; rNDP, ribonucleoside diphosphate; RNR, ribonucleotide reductase

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Table I

Enzyme activities in fractionated rat liver mitochondria

A. Membrane vs. soluble fraction. Mitochondria were divided into a soluble fraction and a membrane fraction, essentially as described by Kang et al (32). Mitochondria were ruptured by sonic oscillation, then centrifuged at 320,000 x g for one hour at 4 oC. The supernatant was the soluble fraction and the pellet, after resuspension, was the membrane fraction. Each value is a mean ± standard deviation of two independent experiments with different rats, involving duplicate assays in each experiment. Both citrate synthase and cytochrome c oxidase activities are recorded in nmol/min/mg protein. Ribonucleotide reductase activities are recorded in pmol/min/mg protein.

Enzymes Mitochondrial Mitochondrial Mitochondrial extract soluble fraction membrane fraction

Citrate synthase 126 ± 21 292 ± 88 31.2 ± 12.7

Cytochrome c 157 ± 13 20.3 ± 1.1 421 ± 69 oxidase

Ribonucleotide 13.6 ± 0.5 49.8 ± 16.3 4.6 ± 1.9 reductase

B. Mitoplasts vs. intact mitochondria. Mitoplasts were prepared as described by Ragan et al (33). Briefly, a mitochondrial suspension at 25 mg/ml protein was mixed with digitonin at a ratio of 1 mg digitonin to 10 mg protein. After incubation for 15 minutes on ice the sample was diluted with isotonic buffer and centrifuged at 15,000 x g for 10 minutes, followed by gentle resuspension of the pellet. Each value is a mean of two independent experiments from different rats. Citrate synthase is a marker enzyme for mitochondrial matrix. Adenylate kinase is a marker enzyme for mitochondrial intermembrane space.

______Enzymes Mitochondrial extract Mitoplasts Per cent total activity ______

Citrate synthase 100 79

Ribonucleotide reductase 100 82

Adenylate kinase 100 2

15

Table II

Immunoprecipitation of Ribonucleotide Reductase Subunits In Rat Liver Mitochondrial Extracts

Antibodies to mouse R1, R2, and p53R2 proteins were used for immunoprecipitation analysis of washed rat liver mitochondrial extracts as described in the text. Peptides were identified with at least 95% probability, and protein identification based upon each individual peptide was at least 95%.

Antibody to Rat R1 Peptide in Immunoprecipitate Molecular Mass of Peptide

Theoretical Observed

R1 SYLLKINGK 1177.8 1175.6

NTGKEEQRAR 1231.7 1229.6

SNQNLGTIKSNLCTEIVEYTSK 2774.3 2771.2

NTAAMVCSLENR 1366.7 1364.6

R2 AQQLWYAIIESQTETGTPYMLYK 2933.4 2930.4

NQIIACNGSIQSIPEIPEDLK 2424.1 2421.1

ALKEEEEK 1018.5 1016.5

NTAAMVCSLENR 1366.7 1364.6

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FIGURE LEGENDS

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 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. Values for the remaining assays represent average of duplicate assays on single preparations.

Figure 3. Sucrose gradient centrifugal analysis of ribonucleotide reductase activity in pig liver cytosol and mitochondrial extracts. Details are in 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.

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.

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.

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.

17

ATP

Unidentified ATP Unidentified

dGTP GTP dGTP

[dGTP] pmol/mg protein Before boronate column After boronate column

Enzymatic assay 298 258 HPLC peak area 300 333

Ribonucleotide reductase

min/mgprotein mol/ p

Lactate dehydrogenase protein

/mg mol/min µ

A. Pig liver cytosol )

RNR activity pmol dCDP/min pmol (

Fraction number B. Pig liver mitochondria )

RNR activity pmol dCDP/min pmol (

Fraction number

C. Hemoglobin standard (A540) Absorbance at 540 nm

Bottom Fraction number Top 240 5.0

200 Protein concentration 4.0 160 (mg/ml) /ml) Fraction # 3.0 ng

( 120 1 2 18 19 20 36 37 38 HEK293 2.0 80 DNA concentration DNA R1 1.0 40

R2 0 0.0 0 5 10 15 20 25 30 35 40 45 HSP60 Fraction number

TFAM b) Bradford and Quant-iT PicoGreen assays

RPOL 25 20 a) Immunoblotting 15 10

/min./mg. protein 5 0 nmo -5 0 5 10 15 20 25 30 35 40 45 RNR, Fraction number c) RNR activity assay

6 30 20

5 25 16

4 CMP 20 12 dCMP 3 15 d(T/U)MP

8 2 10

4 1 CDP 5 d(T/U)DP dCDP [nucleotide] pmol/mg mitochondrial protein d(T/U)TP CTP dCTP 0 0 0 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40

Minutes of incubation 35 20

GDP 18 30 16

25 14 GMP dGMP 12 20 10 15 8

6 10 GTP 4 dGTP dGDP 5 2 [nucleotide] pmol/mg mitochondrial protein 0 0 0 10 20 30 0 10 20 30

Minutes of incubation