©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intracellular Localization of 8-Oxo-dGTPase in Human Cells, with Special Reference to the Role of the Enzyme in Mitochondria (*)

Dongchon Kang (1)(§), Jun-ichi Nishida (2), Akihiro Iyama (2), Yusaku Nakabeppu (2), Masato Furuichi (2), Toshiyuki Fujiwara (3), Mutsuo Sekiguchi (2), Koichiro Takeshige (1)

From the (1)Department of Biochemistry, Kyushu University School of Medicine, Fukuoka 812, the (2)Medical Institute of Bioregulation, Kyushu University, Fukuoka 812, and the (3)Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka 814-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We examined the intracellular distribution of 8-oxo-dGTPase (8-oxo-7,8-dihydrodeoxyguanosine triphosphatase) encoded by the MTH1 gene, a human mutator homologue. The activity of 8-oxo-dGTPase mainly located in cytosolic and mitochondrial soluble fractions of Jurkat cells, a human T-cell leukemia line. Electron microscopic immunocytochemistry, using a specific antibody against MTH1 protein, showed localization of MTH1 protein in the mitochondrial matrix. Activity in the mitochondria accounted for about 4% of the total activity. The specific activity in the mitochondrial soluble fraction (8093 units/mg protein) was as high as that in the cytosolic fraction (8111 unit/mg protein). The 8-oxo-dGTPase activities in cytosolic and mitochondrial soluble fractions co-eluted with MTH1 protein by anion-exchange chromatography, and the molecular mass of the mitochondrial MTH1 protein was much the same as that of the cytosolic MTH1 protein (about 18 kDa). HeLa cells expressing MTH1 cDNA showed an increased cytoplasmic signal together with a weak signal in the nucleus in in situ immunostaining of MTH1 protein, and the overexpressed MTH1 protein was recovered from both cytosolic and mitochondrial fractions. Thus, the 8-oxo-dGTPase encoded by MTH1 gene is localized in mitochondria and cytosol.


INTRODUCTION

Oxygen radicals generated through the process of oxidation-reduction reactions in living cells attack many reactive moieties of DNA. When DNA is subjected to such oxidative lesions, strand breaks of DNA occur as does modification of bases, and cellular dysfunction, mutagenesis, and carcinogenesis follow(1, 2, 3) . Among the oxidative lesions of DNA, 8-oxoguanine()(8-oxo-7,8-dihydroguanine), an oxidized form of guanine, is a major causative lesion for mutagenesis by oxygen radicals, since during DNA replication it can pair with adenine as well as cytosine, with almost equal efficiency(4, 5) . Thus, 8-oxoguanine would cause A:T to C:G and G:C to T:A transversion mutations.

Organisms are equipped with elaborate mechanisms to counteract such mutagenic effects of 8-oxoguanine. In Escherichia coli, two DNA glycosylases encoded by mutM and mutY genes function to repair 8-oxoguanine. MutM protein removes 8-oxoguanine paired with cytosine (6) and MutY protein removes adenine paired with 8-oxoguanine in DNA(7) . The oxidized form of guanine is also formed in the nucleotide pool of the cell and can be eliminated by the mutT gene product. MutT protein hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing misincorporation of 8-oxo-dGMP into DNA (5, 8). In a mutT-deficient strain, the rate of spontaneous occurrence of A:T to C:G transversion increases hundreds to thousandsfold compared to the wild type(9, 10, 11) . While the spontaneous mutation rate in mutM or mutY-deficient strain is 10-50 times higher than that in wild type strain(7, 11) , a rate of mutation in the double mutants of mutM and mutY is equivalent to that of the mutT mutant(11) .

Mammalian cells contain enzyme activities similar to those E. coli enzymes. 8-Oxo-dGTPase has been purified from human Jurkat cells, a T cell leukemia cell line(12) . The cDNA was isolated, and the genomic sequence was determined(13, 14) . The human 8-oxo-dGTPase shows a considerable degree of amino acid sequence homology with the E. coli MutT protein(13) , and expression of the human cDNA in mutT-deficient E. coli cells efficiently reduced the increased frequency of A:T to C:G transversion to the level seen in wild type strain(14) . Hence, the enzyme is considered to be a human counterpart of MutT protein, and the gene was named MTH1 (for mutT homologue 1)(14) .

In eukaryotic cells, a pool of dNTP for nuclear DNA replication is present mainly in the cytosol(15) . For mitochondrial DNA synthesis, mitochondria preserve a pool of dNTP, consisting of more than 10% of the total intracellular dNTP. The amount of the mitochondrial DNA is roughly 1% of the total DNA in the cell(15, 16) . Judging from the different behavior of nucleotides in mitochondrial and cytosolic pools (15-17), nucleotides in the former seem to be synthesized in the mitochondria and are not derived from the cytosolic pool. The activity of ribonucleotide reductase that catalyzes synthesis of deoxyribonucleoside 5`-diphosphate from ribonucleoside 5`-diphosphate is present in both the cytpolasm and mitochondria(17) .

The mitochondrial respiratory chain located on inner membranes is a major site for the initiation of lipid peroxidation (18, 19) which can lead to oxidation of the guanine base to 8-oxoguanine(20) . In addition, the mitochondrial respiratory chain produces superoxide (21) which can be converted to hydroxyl radical via hydrogen peroxide. The hydroxyl radical is the main species of active oxygens that attack the guanine base(22) . Thus, DNA and dNTP in the mitochondrial pool may be exposed to a greater oxidative stress. The repair of oxidized mitochondrial DNA and elimination of 8-oxo-dGTP from the mitochondrial dNTP pool may be crucial to maintain integrity of mitochondrial DNA. A system for repair of oxidatively damaged DNA in the mitochondria has been described(23) . However, it is uncertain whether the mitochondria possess mechanism(s) for eliminating 8-oxo-dGTP from dNTP pool. We examined the intracellular distribution of 8-oxo-dGTPase in human cells with special reference to role of the enzyme 8-oxo-dGTPase in the mitochondria. Several lines of evidence obtained in this study show that the mitochondrial matrix possesses 8-oxo-dGTPase and suggest that the 8-oxo-dGTPase present in mitochondria and cytosol fractions is the product of the same gene.


EXPERIMENTAL PROCEDURES

Materials

8-Oxo-dGTP was synthesized from dGTP as described previously(5) . [-P]GTP (>15TBq/mmol) and I-protein A (1.1 GBq/mg) were purchased from Amersham International plc (Buckinghamshire, United Kingdom). Other reagents were of analytical grade.

Fractionation of Cultured Cells

Jurkat cells, a human T cell leukemia line, were grown in RPMI 1640 medium containing 10% fetal calf serum. Cells in midlog growing phase were harvested, washed in an isotonic sucrose buffer (TES) composed of 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.25 M sucrose, 15 µg/ml leupeptin, 5 µg/ml (p-amidinophenyl)methanesulfonyl fluoride hydrochloride (APMSF) and 50 ng/ml pepstatin, and suspended in the same buffer (5 10 cells/ml). The following procedures were performed at 4 °C. The cells were homogenized in a Potter-Elvehjem homogenizer, the homogenate was centrifuged at 600 g for 10 min, and the supernatant (post-nuclear supernatant) was centrifuged again at 600 g for 10 min to minimize contamination of the post-nuclear supernatant with nuclei and intact cells. The post-nuclear supernatant was centrifuged at 7,000 g for 10 min. The pellet (crude mitochondrial fraction) was used for further purification of mitochondria. The 7,000 g supernatant was centrifuged at 320,000 g for 1 h and the resulting supernatant was used as the cytosolic fraction. The 320,000 g pellet was washed three times with TES and served as the microsomal fraction. The crude mitochondrial fraction was layered on discontinuous sucrose gradient made by successive layering 4.5 ml of 1.5 M and 1.0 M sucrose from the bottom and then centrifuged at 80,000 g for 1 h. The phase between the layers of 1.5 and 1.0 M sucrose was collected and washed three times with TES (mitochondrial fraction).

For preparation of the nuclear fraction, cells were suspended in hypotonic buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 15 µg/ml leupeptin, 5 µg/ml APMSF, and 50 ng/ml pepstatin) containing 5 mM CaCl which stabilizes nuclear membranes and was then kept on ice for 20 min. After homogenization of the cells, the tonicity of the homogenate was made isotonic by adding 2.2 M sucrose, then the homogenate was centrifuged at 600 g for 10 min. The supernatant was centrifuged at 320,000 g for 1 h. The supernatant showed essentially the same levels of activities of lactate dehydrogenase and 8-oxo-dGTPase as seen in the cytosolic fraction prepared without CaCl (results not shown). The 600 g pellet was recentrifuged at 600 g for 10 min. The resulting pellet was homogenized in TES containing 5 mM CaCl, layered on discontinuous sucrose gradient made by successive layering of 4.5 ml of 2.3 and 1.5 M sucrose containing 1 mM MgCl and centrifuged at 80,000 g for 1 h. The pellet at the bottom was collected and washed three times with TES with 0.25 M sucrose containing 1 mM MgCl (nuclear fraction).

Subfractionation of Mitochondria

To separate mitochondria into membrane and soluble fractions, 4 mg of the mitochondrial fraction in 1 ml of TES was sonicated at output 3 for three 1-min cycles, using a Branson sonifier and then centrifuged at 320,000 g for 1 h at 4 °C. The supernatant was used as the mitochondrial soluble fraction. The pellet was homogenized in TES and served as the mitochondrial membrane fraction.

Mitoplasts were prepared as follows. Freshly prepared mitochondria (3 mg) in 0.3 ml of TES were diluted 10-fold with buffer containing 5 mM Tris-HCl, pH 7.4, and 1 mM EDTA and centrifuged at 14,000 g for 20 min at 4 °C. The pellet was resuspended in 3 ml of the same hypotonic buffer and left to stand on ice for 20 min. The suspension was centrifuged at 14,000 g for 20 min at 4 °C and washed three times with 3 ml of the hypotonic buffer (mitoplasts). The supernatant at each step was also stored until measurement of enzyme activities.

8-Oxo-dGTPase Assay

8-Oxo-dGTPase activity was assayed by measuring the hydrolysis of 8-oxo-dGTP to 8-oxo-dGMP(5) . The reaction mixture (12.5 µl) contained 20 mM Tris-HCl, pH 8.0, 4 mM MgCl, 40 mM NaCl, 20 µM 8-oxo-dGTP, 80 µg/ml bovine serum albumin, 8 mM dithiothreitol, 10% glycerol, and the enzyme fraction to be examined. The reaction was run at 30 °C for 20 min and stopped by spotting 2 µl of the reaction mixture onto a polyethyleneimine-cellulose plate (Merck, Darmstadt, Germany). The product was separated from the substrate by thin layer chromatography with 1 M LiCl for 1 h, and the radioactivity was measured using a Fujix 2000 Bio-image analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan). One unit of activity was defined as amount of enzyme that produced 1 pmol of 8-oxo-dGMP/min.

Antibody Preparation

A peptide corresponding to Lys to Val (M78) was synthesized and purified by HPLC. The polypeptide was coupled with bovine serum albumin and hemocyanin by 0.1% glutaraldehyde(24) . To obtain polyclonal antibodies against the peptide, each of the coupled peptides (200 µg) was emulsified with adjuvant, Titer Max (Vaxel, Inc., Norcross, GA) and injected into a Japanese white rabbit. Four weeks later, the first booster injection (100 µg) was given, followed by three booster injections at 2-week intervals. Sera were obtained 2 weeks after the last booster injection, and antibodies against the peptides were purified on immunoaffinity columns in which the peptide was covalently linked to activated CH-Sepharose 4B (Pharmacia LKB)(24) . The purified antibody was designated anti-M78.

Polyclonal rabbit antibodies against human MTH1 protein were prepared using TrpE-MTH1 fusion protein, as described by Nakabeppu and Nathans (25). E. coli BL21(DE3) carrying pET:TrpE-MTH1 was cultured at 37 °C in 250 ml of broth containing 50 µg/ml of ampicillin to OD = 0.6 and then isopropyl--D-thiogalactoside was added at a final concentration of 1 mM(26) . After cultivation for 6 h, the cells were harvested by centrifugation, washed with Tris-buffered saline, and disrupted by sonication on ice in 50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, and 0.3 M NaCl.The insoluble protein fraction contained about 10 mg of TrpE-MTH1 fusion protein and was electrophoresed on SDS-12.5% polyacrylamide gels. A region of the gel containing the fusion protein was excised (approximately 200 µg), emulsified with adjuvant (Titer Max, Vaxel, Inc., Norcross, GA), and injected into a Japanese white rabbit. Four weeks later, the first booster injection (approximately 100 µg) was given, followed by booster injections at 2-week intervals for 1 year. Sera were obtained after the second injection of booster, and antibodies were purified with the aid of TrpE-MTH1 fusion protein-Sepharose and TrpE protein-Sepharose affinity columns(25, 27) . This antibody preparation was designated as anti-hMTH1.

Immunoblotting Analysis of Subcellular Fractions

Proteins were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE)(28) , and immunoblotting analysis was performed as described elsewhere(27, 29) . Briefly, a blot was blocked in Tris-buffered saline (10 mM Tris-HCl, pH 7.6, and 150 mM NaCl) containing 0.05% Tween 20 and 5% bovine serum albumin at 52 °C for 1 h, probed with 10 µg/ml anti-hMTH1 antibody in the blocking solution at 4 °C overnight, washed with Tris-buffered saline containing 0.05% Tween 20, and reacted with I-protein A in the blocking solution at 4 °C for 1 h. The antibody-reactive protein was visualized, and the radioactivity was measured using a Fujix 2000 Bio-image analyzer. In the case of Fig. 1A and Fig. 3, the protein reactive to the antibody was probed with biotinylated anti-rabbit IgG and the avidin-biotin complex (Vector Laboratory, Burlingame, CA) and detected using the chemiluminescence reagent (DuPont NEN).


Figure 1: Immunoblotting of subcellular fractions with the anti-hMTH1. A, proteins of the total homogenates (14.6 µg which corresponds to 2.5 µg of the cytosol) were separated on 15% SDS-PAGE and immunoblotted with 10 µg/ml of non-immune IgG or the anti-hMTH1 as described under ``Experimental Procedures.'' B, proteins were separated on 15% SDS-PAGE and analyzed. MTH1 in each fraction was quantified and is expressed as radioactivity/mg of protein (arbitrary unit). Applied protein contents per lane are shown.




Figure 3: Analyses by anion-exchange chromatography. The mitochondrial soluble (0.85 mg) and cytosolic (2 mg) fractions were analyzed by MonoQ anion-exchange chromatography. The activity hydrolyzing 8-oxo-dGTP to 8-oxo-dGDP () or to 8-oxo-dGMP () was assayed using 2 µl from 1 ml of each fraction. The proteins reactive to the anti-hMTH1 in each fraction (10 and 2.5 µl/lanes in upper and lower panels, respectively) were also analyzed. Upper panel, mitochondrial soluble fraction; lower panel: cytosolic fraction.



Immunocytochemistry

Colloidal gold with a diameter of 8 nm was prepared by reducing tetrachloroauric acid with tannic acid and sodium citrate(30) . Goat antibody to rabbit IgG was conjugated to the colloidal gold at pH 9.0 according to De Mey et al.(31) . The resulting IgGgold complex was centrifuged and resuspended in 20 mM Tris-HCl, pH 8.2, containing 1% bovine serum albumin and 50% glycerol.

For electron microscopic immunocytochemistry, the washed fraction of crude mitochondria was used because the hypertonic procedure had resulted in mitochondria with a highly condensed configuration. Isolated mitochondria were pelleted, fixed with 8% paraformaldehyde, and embedded in LR white resin at 50 °C. Thin sections were cut with a diamond knife, treated with 1% bovine serum albumin for 10 min, and incubated with anti-hMTH1 antibody at a concentration of 25 µg/ml for 2 h at room temperature. After washing with phosphate-buffered saline, the sections were incubated with anti-rabbit IgG-gold (OD = 0.08) for 1 h. Immunolabeled sections were then stained with uranyl acetate and lead citrate and examined under a Hitachi HU12 electron microscope at 100 kV. Control experiments were done using non-immunized rabbit IgG instead of anti-hMTH1 antibody.

Light microscopic immunocytochemistry of HeLa MR cells was done as described by Ishibashi et al.(29, 32) after the cells had been fixed with 0.2% glutaraldehyde for 1 h at 4 °C and permeabilized with 50% methanol and 50% acetone for 2 min at room temperature.

MonoQ Anion-exchange Chromatography

The cytosolic fraction (2 mg) and the mitochondrial soluble fraction (0.85 mg) were analyzed by MonoQ anion-exchange chromatography, as described(13) . The activity of 8-oxo-dGTP hydrolysis in each fraction was assayed.

Plasmids

A mammalian expression vector pcDEB which carries the SR promoter and the hph gene for selection with hygromycin B has been described(27) . An entire cDNA for human MTH1 protein (13) was inserted downstream the SR promoter to generate pcDEB-MTH1. A coding region (NarI-BamHI fragment of the cDNA) corresponding to amino acid residues 3-156 of MTH1 protein was fused to the TrpE coding region at the PstI-BamHI site of pYN3103-TrpE vector(25) . The NcoI-BamHI fragment of the plasmid, yielding TrpE-MTH1 fusion protein, was subcloned into NcoI- and BamHI-digested T7 promoter expression vector pET8c(26) , resulting in pET:TrpE-MTH1.

Culture of HeLa MR Cells

HeLa MR cells (32) were maintained in Dulbecco's modified Eagle's medium, supplemented with 100 µg/ml of streptomycin, 100 units/ml of penicillin, and 5% each of fetal bovine serum and horse serum. Cells carrying pcDEB or pcDEB-MTH1 were maintained in medium containing 150 µg/ml of hygromycin B.

DNA Transfection

HeLa MR cells were transfected with pcDEB or pcDEB-MTH1, using the procedure of Chen and Okayama (33). Transformants were selected in medium containing 300 µg/ml of hygromycin B. Established transformant with pcDEB was designated MRV11, and three transformants with pcDEB-MTH1 were designated MR11, MR51, and MR81.

Other Methods

The activity of lactate dehydrogenase was measured by the method of Bergmeyer et al.(34) . One unit of the activity was defined as the absorbance change of 0.001/min. The activities of succinate-cytochrome c reductase(35) , adenylate kinase(35) , and fumarase (36) were measured as described. One unit of the activities for adenylate kinase and fumarase was defined as the absorbance change of 0.01/min. Protein concentration was determined using a Bio-Rad DC protein assay kit with bovine serum albumin as a standard, according to the manufacturer's instruction.


RESULTS

Intracellular Distribution of 8-Oxo-dGTPase

Jurkat cells were separated into cytosolic, mitochondrial, microsomal, and nuclear fractions characterized by the existence of specific marker enzymes; most lactate dehydrogenase was present in the cytosolic fraction while almost all of succinate-cytochrome c reductase was in the mitochondria (). The specific activity of 8-oxo-dGTPase in the mitochondrial fraction was about 17% of that in the cytosolic fraction. Since the specific activity of lactate dehydrogenase, a marker enzyme of cytosol, in the mitochondrial fraction was less than 1% of that in the cytosolic fraction, it is unlikely that the presence of 8-oxo-dGTPase in the mitochondrial fraction was due to contamination with the cytosolic fraction. On the other hand, 8-oxo-dGTPase activities in the microsomal and nuclear fractions may be explained by contamination with the cytosol, since the values were essentially the same as those of lactate dehydrogenase in the microsomal and nuclear fractions ().

The total activities of 8-oxo-dGTPase in cytosol and mitochondria fractions were calculated on the basis of recovery of lactate dehydrogenase and succinate-cytochrome c reductase activities in those fractions. The combined activities of 8-oxo-dGTPase in the cytosolic and mitochondrial fractions almost correspond with the total activities (). The 8-oxo-dGTPase activity in the mitochondria was about 4% of that in the entire cell. In the case of promyelocytic leukemia HL60 cells, mitochondrial 8-oxo-dGTPase occupied 9.6% of the total activities of the cells.

Immunological Detection of MTH1 Protein

To determine whether the activity of 8-oxo-dGTPase measured with subcellular fractions actually represents the amount of MTH1 protein itself, we prepared an antibody against TrpE-MTH1 fusion protein and used them for immunological analyses. When the total homogenate was immunoblotted with the anti-hMTH1 (Fig. 1A), the antibody reacted with a single protein with a molecular mass of 18 kDa, a mass corresponding to the molecular mass deduced from the MTH1 cDNA. When non-immune IgG was used, signals were hardly visible.

Each subcellular fraction was analyzed by immunoblotting using the anti-hMTH1 (Fig. 1B). A single band corresponding to the 18-kDa polypeptide was detected in the cytosol and in the mitochondria. The intensity of the signal/mg of protein in each fraction essentially paralleled to the activity of 8-oxo-dGTPase (see Fig. 1B and ). This would suggest that a single molecular species of MTH1 protein is responsible for 8-oxo-dGTPase activities present both in the cytosolic and mitochondrial fractions.

Localization of 8-Oxo-dGTPase in the Mitochondrial Matrix

The sublocalization of 8-oxo-dGTPase (MTH1 protein) in the mitochondria was next examined. When mitochondria were subfractionated into membrane and soluble fractions, more than 80% of the activity was recovered in the soluble fraction. The specific activity of 8-oxo-dGTPase in the mitochondrial soluble fraction was five times higher than that of the intact mitochondrial fraction, the level being as high as that of the cytosolic fraction (Tables I and III). This value was higher than a factor of enrichment of fumarase, a marker enzyme for the mitochondrial matrix. This would imply that 8-oxo-dGTPase is present in soluble form in the mitochondria while fumarase may have a weak association with mitochondrial membranes.

To see whether mitochondrial 8-oxo-dGTPase is localized in the intermembrane space or in the matrix, we prepared mitoplasts which do not have components of intermembrane space. The specific activities of fumarase and 8-oxo-dGTPase in the mitoplasts were 76 and 65% of those in the intact mitochondrial fraction, respectively, while the specific activity of adenylate kinase, a marker enzyme of the intermembrane space, was only 6% of that in the mitochondrial fraction (I). This low, specific activity of adenylate kinase in the mitoplasts is not due to inactivation of the enzyme during the preparation of mitoplasts since about 70% of the total activities was recovered in the post-mitoplast supernatant. These results would suggest that 8-oxo-dGTPase is mainly localized in the mitochondrial matrix.

The localization of 8-oxo-dGTPase in the matrix was further confirmed by electron microscopic immunocytochemistry of mitochondria stained with anti-hMTH1 (Fig. 2). To rule out the possibility that the cytosolic 8-oxo-dGTPase might give a false positive signal on the staining of mitochondrial 8-oxo-dGTPase, we used isolated mitochondria for the immunostaining experiments. The mitochondria showed a condensed configuration usually observed in mitochondria isolated by the method used here(37) . Proteins reactive to the anti-hMTH1 were predominant on the matrix (electron dense area), not on the intermembrane space (Fig. 2). When non-immune IgG was used, signals were nil (results not shown).


Figure 2: Electron microscopic immunocytochemical localization of MTH1 in mitochondria. Isolated mitochondria from Jurkat cells were stained with 20 µg/ml of the anti-hMTH1. The length of a bar indicates 0.1 µm.



Chromatographic Resolution of 8-Oxo-dGTPase Protein

We analyzed 8-oxo-dGTPase activities in cytosolic and mitochondrial fractions by anion-exchange chromatography, using a MonoQ column, the objective being to further confirm that the MTH1 protein is actually responsible for activity seen in those fractions. The 8-oxo-dGTPase activity eluted as a single peak and essentially the same elution patterns were obtained with the enzyme from cytosolic and mitochondrial fractions (Fig. 3). The activity of 8-oxo-dGTPase co-eluted with proteins reactive to the anti-hMTH1 both in the cytosolic and mitochondrial soluble fractions, thereby indicating that 8-oxo-dGTPase present in the cytosolic and mitochondrial fractions is the same or similar molecular species of protein, coded by the MTH1 gene.

In the cytosolic fraction, we detected activity converting 8-oxo-dGTP to 8-oxo-dGDP, which eluted more slowly than did the actual 8-oxo-dGTPase peak (Fig. 3, lowerpanel). This activity represents a nonspecific nucleotide triphosphatase that was not found in the mitochondrial soluble fraction (Fig. 3, upper panel). This evidence further supports the notion that 8-oxo-dGTPase in the mitochondrial fraction is not due to the contamination with the cytosolic fraction.

Overexpression of MTH1 Protein in HeLa Cells

It was of interest to determine whether the MTH1 protein would locate in cytosolic and mitochondrial fractions when the protein in a cell is overproduced. We transfected MTH1 cDNA into HeLa cells and selected several transfectants overproducing the MTH1 protein. We established three clones of MR11, MR51, and MR81 which produced about 10-300 times larger amounts of MTH1 protein ( Fig. 4and ). In MRV11, a clone transfected with the vector alone, the signal for the immunoreactive protein represents the level of endogenous MTH1 protein. When the mitochondrial fraction was prepared from each of these cells, the activity of lactate dehydrogenase in these materials contained less than 1% of the activity found in the cytosolic fraction (), thereby indicating that these mitochondrial fractions were not contaminated with cytosolic materials. The amount of MTH1 protein in the mitochondrial fraction of various strains varied considerably. Compared to values in the cytosolic fraction, the range was from 4 to 10% ( Fig. 4and ). Thus, the relative distribution of 8-oxo-dGTPase was much the same even with overproduction of the protein. Such being the case, the sequence encoded by the MTH1 cDNA probably has the full information leading to localization of the protein in mitochondria as well as in cytoplasm.


Figure 4: Immunoblotting analyses of MTH1 overexpressed in HeLa cells. HeLa cells were transfected with pcDEB-MTH1 to overproduce MTH1 protein. The cytosolic and mitochondrial fractions were prepared from HeLa cells and analyzed as for Fig. 2. Applied proteins/lane were 1.25 and 10 µg for the cytosolic and mitochondrial fractions, respectively. C, cytosol; M, mitochondria.



Light Microscopic Immunocytochemistry

Intracellular distribution of MTH1 protein was further examined by light microscopic immunocytochemistry. Immunostaining signals were rare in case of normal cells, thus we analyzed HeLa MR51 cells which overproduce MTH1 protein. When the cells were stained with the anti-hMTH1 antibody, there was a typical cytoplasmic staining and a slightly weaker nuclear staining (Fig. 5B). A similar staining was observed when anti-peptide antibody for MTH1, anti-M78, was used (Fig. 5D). Such a signal was hardly visible in HeLa MRV11 cells which carry the vector alone (Fig. 5, A and C).


Figure 5: Light microscopic immunocytochemistry of HeLa cells. HeLa MRV11 (A and C) and MR51 (B and D) cells were immunostained with 0.33 µg/ml of the anti-hMTH1 (A and B) and the anti-M78 (C and D).




DISCUSSION

The spontaneous oxidation of dGTP forms 8-oxo-dGTP, which can be inserted opposite dA and dC residues of template DNA with almost equal efficiency, and the MutT protein of E. coli specifically degrades 8-oxo-dGTP to the monophosphate(5) . Since defects in the mutT gene increase the occurrence of A:T to C:G transversions 100-10,000-fold over the wild type level(9, 10, 11) , elimination of the oxidized form of guanine nucleotide from the nucleotide pool is important for the high fidelity of DNA replication. An enzyme similar to the MutT protein was detected in human cells (12) and the cDNA was cloned(13) . As expression of human cDNA in E. coli mutT mutant cells suppresses almost completely the occurrence of specific A:T to C:G mutation(14) , the human 8-oxo-dGTPase probably has the same antimutagenic capacity as the MutT protein. The human gene was named MTH1 (for mutT homologue 1) and locates on chromosome 7p22(14) .

We have shown in the present study that the MTH1 protein is mostly present in the cellular cytosolic fraction. In subcellular fractionation, little MTH1 protein was recovered in the nuclear fraction. In situ immunostaining of MTH1-overexpressing HeLa cells revealed a typical cytoplasmic staining with slightly weaker nuclear staining. MTH1 protein present in nuclei may be lost into the cytosolic fraction during isolation of nuclei since the MTH1 protein does not have a typical nuclear localization signal (13) and size is small enough to freely traverse nuclear pores(38) . Even taking this into account, it is reasonable to conclude that a large part of the 8-oxo-dGTPase is localized in the cytosol. Larger pools of dNTP are present in cytosol than in nucleus, the former being supply of materials for the replication of chromosomal DNA(15, 16) .

We have shown that a considerably high level of the 8-oxo-dGTPase protein is present in the mitochondrial matrix. Mitochondria synthesize their own dNTPs (17) and form an independent pool of dNTP from pools in the cytosol and the nucleus(15, 16) . The mitochondrial dNTP pool is probably exposed to a stronger oxidative stress than are the cytosolic and nuclear pools because the respiratory chain producing a large amount of active oxygen species is present. DNA polymerase , the replicative polymerase for mitochondrial DNA, readily misincorporates 8-oxo-dGMP opposite adenine(39) . Thus, the 8-oxo-dGTPase in the mitochondrial matrix may play an important role in maintaining genetic integrity of the mitochondria DNA. In mitochondria, naked DNA is surrounded by membranes containing proteins for the respiratory chain which catalyze lipid peroxidation reaction yielding active oxygens(18, 19, 21) . Such a situation is close to that of bacterial DNA, in which mutT deficiency causes a high frequency of A:T to C:G transversion(9, 10) .

Mitochondria are central for aerobic energy production in eukaryotic cells and, therefore, the proper function of mitochondria is critical for cell survival. Many cases of mitochondrial neuromyopathy caused by dysfunction of mitochondrial respiration have been noted(40) . Since the integrity of mitochondrial DNA is essential for the normal functions of mitochondria(41) , it is important to protect mitochondrial DNA from attack by oxygen radicals inevitably produced by the mitochondrial own activity. Accumulation of mutations in mitochondrial DNA has been correlated with the decline of the oxidative phosphorylation with aging and with the impairment of the respiratory chain in degenerative diseases(1, 42) . It has also been reported that content of 8-oxoguanine in mitochondrial DNA increases with aging(1, 43) . Therefore, some portion of mutations leading to dysfunction of the mitochondrial respiratory chain may be caused by 8-oxoguanine. Involvement of 8-oxoguanine in deletion of mitochondrial DNA which is often observed in the Parkinson disease has also been discussed(43) . The content of 8-oxoguanine in DNA may be determined by a dynamic equilibrium between generation of 8-oxoguanine in DNA and excision repair of base from DNA. It is an intriguing question whether impairment of the 8-oxo-dGTPase or other repair enzymes is one of the causes for mitochondrial dysfunction in aging as well as in some degenerative diseases.

We have shown that an identical molecular form of MTH1 protein is present both in cytosol and mitochondria. There are several examples that isozymes exist in the cytosol and in the mitochondria(44) . Enzymes which locate in the two sites are classified into three categories: (i) enzymes coded by two separate genes, such as alcohol dehydrogenase; (ii) enzymes derived from alternatively spliced mRNAs, e.g. 2-isopropylmalate synthase; and (iii) enzymes differentially translated from single mRNA, for example, fumarase. Prefumarase carrying a signal sequence is imported into mitochondria, processed, and eventually takes on the same size as cytosolic fumarase (45). The last mechanism might relate to 8-oxo-dGTPase but there is no possible alternative initiation sites at the 5` region of cDNA with a signal sequence which would be processed(13) . There is the possibility that the intracellular localization of MTH1 protein is determined by post-translational modification of the protein.

  
Table: Enzyme activities in subcellular fractions of Jurkat cells

Subcellular fractions of Jurkat cells were prepared as described under ``Experimental Procedures.'' Activities of lactate dehydrogenase, succinate-cytochrome c reductase, and 8-oxo-dGTPase in subcellular fractions are expressed as percent of those in cytosol, mitochondria, and cytosol, respectively. Each value is a mean ± S.D. (n 3). Values without S.D. are means of two independent experiments.


  
Table: Distribution of 8-oxo-dGTPase in cytosol and mitochondria

The total activities of 8-oxo-dGTPase in cytosol and mitochondria fractions were calculated on the basis of recovery of the lactate dehydrogenase and succinate-cytochrome c reductase activities present in the fractions. The activities are expressed as percent of those in the homogenate. Each value in Jurkat cells is a mean ± S.D. of three independent experiments. Values in HL60 cells are from a single experiment.


  
Table: Enzyme activities in submitochondrial fractions

Submitochondrial fractions were prepared from mitochondria of Jurkat cells. Activities are expressed as percent of those in mitochondria. Values are means of two independent experiments.


  
Table: Mitochondrial localization of MTH1 overexpressed in HeLa cells

hMTH1 protein in cytosol and mitochondrial fractions of each strain was quantified with immunoblotting by anti-hMTH1. Each value in parenthesis indicates percent of that in the cytosol.



FOOTNOTES

*
This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Kyushu University School of Medicine, 3-1-1 Maidashi, Fukuoka 812, Japan. Fax: 81-92-632-2373.

The abbreviations used are: 8-oxoguanine, 8-oxo-7,8-dihydroguanine; 8-oxo-dGTPase, 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase; APMSF, (p-amidinophenyl)methanesulfonyl fluoride hydrochloride; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We extend special thanks to Dr. H. Sumimoto for helpful discussions and M. Ohara for useful comments on the manuscript.


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