Localization of the deoxyribonucleotide biosynthetic enzymes ribonucleotide reductase and thymidylate synthase in mouse L cells

Experimental

Cell Research 167 (1986) 417-428

Localization of the Deoxyribonucleotide Biosynthetic Enzymes Ribonucleotide Reductase and Thymidylate Synthase in Mouse L Cells REBECCA KUCERA,‘,*

and HENRY PAULUS1,2

‘Department of Metabolic Regulation, Bosron Biomedical Research Instituie, Boston, MA 02114, and ‘Department of Biological Chemistry Harvard Medical School, Boston, MA 02115, USA

Two different approaches were used to define the intracellular localization in mouse L929 cells of two deoxyribonucleotide biosynthetic enzymes: ribonucleoside diphosphate reductase (ECl. 17.4.1) and thymidylate synthase (EC2.1.1.45). The first involved treatment with saponins, which render the plasma membrane permeable to proteins without disrupting intracellular organelles. Under conditions where nuclear DNA synthesis and the activity of the nuclear enzyme NMN adenylyltransferase were unaffected, the entire cellular complements of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, and of ribonucleotide reductase and thymidylate synthase were released at the same rate and with similar dependence on saponin concentration. The second approach involved centrifugal enucleation of cells treated with cytochalasin B (CB) and measurement of the distribution of enzyme activities in the resulting cytoplast and karyoplast fractions. Whereas most NMN adenylyltransferase activity remained with the karyoplasts, glucose-6-phosphate dehydrogenase, ribonucleotide reductase, and thymidylate synthase were almost exclusively associated with the enucleated cytoplasts. These results indicate that, under conditions where nuclear DNA synthesis is apparently unperturbed, the intracellular distribution of the deoxyribonucleotide biosynthetic enzymes studied is the same as that of glucose-6-phosphate dehydrogenase, a typical cytosol enzyme, and clearly differs from that of NMN adenylyltransferase, a nuclear enzyme. @ 1986 Academic PES, IW.

The fact that the only known metabolic function of deoxyribonucleotides is to serve as precursors for DNA has led to the suggestion that their synthesis and utilization may be physically linked in the form of a multienzyme complex. Such a complex, or replitase [l], would of course reside in the nucleus and include the enzymes of deoxyribonucleotide biosynthesis as well as of DNA replication. The first committed enzyme of deoxyribonucleotide biosynthesis, ribonucleotide reductase, has been much studied in the context of this hypothesis, especially in relation to the possible channelling of ribonucleoside diphosphates into DNA [l-6] and its association with other DNA biosynthetic enzymes [l, 3, ‘7, 81. The interpretation of these relatively complex experiments has not always been straightforward [9]. On the other hand, a simpler test of the replitase hypothesis was provided by determining whether ribonucleotide reductase is a nuclear or a cytoplasmic enzyme. Although enucleation experiments had initially indicated a * To whom offprint requests should be addressed. Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/86 $03.00

418 Kucera and Paulus

migration of ribonucleotide reductase to the nucleus during the DNA replicative phase [I], subsequent experiments involving immunofluorescence studies [lo] and rapid subcellular fractionation [ 111suggested that the enzyme was confined to the cytoplasm at all times. When defining the intracellular localization of an enzyme, considerable care must be exercised not to perturb the intracellular macromolecular milieu. We had earlier developed a procedure for studying the properties of intracellular ribonucleotide reductase under conditions of minimal perturbation by selectively permeabilizing the plasma membrane of mouse L929 cells with dextran sulfate [12-141. In this paper, we have extended the cell permeabilization approach to study the intracellular location of enzymes by comparing the action of dextran sulfate with that of more powerful permeabilizing agents, the saponins, which render the plasma membrane permeable to larger molecules such as proteins without perturbing nuclear fractions. Our experiments revealed that ribonucleotide reductase and thymidylate synthase resided exclusively in the cytoplasm. This conclusion was confirmed by examining the distribution of enzyme activities in cytoplast and karyoplast fractions obtained by exposing cytochalasin B (CB)treated L929 cells to a centrifugal field.

MATERIALS

AND METHODS

Materials Culture media, calf serum and antibiotics were obtained from Gibco, N.Y.; cytochalasin B (CB), saponin (Gypsophila &guns), Ficoll (Type 400), and polyethylene glycol (PEG, approx. M,8000) were from Sigma. The PEG was recrystallized from acetone/ether 1151.Digitonin was obtained from Calbiochem-Behring. Isotopically labelled compounds were either from New England Nuclear or Amersham. Sources of materials used in ribonucleotide reductase assays and subsequent chromatography have been described previously [12].

Cell Culture Mouse L929 cells were maintained as suspension cultures in minimum essential medium (Eagle) for suspension culture supplemented with 10% calf serum, 100 U/ml penicillin G, and 100 kg/ml streptomycin. Cultures were inoculated at a density of lo5 cells/ml and harvested during midexponential growth (5-8X105 cells/ml). The cells have been karyotyped (Institute for Medical Research, Camden, NJ.) and were periodically tested for the presence of mycoplasma using a DNA-fluorescent dye binding assay (Bionique Laboratories, Inc., Saranac Lake, N.Y.); all tests were negative.

Cell Permeabilization Unless otherwise indicated, cells were permeabilized by a 20-min exposure at 4°C to either 500 &ml sodium dextran sulfate, 0.04% saponin, or 0.04% digitonin, in a buffer consisting of 93 mM NaCl, 5 mM KC], 2 mM MgCll and 50 mM tricine, pH 7.4 (NaCVtricine buffer). Saponin and digitonin solutions were made immediately before use in the NaCl/tricine buffer. The permeabilized cells were washed with 50 vol of the NaCVtricine buffer and then with a buffer consisting of 80 mM KC], 50 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) pH 7.2, before resuspension in the latter at lo8 cells/ml. For experiments where released enzymic material was assayed, saponin or digitonin permeabilization was done directly in the KCliHEPES buffer, and the permeabilized cells were removed by 30-set centrifugation at 9000 g. Exp Cell Rrs 167 (1986)

Cytoplasmic Preparation

of Cytoplasts

deoxyribonucleotide

biosynthesis

419

and Karyoplasts

L cells were enucleated in suspension by centrifugation in a discontinuous Ficoll density gradient in the presence of 10 ug/ml cytochalasin B, using a modification of the procedure described by Wigler & Weinstein [16]. Cells in suspension culture were collected by centrifugation, resuspended at a density of 2~ 10’ cells/ml in 14 mls of 15% (w/v) Ficoll, and placed on a step gradient consisting of the following layers: 9.2 ml of 15%, 2.3 ml of 18%, 2.3 ml of 19%. 9.2 ml of 20%, and 9.2 ml of 29.5% Ficoll, followed by an overlay of 14 ml without Ficoll. All solutions were prewarmed at 37°C and contained 10 Kg/ml CB and 0.5 % DMSO in suspension medium. They were prepared by appropriate dilution of 29.5 % (w/v) Ficoll, which results from the 1 : 1 dilution of 50 % (w/w) Ficoll. The gradients were made immediately before centrifugation of the cell preparations, which took place at 25000 rpm in a Beckman SW25.2 rotor (prewarmed to 37°C) in a Beckman L5-50 ultracentrifuge at 34°C for 60 min. After centrifugation, the bottoms of the tubes were punctured and l-ml fractions were collected. Karyoplasts sedimented to the 20-29.5% Ficoll interface, and cytoplasts were distributed over the l&20% Ficoll area. A small amount of cellular debris was present at the top of the gradient, indicating some cell lysis. The recovery of cytoplasts was typically 60 %, while that of karyoplasts was 20-30%. The cytoplasts and karyoplasts were washed with 100 vol of the Na-tricine buffer to remove both Ficoll and CB prior to use in enzyme assays. To screen for possible whole cell and cross-contamination, samples of the cytoplast and karyoplast fractions were placed in T25 flasks with minimum essential medium (monolayer formulation) and 10% calf serum. Consistently, cytoplasts could spread out on the surface in a limited manner but less than 0.1% of the cytoplasts assumed the morphology of a nucleated cell, whereas less than 0.1% of the karyoplasts had the ability to spread out over the surface. This indicated a very low level of whole ceil or cross-contamination.

Preparation

of Cell Extracts

Whole cells, cytoplasts, and karyoplasts were disrupted by sonication as previously described for whole cells [14]. The response of the cytoplast and karyoplast cell derivatives to sonication was nearly identical with that of whole cells. When indicated, the particulate debris was removed by a brief microcentrifugation at 9000 g, and the resulting supematant was utilized for enzymic studies.

Assay of Ribonucleotide

Reductase

CDP reduction was assayed essentially as previously described. Briefly, permeabilized cells (2x 106)or cell-derived material was incubated in 0.1 ml buffer (80 mM KC1 and 50 mM HEPES. pH 7.2) containing 0.022 mM [5-3H)CDP (0.55 uCi), 4 mM adenyl-5’-yl imidodiphosphate (AMP-PNP), 6 mM dithiothreitol (DTT), and 1 mM Fe&. If cell-free material was being assayed (sonic extracts or supematants from permeabilized cell incubations), PEG was present at 15% during the assay. PEG at that concentration assured a linear relationship between extract concentration and enzyme activity but otherwise had no effect on ribonucleotide reductase. After 10 min at 37°C the reaction was ended by adding an equal voiume of ethanol. After removal of the cells by centrifugation and the addition of dCMP or dCTP as carrier, the supematant solutions were chromatographed via a two-step procedure involving chromatography on phenyl(dihydroboryl)polyacrylamide and ion exchange chromatography with AGl-X8 (formate), as described earlier [12]. Radioactivity was determined in Liquiscint (National Diagnostics) with a Searle Mark III scintillation counter.

Assay of Thymidylate

Synthase

Thymidylate synthase (EC2.1.1.45) was assayed using a modification of the procedure described by Rode et al. [17]. In a 0.1 ml volume, 2x lo6 cell equivalents were incubated with 8 mM DTT, 0.1 mM NADPH, 2.5 mM formaldehyde, 0.13 mM tetrahydrofolate, and 45 uM [5-3H]dUMP (0.5 nCi) in 80 mM KCI, 50 mM Hepes buffer, (pH 7.2). After a IO-min incubation at 37°C. the reaction was stopped by the addition of an equal volume of 1.2 M trichloroacetic acid (TCA) and 10 VOI of 50 mg/ml charcoal (Norit A). After a brief incubation, radioactivity in 0.2 ml of the supematant solution was measured using Liquiscint and a Searle Mark III scintillation counter. Exp Cell h!es 167 (1986)

420 Kucera and Paulus Other Assay Methods The activity of glucose-6-phosphate dehydrogenase (EC I. 1.1.49) was measured spectrophotometritally at 340 nm by incubating 2~ lo6 cell equivalents with 10 mM MgClz, 1 mM glucose-6-phosphate and 0.4 mM NADP in 80 mM KCI, 50 mM HEPES (pH 7.2). NMN adenylyltransferase (EC2.7.7.1) was measured by a minor modification of the procedure of Atkinson et al. [18]. In a 0.5 ml volume, 1x 10’ cell equivalentswere incubated with 4 mM ATP, 15 mM MgCl,, and 1 mM NMN in glycylglytine buffer, pH 7.5, for 30 min at 37°C. The reaction was stopped by the addition of 0.5 ml of 0.5 M TCA, followed by brief centrifugation. A sample (0.4 ml) of the supernatant was added to 0.58 ml of a mixture containing 0.25 mmoles each of NaOH and glycine in 5% ethanol. The increase of absorbance at 340 nm upon addition of 0.02 ml alcohol dehydrogenase (5 mg/ml) was then measured, a maximum value generally being reached after 6 min. The incorporation of L3H]dTTP into DNA was measured as previously described [12], except the assay time was shortened to 15 min. Protein concentration was measured by the Coomassie Brilliant Blue G-250 procedure 1191using a commercial reagent (Pierce Chemical Company).

Cell Volumes Cell volumes were estimated using 15-urncalibration beads and a Coulter model ZF particle counter (Coulter Electronics), according to instructions supplied with the Coulter particle counter manual. The procedure has as its basis a proportionality between particle volume and the instrument threshold setting that yields half-maximal count, provided that the cell population is relatively homogeneous in size and the instrumental parameters remain unchanged during the volume determination.

RESULTS Effect of Saponins on L929 Cells

Studies from several laboratories [20-221 have shown that steroid glycosides such as digitonin and other saponins can be used to disrupt the integrity of the plasma membrane preferentially with little effect on intracellular membrane structures. This specificity provides the opportunity to distinguish between enzymes localized in the cytosol and those in organelles by examining their release from saponin-treated cells. The applicability of such an approach to mouse L cells was tested by comparing the effects of dextran sulfate and saponins. We had shown earlier that treatment of L929 cells with dextran sulfate made the cells permeable to small molecules such as nucleotides, but that such cells retained almost all their protein (~97%) and could resume growth under appropriate conditions after the removal of dextran sulfate [12]. In contrast, treatment with digitonin under equivalent conditions (20 min at 4°C) led to the loss of 30 % of the cellular proteins and all attempts to restore viability proved unsuccessful. On the other hand, gross external morphology was similar after both kinds of treatment, indicating that the effect of digitonin was not due to cell disruption but to an alteration in the permeability characteristics of the plasma membrane. This is in agreement with the observations on other cell types which indicated that saponins cause the selective loss of cytosolic proteins [22, 231. Nuclear Functions

in Saponin-treated

L929 Cells

To ascertian whether the protein loss resulting from treatment with saponin was confined to the cytosolic compartment, we compared the effects of saponin ExpCell

Res 167 (1986)

Cytoplasmic deoxyribonucleotide biosynthesis

1

2

3

421

Fig. 1. DNA synthesis in permeabilized 1,929 cells as a function of cell concentration. Cells were permeabilized - by a 20-min exposure at 4°C to either dextran sulfate (0) or saponin (O), washed extensively, then incubated at 37°C for 15 min with [3H]dTTP and other assay compo_ nents as described in Materials and Methods.

Cell Number (1 06cells)

and dextran sulfate on nuclear functions. We had found earlier [ 121that treatment with dextran sulfate allowed L929 cells to utilize [3H]dTTP for DNA synthesis at a rate close to that of DNA replication in intact cells. As shown in fig. 1, dextran sulfate- and saponin-treated cells incorporated [3H]dTTP into DNA at similar rates. The loss of 30% of cellular protein occasioned by saponin treatment thus had no effect on DNA synthesis capacity, consistent with the notion that the loss was confined to cytosolic proteins. To study more directly the effect of saponin treatment on nuclear proteins, we examined the retention of the nuclear enzyme NMN adenylyltransferase. The level of NMN adenylyltransferase in sonic extracts of digitonin-treated cells was 95 % of that seen in extracts of intact cells (2.7 nmoles per 30 min per 10’ cells), demonstrating that treatment with saponin caused no significant loss of this nuclear enzyme. Effect of Saponins on the Release of Glucose6Phosphate Dehydrogenase, Ribonucleotide Reductase, and Thymidylate Synthase from L929 Cells The observation that treatment with saponins had no effect on nuclear functions while causing significant protein loss, presumably cytosolic, suggested an approach to determine whether an enzyme is primarily associated with the cytosol or the nucleus. This approach involved the comparison of the release of the enzyme in question with that of a typical cytosolic enzyme as a function of saponin concentration and time. A comparison was made between the release characteristics of the known soluble cytoplasmic enzyme, glucose-6-phosphate dehydrogenase, and that of ribonucleotide reductase and another enzyme involved in DNA precursor synthesis, thymidylate synthase. If the enzymes involved in DNA precursor synthesis had a nuclear location or were physically constrained in the cytoplasm through association with either the cytomatrix or elements of the nuclear membrane, one would expect a delay in release when compared with the release of glucose-6-phosphate dehydrogenase. Exp Cell Res 167 (1986)

422 Kucera and Paulus

0

0.01 0.02 0.03 0.04 0.05 Saponin

Concentration

(%I

0

5 Exposure

10

15 Time

20

25

(min)

2. Release of enzymes from permeabilized L929 cells upon exposure to saponin. Cells (IO’ per ml) were treated for (A) 20 min with various concentrations of saponin; (B) with 0.04% saponin for various exposure times. Cells were then removed by centrifugation and enzymic activities in the supematant were measured as described in Materials and Methods. The values for ribonucleotide reductase (O), thymidylate synthase (H) and glucose G-phosphate dehydrogenase (A) are expressed as % of the activity seen in the supematant of sonically disrupted intact cells (48.3, 399 and 214000 pmoles product per 3x IO6 cells per IO min, respectively, in (A) 15.1, 405; (B) 294000 pmoles

Fig.

product per 3x IO6 cells.

Enzyme release was monitored by assaying the supernatants from cells treated for 20 min with various concentrations of saponin. The release of glucose-6phosphate dehydrogenase, ribonucleotide reductase, and thymidylate synthase were closely parallel and reached a maximum value at 0.03-0.04% saponin (fig. 2A). Comparison with enzyme activities in sonic extracts of intact cells showed that the release of the three enzymes was complete, and no residual activity was found associated with the saponin-treated cells (data not shown). The time course of release of the three enzymes in the presence of 0.04 % saponin was also almost the same (fig. 2B). Appropriate control experiments showed that saponin had no effect on enzyme activity at the concentrations used and that enzyme activity was a linear function of enzyme concentration. The latter was especially important with respect to ribonucleotide reductase, which has been found to exhibit an anomalous concentration response in dilute extracts [24, 251. We have found that assay in the presence of 15% PEG could largely eliminate this non-linear behavior (R. Kucera & H. Paulus, unpublished experiments). Similar enzyme release results were obtained when digitonin was used in the place of saponin (data not shown). On the other hand, all enzyme activities, including NMN adenylyltransferase, were retained by cells permeabilized by dextran sulfate. The observation that glucose-6-phosphate dehydrogenase, ribonucleotide reductase, and thymidylate synthase activities were completely released by treatment with saponins at nearly identical rates and with similar dependence on saponin concentration suggested strongly that the three enzymes resided entirely in the same subcellular compartment. It should be noted that these experiments were done with exponentially growing cells of which about Exp Ceil Res 167 (1986)

Cytoplasmic deoxyribonucleotide biosynthesis

423

Table 1. Characteristics of intracellular and released ribonucleotide reductase Relative rates of CDP reduction

Addition or deletion” None -AMP -PNP -AMP -PNP, +4 mM ATP, +5 mM MgCIZ - DTT -DTT. + 1 mM NADPH -FeCI, +I mM hydroxyurea + 100 urn dATP

Intracellular Activityh m’c)

Released activity’ (%I

100 0

100 20

21 66 125 78 24 1

70 73 88 68 21 15

’ Ribonucleotide reductase assay was performed as described in Materials and Methods. b Intracellular activity was measured in dextran sulfate-permeabilized cells: 100% activity corresponds to 30.8 pmoles dCDP produced per 10 min per 3~10~ cells. ’ Released activity was measured in the supernatant from saponin-permeabilized cells; 100% activity corresponds to 56.6 pmoles dCDP produced per 10 min per 3x IO6cells.

45% were in S phase. When cells were used that were enriched in GI phase by unit-gravity sedimentation or arrested in GO by isoleucine deprivation [14], similar amounts of glucose-6-phosphate dehydrogenase but no ribonucleotide reductase activity were detected in the release fraction (data not shown). Comparison of Intracellular

and Released Ribonucleotide Reductase

In view of the structural complexity of ribonucleotide reductase, it was of interest to compare the properties of the enzyme when retained in cells treated with dextran sulfate and when released from cells treated with saponin. Both forms of the enzyme were dependent on the presence of either ATP or the ATP analog, AMP-PNP, for activity (table 1). The released ribonucleotide reductase could utilize its natural allosteric effector ATP more efficiently than its intracellular counterpart, presumably due to reduced competition with ongoing cellular RNA synthesis [ 131.Both the released and intracellular ribonucleotide reductase activities were enhanced by the presence of iron and the presence of hydrogen donors, with the intracellular form able to utilize the endogenous hydrogen donor NADPH more efficiently, probably due to the presence of an intact hydrogen donor system in the dextran sulfate permeabilized cell [13]. The inhibition of ribonucleotide reductase activity by hydroxyurea and the negative allosteric effector dATP was similar whether the enzyme was assayed intracellularly or extracellularly. 2X-868342

EXP Cdl

.Res 167 (1986)

424

Kucera and Paulus

Table 2. Distribution

of enzyme activities

in cell derivatives produced by enuclea-

tion Specific activity

(pmolesiminimg

Cell fraction”

Soluble protein content (mg/lO’ cells)

protein)

NMN adenylyl transferase

Glc-6-P dehydrogenase

Ribonucleotide reductase

Thymidylate synthase

Intact cells Karyoplasts Cytoplasts

14.1 1.2 7.1

487 6 510 346

37 100 5 940 38 600

7.8 0.2 5.9

91 5 64

0 Cells were harvested and washed with buffer (intact cells) or subjected to CB enucleation (cytoplasts and karyoplasts) as described in Materials and Methods. All enzyme assays were performed on the supernatants from sonic extracts of the intact cells and their derivatives. The data are averages of three separate experiments.

Effect of Enucleation

on the Location

of Ribonucleotide

Reductase

Another approach to resolving the location issue of enzymes involved in DNA precursor synthesis was the use of CB to produce cell derivatives containing either intact nuclei (karyoplasts) or the bulk of the cytoplasm (cytoplasts). Using an isopycnic sedimentation procedure that allowed the enucleation of cells in suspension, we obtained a 60% yield of cytoplasts and a 30% yield of karyoplasts. The intact cells, cytoplasts and karyoplasts differed significantly both in their size (1440, 720, and 400 pm3, respectively) and their protein content (15.7, 9.2 and 2.6 mg per lo7 cells, respectively). When the cells and cell derivatives were sonicated, centrifuged, and their supernatants assayed for ribonucleotide reductase, virtually all the activity was present in the cytoplasts, together with thymidylate synthase and glucose-6-phosphate dehydrogenase, while the bulk of NMN adenylyltransferase was associated with the karyoplasts (table 2). Similar results, except for slightly higher NMN adenylyltransferase activity in the karyoplast fraction, were obtained when uncentrifuged sonic extracts rather than supernatants were assayed (data not shown). DISCUSSION The two approaches used to define the intracellular localization of the deoxyribonucleotide biosynthetic enzymes ribonucleotide reductase and thymidylate synthase in mouse L929 cells are summarized in table 3. Both approaches involved the comparison of the behavior of these enzymes with that of NMN adenylyltransferase and glucose-6-phosphate dehydrogenase, known to be localized primarily in the nucleus and the cytosol, respectively [26. 271. One method measured the release of enzymes from selectively permeabilized cells, whereas the other examined their distribution in enucleated and cytosol-depleted cell derivatives. Exp Cell Res 167 11986)

Cytoplasmic 3. Summary enucleation

Table

Cell preparations A. Permeabilized

of localization

deoxyribonucleotide

biosynthesis

425

studies. Enzymic activities following permeabilization

[3H]dTTP incorporation into DNA

NMN adenylyltransferase

Glc-6-P dehydrogenase

Ribonucleotide reductase

Thymidylate synthase

Present

Present

Present

Present

Present

Present

Present

Absent

Absent

Absent

NAb NA

Present Absent

Absent Present

Absent Present

Absent Present

cells

Dextran sulfatepermeabilized cells (low level of permeabilization) Saponin permeabilized cells (high level of permeabilization) B. Cell derivatives” Karyoplasts Zytoplasts

or

’ Assays performed on sonicated cell preparations after centrifugation to remove particulate matter. ’ Not applicable.

When L929 cells were treated with low concentrations of saponins (about 25 ug/mg protein), essentially all cellular glucose-6-phosphate dehydrogenase was released into the extracellular medium, while NMN adenyltransferase was completely retained, consistent with the notion that saponin selectively perturbed the plasma membrane. Under the same conditions, ribonucleotide reductase and thymidylate synthase activities were also completely released from the cells, suggesting that, like glucose 6-phosphate dehydrogenase, these were freely soluble cytosolic enzymes. The fact that all three enzymes were released at identical rates argued strongly against any sort of association with the nucleus or cytoplasmic structural elements, which would cause a delay in release. It is of course difficult to rule out the possibility that saponins might also have an effect on the nuclear membrane. However, since the cholesterol content of the nuclear membrane is less than one-third that of the plasma membrane [28], one would expect the relative effects on the two membranes to be highly dependent on saponin concentration. As a result, the release of a nuclear enzyme should require relatively higher concentrations of saponin than that of a cytoplasmic component. Our observation that glucose-6-phosphate dehydrogenase, ribonucleotide reductase, and thymidylate synthase were released at identical rates over a wide range of saponin concentrations suggested that their release was determined by the action of saponin on a single type of membrane. A comparison of the properties of ribonucleotide reductase activity assayed intracellularly in dextran sulfate-permeabilized cells and in the extracellular material obtained after treatment with saponin showed that both activities resemExp Cell Res

426 Kucera and Paulus

bled purified ribonucleotide reductase in their response to activators and inhibitors. The difference in their response to NADPH was due to the fact that treatment with dextran sulfate caused little damage to cellular integrity [ 121,thus preserving the ability of intracellular ribonucleotide reductase to utilize NADPH as a hydrogen donor. The small differences observed in the response to ATP and ATP analogs were probably due to the metabolism of ATP in the dextran sulfate-permeabilized cells and the presence of residual ATP in the saponin extracts. There is thus little question that the enzyme released by saponin is indeed ribonucleotide reductase. The other approach to study intracellular localization of enzymes measured their distribution in the cytoplast and karyoplast fractions obtained by exposing CB-treated L929 cells to a centrifugal field. The specific activity of the nuclear enzyme NMN adenylyltransferase was 20 times as high in the karyoplasts as in the cytoplasts, indicating a very low level of nuclear leakage or nuclear contamination of the cytoplast fraction. The specific activity of glucose-6-phosphate dehydrogenase in the karyoplast fraction was 15% of that in the cytoplasts, which corresponded to 3 % of the total activity, suggesting that the karyoplasts had retained about 3 % of the cytoplasm. This level of cytoplasmic carry-over to the karyoplast fraction is typical of the enucleation method [29]. Accordingly, the observation that the specific activities of the deoxyribonucleotide biosynthetic enzymes ribonucleotide reductase and thymidylate synthase were 12-30 times as high in the cytoplast as in the karyoplast fraction indicated that these enzymes were localized exclusively in the cytosol. Our results parallel the findings of Herrick and co-workers [30], who found that 90 % of thymidine kinase activity of mouse L929 cells was associated with cytoplasts under conditions where DNA polymerase a remained entirely with the karyoplast fraction. On the other hand, our results contrasted with those of Reddy & Pardee [l , 21, who found considerably higher specific activities of thymidylate synthase and ribonucleotide reductase in karyoplasts derived from S-phase CHEF/18 Chinese hamster cells than in cytoplasts. The reason for this discrepancy is not known. The possibility that treatment with CB weakens the nuclear association of enzymes is not a likely explanation, since both experiments involved the use of that drug. Other possibilities are differences in cell lines and culture conditions, our experiments involving cells grown in suspension culture, whereas Reddy & Pardee [l , 21 used cell monolayers. The unambiguous definition of the intracellular localization of an enzyme by any single technique is virtually impossible. This structure also applies to the results in this paper, which must be viewed as links in the chain of evidence for a cytoplasmic localization of ribonucleotide reductase. Earlier evidence in support of that notion came from the immunocytochemical studies of Engstrom et al. [lo] and the subcellular fractionation experiments by Mathews and co-workers [l 11. The former involved the use of monoclonal antibodies against the Ml subunit of ribonucleotide reductase and showed that immunoreactivity was confined to the Exp Cell Res 167 (1986)

Cytoplasmic

deoxyribonucleotide

biosynthesis

427

cytoplasm of mouse fibroblast (3T6) and bovine kidney (MDBK) (cells [IO]. Similar results were obtained with polyclonal antibodies against the M2 subunit (quoted in [9]). Although these observations constitute very strong evidence that ribonucleotide reductase is indeed a cytoplasmic enzyme, the possibility that a subfraction of the enzyme occurs in a nuclear complex in which the appropriate antigenic determinants are masked cannot be eliminated. The subcellular fractionation experiments of Leeds et al. [ll] involved the rapid isolation of nuclei from Chinese hamster ovary (CHO) cells and demonstrated the absence of nuclear ribonucleotide reductase acitvity at all stages of the cell cycle. Studies of nuclear dNTP levels indicated minimal nuclear perturbation during the fractionation procedure, making the selective loss of ribonucleotide reductase unlikely, although not impossible. Our results also are not unambiguous. For example, it might be argued that saponins perturb nuclear structure sufficiently to cause the loss of ribonucleotide reductase but not of NMN adenylyltransferase, which may be more strongly associated with chromatin [31]. The fact that saponin treatment has no effect on DNA replication weakens this objection and certainly speaks against a direct functional involvement of ribonucleotide reductase with the DNA replicative machinery. The enucleation experiments with CB could be criticized because disaggregation of microfilaments by the drug [32] might affect the subcellular localization of enzymes. This possibility is unlikely, since the nearly complete absence of ribonucleotide reductase in the karyoplast fraction would have to be explained in terms of an active extrusion of the enzyme from the nucleus. Our results thus serve to strengthen the conclusion that ribonucleotide reductase is an exclusively cytoplasmic enzyme and show that the same applies to thymidylate synthase. The likelihood that two major deoxyribonucleotide biosynthetic enzymes function in a different subcellular compartment than the enzymes which utilize their products raises important questions concerning cellular economy and control mechanisms that are currently the subject of active investigation in a number of laboratories (e.g. [9, 11, 33, 341). The authors wish to thank Patricia Venezia for her technical assistance. This research was supported by grants PCM 83-09161 from the National Science Foundation, GM 32704 from the National Institute of General Medical Sciences, and RR 05711from the Biomedical Research Support Program, National Institutes of Health.

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