Subcellular Localization Sugar Nucleotide Synthetases* of

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A. Vol. 255, No. 19, Issue of October 10, pp. 9225-9229. 1980

Subcellular Localization of Sugar Nucleotide Synthetases* (Received for publication, April 24, 1980)

Stephen W. Coatesf, Theodore Gurney, Jr.8, Linda Wyles SommersS, Mary YehS, and Carlos B. Hirschbergfe From the +E. A. Do& Department of Biochemistv, St. Louis University School ofMedicine, St. Louis, Missouri 63104, and the SDepartment of Biology, University of Utah, Salt Lake City, Utah 84112

Sugar nucleotides are Drecursors of sugar moieties in obtained bv others in brain and kidney (Gielen et al., 1970 glycGlipids and glycoproteins and thus may have a regulatory role in the metabolism of these macromolecules. We have therefore studied the subcellular localization of four sugar nucleotide synthetases. The pyrophosphorylases of GDP-fucose, GDP-mannose, and UDP-glucose were detected only in the cytoplasm; in contrast, at least 85% of the CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase was found in the nucleus. "he latter conclusion was reached by assaying CMP- NeuAC synthetase activity in nuclei and cytoplasm of cells fractionated by a nonaqueous procedure and in karyoplasts and cytoplasts from cells enucleated with Cytochalasin B. In addition, cytoplasts were able to synthesize in situ only 20% as much CMP-NeuAc from free NeuAc as nucleated cells; the same enucleated cells, however, synthesized GDP-fucose from free fucose at levels comparable to nucleated cells. The nuclear CMP-NeuAc synthetase activity could not be solubilized by treatment of the nuclei with a mixture of 2% Triton X-100 and 1% sodium deoxycholate, conditions which solubilize both the outer and inner nuclear membranes and the nuclear pores. These results strongly suggest that the activities of CMP-NeuAc synthetase and GDP-fucose pyrophosphorylase which are assayed in vitro are those catalyzing the reactions in vivo.

Sugar nucleotides are intermediates in the biosynthesis of glycoproteins and glycolipids and may therefore play a regulatory role in their metabolism. Current knowledge of the subcellular localization of enzymes that catalyze the synthesis of sugar nucleotides is rather limited, and only recently have studies begun on how sugar nucleotides reach the sites where the sugar moieties are transferred to macromolecules (Carey et al., 1980). We have become interested in the subcellular localization of sugar nucleotide synthetases in general and CMP-NeuAcl synthetase in particular as a result of our recent characterization of this sugar nucleotide from mammalian tissues (Carey and Hirschberg, 1979) and the previous studies of Kean (1970). Kean showed that between 18% and 50% of the CMP-NeuAc synthetase activity of liver, retina, and kidney was associated with nuclei. Similar results were also

This work was supported by Grants CA17504 (T. G.), CAI7015 (C. B. H.), and GM 26137 (T. G.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Faculty Research Awardee (FRA 187) of the American Cancer Society.

' The abbreviation used is: NeuAc, N-acetylneuraminic acid.

and 1971; van den Eijnden et al., 1972; van den Eijnden, 1973; van Dijk et al., 1972 and 1973). The latter studies also showed that only 10 to 37% of the CMP-NeuAc synthetase activity recovered in the nuclear fraction could be removed with Triton X-100. Since Triton X-100 removes the outer nuclear membrane, it appeared that the nuclear synthetase activity was not due to cytoplasmic contamination. Both Kean (1970) and van den Eijnden (1973) also found that treatment of nuclei with low concentrations of salt solubilized the enzyme activity.

The previous observations raised the following questions: Do cells contain distinct nuclear and cytoplasmic CMP- NeuAc synthetase activities and, if so, do both have physiological roles? Is cytoplasmic activity the result of leakage of nuclear activity during cell fractionation? Does nuclear activity result from adsorption of cytoplasmic activity onto nuclear pores and inner nuclear membrane? In addition, we were interested in the subcellular localization of GDP-fucose pyrophosphorylase which catalyzes the synthesis of GDP-fucose. This sugar nucleotide as well as CMP-NeuAc is a substrate for glycosyltransferases localized in the Golgi apparatus (Schacter et al., 1970; Bennett et at., 1974; and Carey and Hirschberg, 1980). Although GDP-fucose pyrophosphorylase had been partially purified from a cytoplasmic fraction of liver (Ishihara and Heath, 1968), the portion of the total activity which was present in this fraction was not determined.

We also chose to study, for comparative purposes, the subcellular localization of GDP-mannose and UDP-glucose pyrophosphorylases. The transfer of the sugar moieties from these sugar nucleotides to macromolecules most likely occurs in the rough endoplasmic reticulum (Czichi and Lennarz, 1977; Robbins et at., 1977; and Tabas and Kornfeld, 1978).

MATERIALS AND METHODS

Cells and Growth Media-The D 51 clonal derivative of mouse L 929 cells (referred to hereafter as L-cells) was kindly provided by Dr. J. J. Lucas (State University of New York at Stony Brook). BHK 21/ 13 (referred to hereafter as BHK cells) were a gift of Dr. Maurice Green (St. Louis University). L-cells were grown in minimal essential medium (ME medium) with 5% fetal calf serum. BHK cells were grown in ME medium containing 4 times the amounts of amino acids and vitamins and 10% fetal calf serum (Sakiyama et al., 1972). Cells were free of mycoplasma contamination based on staining with Hoechst 33258 (Chen, 1977) and also by autoradiography of cultures labeled with [methyG3H]thymidine. Some of the L-cells used in early experiments of nonaqueous fractionation and enucleation described in Table I1 were iater discovered to have been contaminated with mycoplasma. Both of the fractionation procedures were then repeated with cells which were unequivocally free of mycoplasma. No significant differences in the results were obtained.

Radioactive Substrates-N-Acetyl-~-[~H]mannosamine (2.3 Ci/ m o l ) , ~-[6-~H]hcose (13.1 Ci/mmol), ~-[U-'~C]glucose 1-phosphate (181 mCi/mmol), ~ 4 2 % ]mannose (13.8 Ci/mmol) CMP-N-[4-I4C] acetylneuraminic acid (0.9 mCi/mmol), and GDP-[U-14C]~cose (226

9225

9226 Sugar Nucleotide Synthetases

mCi/mmol) were purchased from New England Nuclear. GDP-[U- “C]mannose was a gift of Dr. P. W. Robbins, Massachusetts Institute of Technology. N-[4,5,6,7,8,9-3H]Acetylneuraminic acid was prepared as described by Warren and Glick (1966). Radiochemical purity was at least 99.5% based on the criteria previously described (Hirschberg et al., 1976). [U-‘4C]Mannose l-phosphate was obtained by enzymatic cleavage of GDP-[U-‘?]mannose by Crotalus adumanteus nucleotide pyrophosphorylase. The product was purified using a Dowex I- formate column and 1 M formic acid as eluent. Purity was assayed on descending DE-81 paper chromatography in ethanol/ammonium acetate (0.2 M, pH 7.5) (3~7). One symmetrical radioactive peak which co- migrated with standard mannose l-phosphate was observed. Upon digestion of this material with alkaline phosphatase (Sigma Chemical Co., type III) all the radioactivity co-migrated with mannose in the above chromatographic system. The yield was 86%.

[6-3H]Fucose l-phosphate was prepared using [6-3H]fucose and fucose kinase, which was obtained by a modification of the procedure previously described by Yurchenco and Atkinson (1975). The purity was determined as previously described for radioactive mannose l- phosphate and was found to be greater than 95%. The yield was approximately 86%.

Preparation of Nuclei and 100,000 x g Supernatant from Liuer- Rat liver nuclei were prepared by the procedure of Blobel and Potter (1966). Nuclei of BHK and L-cells were obtained as previously described by Penman (1969). DNA content was measured according to the methods of Ceriotti (1952) or Foster and Gurney (1976). RNA was measured bv the method of Fleck and Munro (1962). The DNA/ RNA ratio of liver nuclei was 0.102 using Sigma yeast transfer RNA (A& nm = 192) as a standard.

Detergent Treatment of Rat Liver Nuclei and Analyses of Lipid Phosphorus-Nuclei obt&ed as described above were-washed once with 0.25 M “STKM” buffer (0.25 M sucrose, 0.05 M Tris-HCI, pH 7.5 at 2O”C, 0.025 M KCl, and 0.005 M MgCb) and resuspended in the same buffer (0.63 mg/g of wet liver). A 20% Triton X-106 solution alone or a mixture of 20% Triton X-100 and 10% sodium deoxycholate was added to the nuclear suspension (0.5 ml) to a final concentration of 2% Triton X-100 and 1% deoxycholate. After chilling unstirred on ice for 10 min with Triton alone or for 90 s with both detergents, the suspension was centrifuged at 1,009 x g for 10 min. Supernatant and pellet were dialyzed against Tris-HCl (0.01 M pH 7.5, 1% P-mercaptoethanol) for subsequent assays of CMP-NeuAc synthetase (Kean and Roseman, 1966) and lipid phosphorus (Rouser et al., 1970). An aliquot of the nuclear suspension which contained approximately 80 nmol of lipid phosphorus catalyzed the synthesis of approximately 40 nmol of CMP-NeuAc.

Preparation of Nuclei by a Nonaqueous Procedure-Nuclei were also obtained by nonaqueous fractionation of approximately 10’ cells as described by Gurney and Foster (1977). Prior to analyses of CMP- NeuAc synthetase, the nuclei and cytoplasm were dialyzed against Tris-HCl (0.01 M pH 7.5, 1% fl-mercaptoethanol). The cytoplasmic fraction was then concentrated approximately lo-fold through an Amicon filter.

Enucleation of L-Cells-L-cells were enucleated by centrifuging monolayers with Cytochalasin B as described by Lipsich et al. (1978). Control cells were seeded at the same density as cells for enucleation and used without further manipulations.

After centrifugation and recovery (see below), cytoplasts (the cytoplasmic fraction or enucleated cells) could be trypsinized and counted in a Celloscope particle counter. Loss of cytoplasta relative to control cells was 5 to 15%. Cytoplasts contained 2 to 4% of the DNA of control cells and 28 to 48% of the protein. The DNA content of L-cells was 11 ? 1 pg/cell. There were 2.8 X 10’ cells/mg of protein.

Determination of Protein, DNA, and CMP-NeuAc Synthetase Activity in Cytoplasts and Karyoplasts-Following enucleation, the Petri plates containing cytoplasta were removed from the centrifuge bottles used to prepare them and washed three times with Solution A (0.14 M NaCI, 0.0066 M KCl, 0.001 M KPO,, pH 7.4) at 4°C and scraped with a rubber policeman into 0.5 to 1 ml of Solution A. For optimum yield of karyoplasta, the bottles with enucleation medium were recentrifuged (10 min at 10,000 rpm). The enucleation medium was slowly decanted and the karyoplasts (nuclear fractions) were resuspended in Solution A (0.5 to 1 ml). Prior to analyses, both fractions were sonicated (two times, 15 s, 4°C at setting of 7 of a Branson sonicator with a microtip).

Preparation of Cytoplasts for Studies in Situ-The above described Petri plates containing cytoplasts were removed from the centrifuge bottles and the medium replaced with ME medium containing 5% fetal calf serum. The dishes were then placed in a 5% CO,

incubator at 37°C for 1 h. The medium was changed every 15 min. During this time, the cells changed from a poorly attached condition characteristic of Cytochalasin treatment to a more uniformly attached appearance. Cells and cytoplasts were then used for different labeling experiments.

Extraction of Sugar Nucleotides from Cells and Cytoplasts-After labeling the cells and cytoplasts the medium was removed and the plates were washed 10 times with Solution A (4”C, 5 ml each time). Ice-cold 70% ethanol (1 ml) was then added to each plate. After allowing the plates to be on ice for 10 min, cells and cytoplasta were scraped with a rubber policeman and the suspension was placed in a 1.5~ml microfuge tube. Following centrifugation, the supernatant solution was saved. The plate was scraped with an additional 1.4 ml of 70% cold ethanol and the suspension added to the previous pellet. After resuspension and centrifugation, the supematant solutions were combined and dried under nitrogen.

Assays of Sugar Nucleotide Pyrophosphorylases-GDP-mannose- pyrophosphorylase was assayed by adding a mixture of 4-(2-hydrox- yethyl)-1-piperaxineethanesulfonic acid (Hepes) buffer (0.05 ml; 0.1 M, pH 7.5, containing 5 mr+r MgCls), bovine serum albumin (0.01 ml; 2 mg/ml), AMP (0.01 ml, 30 mm), NaF (0.01 ml, 0.1 M), inorganic pyrophosphatase (0.01 ml, 0.67 unit, Sigma Chemical Co.), and GTP (0.01 ml; 5 mu) to tubes containing mannose l-phosphate (1.0 X 10’ cpm). After mixing with a Vortex mixer the reaction was started by adding protein (1 to 5 cg). Activity was measured for 15 min at 37°C. GDP-mannose was purified using paper chromatography in an ethanol/ammonium acetate (1 M, pH 7.5) (6:4) system and visualized by UV light (RF = 0.14). UDP-glucose pyrophosphorylase activity was measured in a similar manner but using 0.01 to 0.05,g of protein and incubating for 10 min at 30°C. For GDP-fucose nyrophosphoryl- ase assays, 1% to 206 pg of protein was used and the reaction was stopped after 30 min at 37°C. GDP-fucose was localized on paper by taking advantage of the slightly faster mobility of this sugar nucleotide (RF = 0.20) compared to GDP-mannose.

RESULTS

Subnuclear Localization of CMP-NeuAc Synthetase-Van Dijk et at. (1973) and van den Eijnden (1973) showed that treatment of brain and kidney nuclei with Triton X-100 removed 10 to 37% of the CMP-NeuAc synthetase activity, suggesting that the remaining 63% to 90% of nuclear activity was not the result of cytoplasmic contamination. However, the selective adsorption of the putative cytoplasmic enzyme to the nuclear pores and to the inner nuclear membrane (lamina) could not be ruled out, since these structures are not removed by Triton X-100. Recently, Kirschner et al. (1977) showed that inner and outer nuclear membranes and nuclear pores could be removed by treating nuclei with a mixture of Triton X-100 and deoxycholate. To study the behavior of CMP-NeuAc synthetase under such conditions, rat liver nuclei were prepared as described by Blobel and Potter (1966). In agreement with Kean’s results (Kean, 1970), 34% of the synthetase activity in the homogenate was recovered in the nuclear fraction. This nuclear preparation was then treated with Triton X-100 alone (to remove the outer nuclear membrane) or with a mixture of Triton X-100 and deoxycholate (to remove both membranes and pores). Table I shows that neither detergent treatment removed enzyme activity in amounts above controls without detergents. Proof that the detergents were effective in removing nuclear membranes was obtained by biochemical analyses of nuclei which showed that 90% of the nuclear lipid phosphorus had been removed (Table I). In addition, electron microscopic examination of the nuclei showed the absence of nuclear membranes in the treated samples (not shown). These results strongly suggested that a portion of the cellular CMP-NeuAc synthetase activity was nuclear.

Distribution of the CMP-NeuAc Synthetase Activity in Cells Fractionated by Different Procedures-The above results did not demonstrate whether the cytoplasmic CMP- NeuAc synthetase activity in liver was physiological or the

Sugar Nucleotide Synthetases 9227

result of leakage of enzyme from the nucleus during subcellular fractionation. To answer this question, we decided to use L 929 and BHK cells grown in tissue culture. With these cells, subcellular fractionation procedures employing nonaqueous solvents or conditions less disruptive than homogenization (see below) had been used to show that cytoplasmic DNA polymerase-a activity was the result of leakage from the nucleus during aqueous subcellular fractionation (Foster and Gurney, 1976, and Henick et al., 1976).

Following aqueous fractionation of L 929 and BHK cells, 13 and 41% of the CMP-NeuAc synthetase activity of the homogenate was recovered in the nuclear fraction (not shown) while 86% and 67% was in the cytoplasm. The relative distri- butions of this activity between these fractions and that of DNA are shown in Table 11.

On the assumption that the cytoplasmic CMP-NeuAc synthetase activity in these cells was the result of leakage from the nucleus during aqueous fractionation, cells were lyophi-

TABLE I Rat liver nuclear CMP-NeuAc synthetase activity: effect of

removing inner and outer nuclear membranes and pores with detergents

Nuclei were obtained by the procedure of Blobel and Potter (1966) and were treated with detergents as described under “Materials and Methods.” Values are the average and range of two separate deter- minations.

CMP-NeuAc sWthe- Lipid phosphorus

Supema- Pellet SUET- Pellet

tase activity Treatment

tant relative R relative R

None 2% Triton

1 1 f 3 8 9 f 6 9 + 2 9 1 f 1 2 8 9 f 3 1 1 f 3

None 1 9 + 5 8 1 f 7 2% Triton + 1% DOC” 21 f 4 79 f 1 9 4 f 1 4 6 + 1

DOC, deoxycholate.

TABLE I1 CMP-NeuAc synthetase activity (Synth. Act.)

Cell fractions were obtained as described under ‘‘Materials and Methods.” The total CMP-NeuAc activity recovered in nuclei and cytoplasm of L-cells relative to homogenate was 99 f 7% for the aqueous fractions, 92 + 16% for the nonaqueous fractions, and 90 f 7% for the enucleation. For BHK cells the values were 108 f 5% for the aqueous fractions and 119% for the nonaqueous fractions. Results are the average and range of two separate fractionations.

1 Fractionation

Aqueous Enuci Nonaqueous

CMP- DNA NeuAc DNA NeuAc

CMP- CMP- NeuAc

Synth. Act.

Synth. Act.

Synth. Act.

relative R A. L-cells frac-

tionated by different procedures

oplasts)

toplasts)

Nuclei (kary-

11 + 5 7 f 5 1 5 f 1 7 f 2 8 7 f 5 Cytoplasm(cy-

85 f 5 93 + 5 85 f 1 93 f 1 13 f 5

I lei

T

B. BHK cells fractionated by an aqueous and nonaqueous procedure

Nuclei 15 14 f 7 62 f 16 Cytoplasm 85 86 f 9 38 f 16 87

13 L ation

DNA

95 * 1

6 f l

lized and fractionated in the presence of anhydrous glycerol (Gurney and Foster, 1977). As shown in Table 11, this procedure gave approximately 85% of the CMP-NeuAc synthetase activity of L 929 and BHK cells in the nuclear fraction.

Both previously described fractionation procedures require tissue homogenization. To obtain nuclei and cytoplasmic fractions by a less disruptive procedure, L 929 cells were treated with Cytochalasin B (Lipsich et al., 1978). The resulting karyoplasts (nuclear-containing fraction) and cytoplasts (enucleated cells) were then assayed for CMP-NeuAc synthetase activity. As can be seen in Table 11, 85% of the activity was in the karyoplast fraction (82% relative to homogenate). In a control experiment it was found that Cytochalasin B did not have an effect on the CMP-NeuAc synthetase activity per se (not shown).

Synthesis of CMP-NeuAc and GDP-fucose in Situ by Cells and Cytoplasts-An important question not answered by the previous experiments was whether the small amount of CMP- NeuAc synthetase activity measured in vitro in the cytoplasmic fractions was sufficient for the synthesis in vivo of CMP- NeuAc necessary for sialylation by the cell. An effort was made to resolve this problem by incubating cells and cytoplasts (on tissue culture dishes) with radiolabeled NeuAc. The ratio of radiolabeled CMP-NeuAc synthesized to free NeuAc within the cells and cytoplasts was determined. Table I11 shows that this ratio was 5 times lower in cytoplasts than nucleated cells. Controls in which nucleated cells had been incubated with Cytochalasin B but not centrifuged or had been preincubated with Cytochalasin B but centrifuged in medium without the drug showed that the difference between cytoplasts and nucleated cells was not due to centrifugation of cells or their treatment with Cytochalasin B per se (not shown).

Evidence is presented in the next section which suggests that GDP-fucose pyrophosphorylase is a cytoplasmic enzyme. One might therefore predict that c-ytoplasts would be able to synthesize GDP-fucose in situ upon incubation with radiolabeled fucose. To test this hypothesis, control cells and cytoplasts were incubated with radiolabeled fucose and the ratio of radiolabeled GDP-fucose to free fucose determined in each case. Table I11 shows that this ratio was similar in nucleated and enucleated cells, strongly suggesting that GDP-fucose pyrophosphorylase was a cytoplasmic enzyme. Although in this particular experiment there was an approximate 70% increase in the ratio of GDP-fucose to fucose in enucleated cells, we have seen 10 to 30% increases in other experiments. Since in each case duplicates were very similar, we attribute the variability to combined effects of Cytochalasin and cell recovery.

TABLE I11 Synthesis in situ of CMP-[’H]NeuAc from (’H]NeuAc and GDP-

[’Hlfucose from [’H]ficose Cell preparations were obtained as described under “Materials and

Methods.” In a typical experiment with NeuAc, 1.9 X lo6 cells/60- mm plate were labeled with 1.3 X 10’ cpm of [’HINeuAc for 1% h at 37°C. Nucleated cells contained 3,200 cpm of C3H]NeuAc and 3,000 cpm of CMP-E3H]NeuAc. Enucleated cells had 3,300 cpm of C3H]NeuAc and 540 cpm of CMP-C3H]NeuAc. In a typical experiment with fucose, 1.9 X lo6 cells/60-mm plates were labeled with 2.7 X lo7 cpm of C3H]fucose for 1% h at 37°C. Nucleated cells contained 9,300 cpm of [‘H]fucose and 26,300 cpm of GDP-[”H]fucose. Enucleated cells contained 1,900 cpm of [3H]fucose and 9,200 cpm of GDP-[”HI- fucose. Results are the average and range of two separate incubations.

cpm CMP-[:‘HI- cpm GDP-[;‘H]h- NeuAc/cpm pH]- cose/cpm [‘Hlfu- NeuAc per plate cose per plate

Nucleated cells 0.91 f 0.04 2.8 f 0.0 Enucleated cells 0.19 * 0.03 4.8 f 0.1

9228 Sugar Nucleotide Synthetases

Subcellular Localization of UDP-Glucose, GDP-Fucose, and GDP-Mannose Pyrophosphorylase-We also determined the subcellular distribution of UDP-glucose and GDP-mannose phosphorylases in rat liver. The enzyme activities were measured in the homogenate, in the 100,OOO X g supernatant and pellet fractions, and in nuclei obtained by the procedure of Blobel and Potter (1966). Approximately 60% of both activities (relative to homogenate) were recovered in the l00,OOO x g supematant fraction and less than 4% in the pellet; nuclei contained less than 0.2%. Assays of different fractions mixed together did not suggest the presence of activators or inhibitors of activity. Although the values for GDP-fucose pyrophosphorylase were more variable than the two other pyrophosphorylases only the 100,OOO X g supernatant fraction contained activity (80 to 200% of homogenate).

DISCUSSION

These studies strongly support the conclusion that at least 85% of the CMP-NeuAc synthetase of the cell is within the nucleus. We were able to show that the nuclear activity was not the result of selective adsorption of a cytoplasmic enzyme to the outer or inner nuclear membrane or to the nuclear pores by using the recently developed procedure of Kirschner et al. (1977), by which all these nuclear structures can be removed with a mixture of detergents. Although a similar conclusion had been previously reached by others (Kean, 1970 van Dijk et al., 1973), it is only the above mentioned development (particularly regarding the nuclear pores) which has enabled us to prove this conclusively.

The question of whether the synthetase activity found in the cytoplasm following aqueous fractionation was the result of nuclear leakage had not been addressed in detail previously. Since significant amounts of both CMP-NeuAc synthetase activity and DNA are recovered in the cytoplasm during aqueous fractionation of several tissues, it had been assumed by others that both leaked from the nucleus at similar rates. This assumption, however, has not been proven. The fact that procedures as different as nonaqueous subcellular fractionation and cell enucleation showed that approximately 85% of the CMP-NeuAc synthetase activity was nuclear strongly suggests that synthetase activity is localized exclusively in the nucleus of the cell. These values for CMP-NeuAc synthetase distribution were obtained without correction for leakage and most likely represent a minimum estimate for nuclear activity.

Perhaps the strongest evidence in support of aU the synthetase being nuclear is the marked decrease in relative levels of sugar nucleotide synthesized from free NeuAc by cytoplasts in situ. Since these cytoplasts were able to synthesize GDP- fucose from free fucose at relative levels comparable to nucleated cells it is very unlikely that the results obtained with NeuAc are artifacts, These latter results are also, to our knowledge, the best evidence available suggesting that the CMP-NeuAc synthetase activity which is assayed in uitro is the same activity which catalyzes the synthesis of sugar nucleotide in uzuo. Our results also show that GDP-fucose pyrophosphorylase is cytoplasmic. Although this enzyme had been partially purified from a soluble cytoplasmic fraction, the proportion of the total activity which was soluble had not been determined (Ishihara and Heath, 1968).

We also found that cytoplasts incorporate only 5% as much radiolabeled NeuAc into sialoglycoproteins as nucleated cells, even though the rate of NeuAc uptake did not differ by more than 50% (not shown). Although the lower synthesis of sialoglycoproteins by cytoplasts is consistent with the lower levels of CMP-NeuAc in these preparations, the results must be interpreted with caution since protein synthesis (measured by leucine incorporation) was also decreased by 50%. Fucose

incorporation into fucoproteins by cytoplasts was decreased by approximately 50% (rather than 95% as NeuAc) when compared to nucleated cells; however, we cannot rule out that both sugars were being incorporated into different precursor pools of proteins and that the pool for sialoglycoproteins was smaller than that for fucoproteins.

Our conclusion that both GDP-mannose and UDP-glucose pyrophosphorylases are cytoplasmic enzymes is based on more conventional approaches. Experiments on these sugar nucleotides with cytoplasts are difficult to interpret because uptake of these sugars is markedly reduced in cytoplasts compared to nucleated cells.

Kean (1970) and van den Eijnden (1973) had reported previously that the nuclear associated CMP-NeuAc synthetase activity could be solubilized with buffers of relatively low ionic strength. Nuclear proteins which exhibit this behavior have been called nucleoplasmic proteins, an example of which is DNA polymerase-a.

CMP-NeuAc synthetase is the only known pathway for the synthesis of CMP-NeuAc whereas the other sugar nucleotides described in this paper can be made by alternate pathways. According to Yurchenco and Atkinson (1977), GDP-fucose pyrophosphorylase contributes only approximately 10% to the total GDP-fucose pool in the cell, the rest being derived from GDP-mannose.

We are unable to postulate a specific role for the localization of CMP-NeuAc synthetase in the nucleus at this time. While a few sialoglycoproteins have been detected in the nucleus (Kawasaki and Yamashina, 1972; Keshgegian and Glick, 1973; Buck et al., 1974; and Bhavanandan, 1979), it seems clear that the bulk of the sialic acid addition to proteins and lipids occurs in the Golgi apparatus (Schacter et al., 1970; Morre, 1971; Carey and Hirschberg, 1980). The possibility that the CMP- NeuAc synthetase has another unknown function related to its nuclear localization is being studied currently.

Acknowledgment-We thank Helen Hartzog for typing this man- uscript.

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