From the Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162, Japan
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ABSTRACT |
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Phosphatidylserine (PS) in mammalian cells is synthesized through the exchange of free L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine (PE). The serine base exchange in Chinese hamster ovary (CHO) cells is catalyzed by at least two enzymes, PS synthase (PSS) I and II. A PSS I-lacking mutant of CHO-K1 cells, PSA-3, which exhibits ~2-fold lower serine base exchange activity than CHO-K1, is defective in the conversion of phosphatidylcholine to PS but has the ability to convert PE to PS. The PSA-3 mutant requires exogenous PS or PE for cell growth. In the present study, from PSA-3 mutant cells, we isolated a mutant, named PSB-2, with a further decrease in the serine base exchange activity. The activity in the homogenate of PSB-2 mutant cells was ~10% that of PSA-3 mutant cells and ~5% that of CHO-K1 cells. The PSB-2 mutant exhibited an ~80% reduction in the PSS II mRNA level relative to that in PSA-3 mutant and CHO-K1 cells. These results showed that the PSB-2 mutant is defective in PSS II. Like the PSA-3 mutant, the PSB-2 mutant grew well in medium supplemented with PS. However, in the medium supplemented with PE, the PSB-2 mutant was incapable of growth, in contrast to the PSA-3 mutant. In the medium with exogenous PE, the PSB-2 mutant was defective in PS biosynthesis, whereas the PSA-3 mutant synthesized a normal amount of PS. A metabolic labeling experiment with exogenous [32P]PE revealed that the PSB-2 mutant was defective in the conversion of exogenous PE to PS. This defect and the growth and PS biosynthetic defects of the PSB-2 mutant cultivated with exogenous PE were complemented by the PSS II cDNA. In addition, the cDNA of the other PS synthase, PSS I, was shown not to complement the defect in the conversion of exogenous PE to PS of the PSB-2 mutant, implying that PSS I negligibly contributes to the conversion of PE to PS in CHO-K1 cells. These results indicated that PSS II is critical for the growth and PS biosynthesis of PSA-3 mutant cells cultivated with exogenous PE and suggested that most of the PS formation from PE in CHO-K1 cells is catalyzed by PSS II.
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INTRODUCTION |
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Phosphatidylserine (PS)1 is one of the major phospholipids in mammalian cells, comprising about 10% of the total membrane phospholipids. To elucidate the mechanism underlying PS biosynthesis in mammalian cells, we have isolated Chinese hamster ovary (CHO) cell mutants defective in PS metabolism (1-4). One of the mutants, PSA-3 (3), isolated through screening for PS auxotrophic mutants, has been successfully utilized for studying the PS biosynthetic pathway, as described below. The availability of PSA-3 mutant cells has made it possible to isolate the CHO cDNA of a PS synthase (PSS) gene, pssA, which is able to confer PS prototrophy on the mutant (5, 7). Recently, the CHO cDNA of another PS synthase gene, pssB, was isolated through a computer search for sequences similar to those of the pssA gene product (6).
Both the pssA gene product, PSS I, and pssB gene product, PSS II, catalyze the exchange of free L-serine with the polar head group (base) of preexisting phospholipid for PS formation (3, 5, 6). In addition to serine base exchange, PSS I can catalyze the exchange of the base moiety of phospholipid with choline and ethanolamine, whereas PSS II can catalyze serine and ethanolamine base exchange, but not choline base exchange (3, 5, 6). The serine and ethanolamine base exchange activities in a homogenate of PS auxotrophic PSA-3 mutant cells are decreased to ~50% of those in the parental strain, CHO-K1, and choline base exchange activity is virtually absent in the mutant cell homogenate (3). The PSA-3 mutant has no detectable amounts of pssA mRNA and its product, PSS I (5, 7). These observations indicate that the PSA-3 mutant is defective in PSS I. The PSS I-defective PSA-3 mutant is incapable of converting exogenous phosphatidylcholine (PC) to PS, suggesting that PSS I catalyzes the exchange of the choline moiety of PC with serine for PS formation in CHO-K1 cells (8). When the PSA-3 mutant is cultivated without exogenous phospholipids for 2 days, the cellular levels of PS and phosphatidylethanolamine (PE), the majority of which in CHO-K1 cells is synthesized through the decarboxylation of PS (9, 10), are reduced by ~70 and ~50%, respectively (3). The defects in the PS and PE biosynthesis of the PSA-3 mutant are complemented by the pssA cDNA (5). This cDNA also complements the defect in the conversion of PC to PS of the PSA-3 mutant.2 Thus, PSS I is responsible for the PS formation from PC in CHO-K1 cells, and it functions as an essential enzyme for the production of normal amounts of PS and PE in CHO-K1 cells cultivated without phospholipid supplementation.
The PSS I-defective PSA-3 mutant, as well as CHO-K1, has the ability to convert exogenous PE to PS (8). On the addition of PE to the medium, PSA-3 mutant cells recover the normal abilities to synthesize PS and to grow (8). Because two PS synthases, PSS I and II, exist in CHO-K1 cells, it is possible that PSS II is responsible for the PE-dependent restoration of PS biosynthesis in the PSA-3 mutant. However, the existence of more than two PS synthases in CHO-K1 cells has not been ruled out. In addition, it has not been resolved whether PSS I exclusively catalyzes the conversion of PC to PS or also catalyzes the conversion of PE to PS. Thus, the question of what enzyme or enzymes are responsible for the PS formation from PE in CHO-K1 cells remains to be elucidated. In the present study, we isolated CHO cell mutants defective in PSS II from PSA-3 cells to address the function of this enzyme. The results presented here suggest that the pssB gene product, PSS II, functions as the principal enzyme in the PS formation from PE in CHO-K1 cells.
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EXPERIMENTAL PROCEDURES |
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Cells and Culture Conditions-- CHO-K1 cells were obtained from the American Type Culture Collection and routinely maintained in Ham's F-12 medium (ICN Biomedicals) supplemented with 10% (v/v) newborn calf serum (ICN Biomedicals), 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, and 1.176 g/liter NaHCO3 at 37 °C under a 5% CO2 atmosphere and 100% humidity. PSA-3 cells (3) and PSB-1, PSB-2, and PSB-2/pssB cells obtained in this study were maintained under the same culture conditions except that the medium was supplemented with 30 µM PS (from bovine brain; Sigma) liposomes prepared as described (11). PSB-2/pssA cells obtained in this study were maintained under the same growth conditions as those for CHO-K1 cells. J774.1 cells were obtained from the American Type Culture Collection and maintained under the same culture conditions as those for CHO-K1 cells, except that the medium was supplemented with 10% (v/v) heat-inactivated fetal calf serum (Atlanta Biologicals) instead of newborn calf serum. The suspension of PE (from egg yolk; Sigma) added to the growth medium was prepared as described (11).
Isolation of Mutants with Decreased Serine Base Exchange Activity by Means of in Situ Colony Assaying-- For mutagenesis, exponentially growing cells were treated with 300 µg/ml ethyl methanesulfonate (Sigma) at 33 °C for 18 h, followed by cultivation without ethyl methanesulfonate at 33 °C for 3 days. The mutagenized cells were seeded to a 100-mm-diameter dish containing 10 ml of growth medium supplemented with 30 µM PS and then grown clonally at 33 °C to yield about 300 colonies/dish. On day 7, the cells were overlaid with a polyester disc (12), a filter paper (Whatman No. 50) (13), and glass beads (12), in that order. The cells were further cultivated with a medium change on days 10, 15, and 20. On day 20, the polyester disc bearing immobilized colonies was transferred to a new dish containing 7 ml of the same growth medium and incubated at 33 °C for 2 days. After additional cultivation at 39.5 °C for 1 day, the polyester disc was subjected to freezing and thawing in phosphate-buffered saline as described (2) to lyse the cells. The polyester disc was then incubated in 2 ml of an assay mixture consisting of 50 mM HEPES (pH 7.5), 5 mM CaCl2, cycloheximide (100 µg/ml), and 40 µM L-[U-14C]serine (20 µCi/µmol; Amersham Pharmacia Biotech) for 20 min at 39.5 °C and then soaked in 10% (w/v) trichloroacetic acid to terminate the serine base exchange reaction, followed by four rinses with 5% (w/v) trichloroacetic acid as described (2). After air drying, the radioactivity associated with the colonies was determined with a bioimage analyzer (FUJIX BAS2000). The colonies were subsequently stained with Coomassie Brilliant Blue as described (12). Mutant clones exhibiting decreased serine incorporation, identified through comparison of the radioactivity and staining intensity of colonies, were retrieved using cloning cylinders from the master dish. These clones were subjected to two more cycles of colony screening and then purified by limited dilution. This mutant screening was performed to allow the isolation of temperature-sensitive conditional mutants, because the decrease in the serine base exchange activity might have a lethal effect on PSA-3 mutant cells. However, mutants actually obtained in this study were constitutively defective in the serine base exchange activity and were able to grow at the high temperature of 39.5 °C in the presence of exogenous PS. Therefore, biochemical characterization of the mutants, performed at this temperature, 39.5 °C, are presented in this report.
Assaying of the Serine, Ethanolamine, and Choline Base Exchange Activities in Cell Homogenates-- Cells were suspended in a sonication buffer consisting of 10 mM HEPES (pH 7.5), 0.25 M sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A and then disrupted on ice with a W-225R Ultrasonic disrupter (Heat System Ultrasonics) equipped with a No. 419 microtip. Undisrupted cells were removed by centrifugation at 750 × g for 10 min at 4 °C, and the resultant supernatant was used as the homogenate. The serine, ethanolamine, and choline base exchange activities in the homogenate were determined at 39.5 °C using radioactive substrates, L-[U-14C]serine, [1,2-14C]ethanolamine hydrochloride (ICN Biomedicals), and [methyl-14C]choline chloride (American Radiolabeled Chemicals), respectively, as described (3).
Northern Blot Analysis--
Poly(A)+ RNAs were
isolated from 1-2 × 108 cells using a
FastTrackTM mRNA isolation kit (Invitrogen) according
to the manufacturer's protocol. 2.5 µg of poly(A)+ RNA
was separated on a 1% agarose/formaldehyde gel and transferred to a
Hybond-N membrane (Amersham Pharmacia Biotech) as described (14), with
slight modifications. DNA probes were labeled with [-32P]dCTP (Amersham Pharmacia Biotech) using a Random
Primed DNA labeling kit (Boehringer Mannheim) according to the
manufacturer's protocol. A 1.8-kilobase pair pssB cDNA
fragment was cleaved from a plasmid, pSVpssB/neo
(15), by KpnI/XbaI digestion according to the
standard method as described (14), and used as a pssB probe.
A commercial
-actin cDNA fragment (CLONTECH)
was used as a control probe. The 32P-labeled
pssB probe was incubated with the membrane in the presence of 50% formamide at 42 °C overnight as described (14), with slight
modifications. The final wash of the membrane was performed with 0.1×
SSC (1× SSC consists of 0.15 M NaCl and 15 mM
sodium citrate, pH 7.0.) containing 0.1% (w/v) SDS at 65 °C.
Detection and quantification of the 32P-labeled
pssB probe hybridized with the poly(A)+ RNA were
performed with a bioimage analyzer (FUJIX BAS2000). After removal of
the probe, the membrane was reprobed with the 32P-labeled
actin probe and analyzed by the same methods.
Extraction and Separation of Phospholipids-- Lipid extraction from cells was performed according to the method of Bligh and Dyer (16). One- and two-dimensional thin-layer chromatography for separation of phospholipids were performed as described (11).
Preparation of [32P]PE and [32P]PC-- J 774.1 cells were seeded at 2 × 107 cells in a 150-mm-diameter dish containing 30 ml of phosphate-free modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate and then labeled with 400 µCi of 32Pi (the Japan Atomic Energy Institute, Ibaragi, Japan) per dish at 37 °C for 3 days. The labeled cells were washed with phosphate-buffered saline and then harvested. Phospholipids were extracted from the labeled cells and then separated by two-dimensional thin-layer chromatography (11). After the positions of the spots of [32P]PE and [32P]PC on silica gel plates had been located by autoradiography, these 32P-labeled phospholipids were scraped off and then extracted from the silica gel as described (16). The labeled lipids were dried up under a N2 stream and then sonicated in the growth medium using a bath-type sonicator.
Isolation of the PSB-2 Mutant Transfected with the pssB or pssA cDNAs-- The plasmids pSVpssB/neo and pSVpssA/neo (15), which carry pssB and pssA cDNAs, respectively, from CHO-K1 cells and a G418-resistant gene were individually introduced into PSB-2 mutant cells by calcium phosphate precipitation (17). The resulting G418-resistant tranfectants were selected in growth medium supplemented with 400 µg/ml G418 (Geneticin®; Life Technologies, Inc.) and 30 µM PS. pssB-transfected G418-resistant clones exhibiting serine base exchange activity similar to that of the PSA-3 mutant were screened by means of the in situ colony assay for serine base exchange, as described above, and then purified by limited dilution. pssA-transfected G418-resistant clones were purified by limited dilution.
Other Methods-- The radioactive labeling, detection, and quantification of radioactive phospholipids were performed as described in the legends to Figs. 3, 4, 6, and 7. The amount of protein was determined as described (18), using bovine serum albumin as a standard.
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RESULTS |
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Isolation of PSS II-defective Mutants from PSA-3 Mutant Cells-- A PSS I-defective mutant of CHO-K1 cells, PSA-3, exhibited ~2-fold lower serine base exchange activity than that of CHO-K1 cells when the activity was determined using cell homogenates (3). To isolate mutants with a further decrease in the serine base exchange activity, PSA-3 mutant cells were mutagenized and screened by means of an in situ assay for serine base exchange. Among approximately 50,000 colonies of mutagenized cells, a mutant clone, named PSB-1, with significantly decreased serine base exchange activity was found. The serine base exchange activity in the homogenate of the PSB-1 mutant cells was ~50% of that in the PSA-3 homogenate (Table I). The ethanolamine base exchange activity in the PSB-1 homogenate was also decreased to ~50% of that in the PSA-3 homogenate (Table I). Regardless of the decreases in the serine and ethanolamine base exchange activities, the PSB-1 mutant showed no remarkable alteration in cell growth, PS biosynthesis, or phospholipid composition compared with the parental strain, PSA-3 (data not shown).
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Growth and PS Biosynthesis of the PSB-2 Mutant-- The PSS I-defective PSA-3 mutant and the PSS I- and PSS II-defective PSB-2 mutant were incapable of growth in the medium without phospholipid supplementation, whereas CHO-K1 cells with normal PSS I and II activities were able to grow well in this medium (Fig. 2). Although the growth defect of the PSA-3 mutant was suppressed by the addition of either PS or PE to the medium, exogenous PS, but not exogenous PE, suppressed the growth defect of the PSB-2 mutant (Fig. 2). These results implied that PSS II is required for the exogenous PE-dependent restoration of cell growth of the PSA-3 mutant.
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The PSB-2 Mutant Is Defective in the Conversion of Exogenous PE to PS-- The PSA-3 mutant is defective in the conversion of exogenous PC to PS, because of the defect in PSS I, but this mutant has the ability to convert exogenous PE to PS (8). To determine whether or not the PSB-2 mutant is defective in the conversion of exogenous PE to PS, PSB-2 and PSA-3 mutant cells were cultivated with [32P]PE, and then the radioactivity of [32P]PS formed from exogenously added [32P]PE in these cells was measured as a function of time. As shown in Fig. 4A, the radioactivity of cellular PS in the PSB-2 mutant was strikingly lower than that in the PSA-3 mutant, although the radioactivity of cellular PE in the PSB-2 mutant was similar to that in the PSA-3 mutant (Fig. 4B). This result indicated that the PSB-2 mutant is defective in the conversion of exogenous PE to PS.
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The PSB-2 Mutant Transfected with the pssB cDNA Recovers the Exogenous PE-dependent Growth and PS Biosynthesis-- In contrast to the PSS I-defective PSA-3 mutant, the PSS I- and PSS II-defective PSB-2 mutant was incapable of growth and defective in PS biosynthesis in the medium with exogenous PE, as described above. To verify that these growth and PS biosynthetic defects of the PSB-2 mutant are due to the deficiency in the pssB gene product, PSS II, we transfected PSB-2 mutant cells with a plasmid, pSVpssB/neo, which carries the pssB cDNA from CHO-K1 cells and a G418-resistant gene. From the resultant G418-resistant stable transfectants, a clone that exhibited an increase of serine base exchange activity but did not have much higher serine base exchange activity than the PSA-3 mutant was selected for further characterization, because the overproduction of PSS II might have secondary effects on growth and PS biosynthesis (6). The serine and ethanolamine base exchange activities in the homogenate of the selected transfectant, PSB-2/pssB, were both ~8-fold those in the PSB-2 homogenate and similar to those in the PSA-3 homogenate (Table III), indicating that the PSS II activity in this transfectant was comparable to that in the PSA-3 mutant. The choline base exchange activity in the PSB-2/pssB homogenate was negligible, indicating that the PSB-2/pssB transfectant remained defective in PSS I activity (Table III). In the medium without phospholipid supplementation, the PSB-2/pssB transfectant remained incapable of growth (Fig. 5A), and the PS and PE levels in this transfectant remained low (Table IV). In the medium with exogenous PE, the PSB-2/pssB transfectant, in contrast to the PSB-2 mutant, grew well (Fig. 5A) and exhibited a normal phospholipid composition, similar to that in PSA-3 and CHO-K1 cells (Table IV). Thus, it is very likely that the deficiency in PSS II is the cause of the growth and PS biosynthetic defects of the PSB-2 mutant cultivated with exogenous PE.
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Growth and PS Biosynthesis of pssA-transfected PSB-2 Mutant Cells-- The PSB-2 mutant was defective not only in PSS II but also in the pssA-encoded PSS I. To examine the effects of expression of pssA in the PSB-2 mutant on growth and PS biosynthesis, we transfected this mutant with a plasmid, pSVpssA/neo, which carries the pssA cDNA from CHO-K1 cells and a G418-resistant gene. The resultant G418-resistant transfectant, PSB-2/pssA, exhibited ~7-, ~2-, and >10-fold increases in serine, ethanolamine, and choline base exchange activities, respectively, as compared with those in the PSB-2 mutant (Table III). On comparison of the choline base exchange activity of the PSB-2/pssA transfectant with that of CHO-K1 cells, the PSS I activity of the PSB-2/pssA transfectant seemed to be ~50% of that in CHO-K1 cells. The PSB-2/pssA transfectant grew normally in the medium supplemented with PS or PE (data not shown), and even in the medium without phospholipid supplementation (Fig. 5B). The PSB-2/pssA transfectant grown in the medium without phospholipid supplementation exhibited a normal phospholipid composition, similar to that of CHO-K1 cells (Table IV).
The Defect in the Conversion of PE to PS in the PSB-2 Mutant Is Complemented by the pssB cDNA, but Not by the pssA cDNA-- To determine whether or not the defect in the conversion of exogenous PE to PS of the PSB-2 mutant was complemented by each of the pssB and pssA cDNAs, the PSB-2/pssB and PSB-2/pssA transfectants, together with PSA-3, PSB-2, and CHO-K1 cells, were cultivated with [32P]PE, and then the radioactivity of cellular PS was determined. As shown in Fig. 6, the activity of conversion of exogenous PE to PS of the PSB-2/pssB transfectant was ~6-fold that of PSB-2 mutant cells and intermediate between those of CHO-K1 and PSA-3 cells. On the other hand, the activity of the PSB-2/pssA transfectant remained at a low level, similar to that in the PSB-2 mutant. These results indicated that the defect in the conversion of exogenous PE to PS of the PSB-2 mutant is complemented by the pssB cDNA but not by the pssA cDNA.
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The PSB-2 Mutant Has a Defect in the Conversion of Exogenous PC to PS, Which Is Complemented by the pssA cDNA, but Not by the pssB cDNA-- The PSA-3 mutant was previously shown to be defective in the conversion of exogenous PC to PS because of the PSS I defect (8). Thus, the PSB-2 mutant, which was defective in both PSS I and II, was assumed to also be defective in this conversion. As expected, a metabolic labeling experiment involving [32P]PC revealed that the PSB-2 mutant remained defective in the conversion of exogenous PC to PS, as shown in Fig. 7. To determine whether or not this defect of the PSB-2 mutant is complemented by each of the pssB and pssA cDNAs, the PSB-2/pssB and PSB-2/pssA transfectants were cultivated with [32P]PC, and then the radioactivity of cellular PS was determined. The PSB-2/pssB transfectant showed negligible activity of conversion of exogenous PC to PS, but the PSB-2/pssA transfectant exhibited activity comparable to that of CHO-K1 cells. These result indicated that the defect in the conversion of exogenous PC to PS of the PSB-2 mutant is complemented by the pssA cDNA but not by the pssB cDNA.
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DISCUSSION |
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PS formation in mammalian cells occurs through the exchange of L-serine with the base moiety of PC or PE (8, 20-22). The serine base exchange in CHO-K1 cells is catalyzed by at least two enzymes, the pssA gene product, PSS I, and the pssB gene product, PSS II (Refs. 3 and 5-7; for a review, see Ref. 22). On the characterization of a PSS I-defective mutant of CHO-K1 cells, PSA-3, PSS I was shown to function as a critical enzyme in the production of PS and PE in CHO-K1 cells (3, 5). In the present study, we tried to isolate PSS II-defective mutants to address the function of PSS II in CHO-K1 cells. A CHO cell mutant, named PSB-2, isolated from PSA-3 mutant cells was shown to be defective in PSS II by several lines of evidence. First, the serine base exchange activity of the PSB-2 mutant was decreased to ~10% of that of the PSA-3 mutant and ~5% of that of CHO-K1 cells. Second, the activity of ethanolamine base exchange, which PSS II is capable of catalyzing (6), of the PSB-2 mutant was also decreased, being less than 15% of that of PSA-3 and CHO-K1 cells. Third, the defects of the PSB-2 mutant in the serine and ethanolamine base exchange activities were complemented by the pssB cDNA. Finally, the level of pssB mRNA in PSB-2 cells was ~20% of that in PSA-3 and CHO-K1 cells.
PSS I activity appears to account for ~50% of the total serine base exchange activity in the homogenate of CHO-K1 cells, because the serine base exchange activity in the homogenate of PSA-3 mutant cells, which has no detectable amounts of pssA mRNA and its product, PSS I (5, 7), is ~50% of that of CHO-K1 cells (3). The serine base exchange activity in PSB-2 mutant cells is ~10% of that in PSA-3 mutant cells, indicating that PSS II accounts for at least ~90% of the total serine base exchange activity of the PSS I-lacking PSA-3 mutant. The remaining ~10% of the total serine base exchange activity of the PSA-3 mutant might also be attributed to PSS II, because the decrease in the serine base exchange activity of the PSB-2 mutant is proportional to the decrease in the cellular level of pssB mRNA of this mutant; both the activity and mRNA level in the PSB-2 mutant are 10-20% of those in the PSA-3 mutant. It is therefore likely that the serine base exchange in the homogenate of CHO-K1 cells is catalyzed almost exclusively by PSS I and PSS II, each of which accounts for approximately 50% of the total serine base exchange activity in the cell homogenate.
Both the PSB-2 and PSA-3 mutants are incapable of growth and producing a normal amount of PS when cultivated in a medium without phospholipid supplementation. Although the PSS I-defective PSA-3 mutant is rescued from the growth and PS biosynthetic defects by the addition of exogenous PE to the medium, the PSB-2 mutant deficient in both PSS I and II remains incapable of growth and defective in PS biosynthesis in the medium with exogenous PE. Labeling experiments with [32P]PE revealed that the PSB-2 mutant, but not the PSA-3 mutant, is defective in the conversion of exogenous PE to PS. This defect and the growth and PS biosynthetic defects of the PSB-2 mutant cultivated with exogenous PE are complemented by the pssB cDNA for PSS II. These results indicate that PSS II catalyzes the conversion of PE to PS and functions as a principal enzyme in PS synthesis in PSA-3 mutant cells cultivated with exogenous PE. Furthermore, these results indicate that PSS II is indispensable for the growth of PSA-3 mutant cells in the medium supplemented with PE.
PSS I can catalyze the PS formation from PC (8), but it remained to be elucidated whether PSS I catalyzes exclusively the conversion of PC to PS or can also catalyze the conversion of PE to PS. In the present study, it was shown that the pssA cDNA for PSS I is incapable of complementing the defect of the PSB-2 mutant in the conversion of PE to PS, although the pssB cDNA for PSS II complements this defect. On the other hand, the defect in the conversion of PC to PS of the PSB-2 mutant was shown to be complemented by the pssA cDNA but not by the pssB cDNA. These results suggested that PSS I negligibly catalyzes the conversion of PE to PS in CHO-K1 cells and confirmed our previous finding that PSS I, but not PSS II, catalyzes the conversion of PC to PS (6, 8).
As described above, the results presented here suggested the following. 1) The serine base exchange in CHO-K1 cells is catalyzed almost exclusively by PSS I and PSS II. 2) PSS II functions as the principal enzyme in PS synthesis in PSA-3 mutant cells cultivated with exogenous PE. 3) PSS II, but not PSS I, can catalyze the conversion of PE to PS. Thus, it is very likely that most of the PS formation from PE in wild-type CHO-K1 cells is catalyzed by PSS II.
Transfection of PSS I- and PSS II-defective PSB-2 mutant cells with the pssA cDNA for PSS I rendered the PSB-2 mutant capable of growth in the medium without phospholipid supplementation. This restoration of the cell growth of the PSB-2 mutant does not require overproduction of PSS I, because one of the resultant phospholipid-prototrophic transformants, PSB-2/pssA, exhibited less PSS I activity than that of CHO-K1 cells. In the medium without phospholipid supplementation, the PSB-2/pssA transfectant exhibits a normal phospholipid composition. These results raise the possibility that PSS II is dispensable for the cell growth and PS biosynthesis of CHO-K1 cells cultivated without phospholipid supplementation. However, the PSS II mutation in the PSB-2 mutant seems to be leaky, because this mutant exhibits reduced but significant serine base exchange activity and pssB mRNA. Thus, elucidation of the significance of PSS II in cell growth and PS biosynthesis under ordinary growth conditions without phospholipid supplementation awaits the isolation of PSS II-null mutants.
The cellular levels of PS and its decarboxylation product, PE, in PSS I-defective PSA-3 mutant cells immediately decreased upon cultivation in the medium without phospholipid supplementation (23), in which this mutant is incapable of growth. The addition of either PS or PE to the medium restored the PS and PE levels of the PSA-3 mutant to normal and suppressed the growth defect of this mutant. These previous observations suggest that PS production by PSS I is critical for the growth of CHO-K1 cells in the medium without phospholipid supplementation. However, it has not been resolved whether PS, PE, or both are critical for cell growth. In the present study, we have shown that exogenous PS, but not PE, complements the growth defect of the PSB-2 mutant. The PSB-2 mutant cultivated with exogenous PE exhibited a reduction in the cellular PS level. The levels of other major phospholipids, including PE, in these mutant cells are normal upon cultivation with exogenous PE. These results suggest that PS is indispensable for the growth of CHO-K1 cells.
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ACKNOWLEDGEMENTS |
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We thank Drs. Yoshimasa Sakakibara and Kentaro Hanada (National Institute of Infectious Diseases) for helpful comments during the preparation of the manuscript and Dr. Hisanori Yamamoto (National Institute of Infectious Diseases) for help in the preparation of 32P-labeled phospholipids.
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FOOTNOTES |
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* This work was supported in part by the Human Sciences Basic Research Project and the Integrated Study Projects on Drug Innovation Science of the Japan Health Sciences Foundation, research grants from the Naito Foundation, Grants-in-Aid for General Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-3-5285-1111, ext. 2125; Fax: 81-3-5285-1157; E-mail: kuge{at}nih.go.jp.
1 The abbreviations used are: PS, phosphatidylserine; CHO, Chinese hamster ovary; PSS, phosphatidylserine synthase; PC, phosphatidylcholine; PE, phosphatidylethanolamine.
2 K. Saito, M. Nishijima, and O. Kuge, unpublished data.
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REFERENCES |
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