(Received for publication, October 21, 1996, and in revised form, April 30, 1997)
From the Department of Biochemistry and Cell Biology, National Institute of Health, Toyama 1-23-1, Shinjuku-ku, Tokyo 162, Japan
Phosphatidylserine (PtdSer) in mammalian cells is synthesized through the exchange of free L-serine for the polar head group (base) of preexisting phospholipid. We previously showed the presence of two different enzymes catalyzing the serine base exchange in Chinese hamster ovary (CHO) cells and isolated the cDNA of one of the enzymes, PtdSer synthase (PSS) I, which also catalyzes the exchange of the base moiety of phospholipid(s) for ethanolamine and choline. In this study, we cloned a CHO cDNA, designated as pssB, which encodes a protein exhibiting 32% amino acid sequence identity with CHO PSS I. Introduction of the pssB cDNA into CHO-K1 cells resulted in striking increases in both the serine and ethanolamine base exchange activities. In contrast to the PSS I cDNA, the pssB cDNA was incapable of increasing the choline base exchange activity. The expression of the pssB gene in Sf9 insect cells also results in striking increases in both serine and ethanolamine base exchange activities. The pssB cDNA was found to transform a PtdSer-auxotrophic PSS I-lacking mutant of CHO-K1 cells to PtdSer prototrophy. The PtdSer content of the resultant transformant grown without exogenous PtdSer for 2 days was 4-fold that of the mutant and similar to that of CHO-K1 cells, indicating that the pssB cDNA complemented the PtdSer biosynthetic defect of the PSS I-lacking mutant. These results suggested that the pssB cDNA encoded the second PtdSer synthase PSS II, which catalyzed the serine and ethanolamine base exchange, but not the choline base exchange.
Phosphatidylserine (PtdSer)1 is one of the major membrane phospholipids in mammalian cells, comprising about 10% of the total phospholipids. To elucidate the biosynthetic pathway and biological function of PtdSer, we previously isolated a Chinese hamster ovary (CHO) cell mutant that requires exogenous PtdSer for cell growth (1). This PtdSer-auxotrophic mutant, PSA-3, is strikingly defective in PtdSer biosynthesis, and its cell extract exhibits an about 50% decrease in the activity of the exchange between free L-serine and the polar head group (base) of preexisting phospholipids for PtdSer formation (1). The parental CHO-K1 cells, but not the mutant cells, have the ability to use phosphatidylcholine (PtdCho) as a phosphatidyl donor for serine base exchange (2). In contrast to PtdCho, phosphatidylethanolamine (PtdEtn) is used by both the CHO-K1 and mutant cells as a phosphatidyl donor for serine base exchange (2). Furthermore, the mutant has been shown to grow normally in a growth medium supplemented with PtdEtn, synthesizing a normal amount of PtdSer (2). These findings led us to conclude that PtdSer in CHO cells is synthesized through serine base exchange, which is catalyzed by two kinds of PtdSer synthase (PSS): one, PSS I, which is absent in mutant PSA-3, can use PtdCho as a substrate; and the other, PSS II, uses PtdEtn but not PtdCho as a substrate.
PSS I appears to contribute to the formation of both PtdSer and PtdEtn because mutant PSA-3 grown without exogenous phospholipids exhibited decreased levels of both PtdSer and PtdEtn (1). Since it has been suggested that much of the PtdEtn in CHO cells is synthesized through the decarboxylation of PtdSer (3), PtdSer synthesized by PSS I probably serves as an essential precursor of PtdEtn.
A CHO cDNA (designated as pssA) which complements the PSS I defect of mutant PSA-3 has been isolated (4). With antibodies raised against synthetic pssA peptides, we recently provided several lines of evidence that the pssA cDNA encodes PSS I (5). PSS I deduced from the cDNA sequence is composed of 471 amino acid residues and has several potential membrane-spanning domains (4). In addition to serine base exchange, PSS I catalyzes the exchange of the base moiety of phospholipid(s) for choline and ethanolamine in cell extracts (1, 4, 6). Subcellular fractionation followed by immunoblotting with an anti-(pssA peptide) antibody indicated that PSS I is enriched in mitochondria-associated membranes and microsomal membranes (5).
Although the function and structure of PSS I are being elucidated, as described above, less is known concerning PSS II, since neither the mutant nor the gene has been isolated. To address the role of PSS II in PtdSer metabolism and its biological significance, we have attempted to isolate the cDNA of PSS II. Here, we describe the cloning of the PSS II cDNA.
Strain CHO-K1 was obtained from the American Type Culture Collection. CHO-K1, mutant PSA-3 (1), and transformant PSA-3/pssB obtained in this study were maintained as described (1). For the ethanolamine supplementation experiment, 100 ml of newborn calf serum was dialyzed three times against 2 liters of phosphate-buffered saline for about 12 h and filter sterilized. Spodoptera frugiperda (Sf9) cells were provided by Dr. Yoshiharu Matsuura (National institute of Health, Tokyo, Japan) and maintained in TC-100 medium (Life Technologies, Inc.) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 10 µg/ml gentamycin, and 0.26% (w/v) tryptose phosphate broth (Life Technologies, Inc.) at 27 °C.
Construction of a cDNA LibraryPoly(A)+ RNA was prepared from CHO-K1 cells as described (7) and used for cDNA synthesis. The cDNA synthesis and construction of a cDNA library in a plasmid vector, pSPORT1, were performed with a SuperScriptTM plasmid system (Life Technologies, Inc.) according to the manufacturer's instructions.
Cloning of a pssB cDNAOligonucleotides corresponding to parts of a human expressed sequence tag (EST) (GenBank number F11951) were used to amplify a pssB cDNA fragment from the CHO cDNA library by means of a two-stage polymerase chain reaction. The primers used for the first round of amplification were TCCAGACTGTCCAGGACGGC (sense) and AGGAACTCGAACATCACGCT (antisense). The same antisense primer and TCCAGGACGGCCGGCAGTTT (sense) were used for the second round of amplification. The amplification reactions were performed for 35 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, and elongation at 72 °C for 90 s, with Taq polymerase (Perkin-Elmer) according to the manufacturer's instructions. For the second round of amplification, the first round reaction mixture was diluted 5,000-fold and used as the template. The 0.3-kilobase pair product of the second round of amplification was subcloned into a plasmid, pBluescript II SK+ (Stratagene), sequenced, and used as a hybridization probe for screening of the CHO cDNA library, after enrichment of hybridizing clones with a GeneTrapperTM cDNA-positive selection system (Life Technologies, Inc.). The enrichment was performed with a biotinylated oligonucleotide, GACTGGTGGATGTGCATGATCATC, corresponding to a part of the 0.3-kilobase pair polymerase chain reaction product, according to the manufacturer's instructions except for omission of the repair reaction. Colony filter hybridization with the 32P-labeled probe was performed as described (8); hybridization was performed for 22 h at 42 °C in 5 × SSPE (1 × SSPE = 0.15 M NaCl, 1 mM EDTA, 10 mM NaH2PO4, pH 7.4), 5 × Denhardt's solution, 0.5% sodium dodecyl sulfate, 50% formamide, and 100 µg/ml denatured salmon sperm DNA; the final wash was performed in 0.2 × SSC (1 × SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.0), and 0.1% sodium dodecyl sulfate at 50 °C for 1 h.
DNA SequencingBoth strands of the pssB cDNA were determined by the dideoxy chain termination method with SequenaseTM (U. S. Biochemical Corp.) according to the manufacturer's instructions, using a series of deletion mutants generated by exoIII/mung bean nuclease treatment (9), in combination with walking primers.
Transient Transfection of CHO-K1 Cells with the pssB and pssA cDNAsA plasmid, pSPORT1/pssB, carrying the pssB cDNA, was cleaved at the SalI and NotI sites, and the resulting pssB cDNA fragment was inserted into these restriction enzyme sites of a mammalian expression plasmid vector, pSV-SPORT1 (Life Technologies, Inc.). The resulting construct, pSVpssB, and pcDPSSA encoding the pssA protein (CHO PSS I) (4) were introduced into CHO-K1 cells using LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's instructions.
Heterologous Expression of the pssB geneThe plasmid, pSPORT1/pssB, was cleaved at the SalI and HindIII sites, and the resulting pssB cDNA fragment was inserted into the XhoI and HindIII sites of a baculovirus transfer vector, pBlueBac4.5 (Invitrogen). A monolayer of Sf9 cells (35-mm-diameter dish) was cotransfected with the resulting construct, pBlueBac4.5/pssB and Bac-N-BlueTM Autographa californica DNA (Invitrogen), using LipofectinTM reagent (Life Technologies, Inc.). For production of control virus, another monolayer of Sf9 cells was cotransfected with the transfer vector pBlueBac4.5 and Bac-N-BlueTM DNA. After 4 days of culture, the transfection supernatants containing pssB recombinant virus or control virus were collected. Fresh monolayers of Sf9 cells (75-cm2 flask) were infected with the transfection supernatant containing pssB recombinant virus or control virus and cultivated in TC-100 insect medium (Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum. After 4 days at 27 °C, cells were harvested, washed two times with ice-cold phosphate-buffered saline, pH 7.4, resuspended in 0.25 M sucrose containing 1 mM EDTA, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM (p-amidinophenyl)methanesulfonyl fluoride, and 10 mM HEPES, pH 7.5, and disrupted by two 15-s sonication bursts on ice. Phospholipid base exchange activity in the preparations was assayed as described (1).
Isolation of Transformant PSA-3/pssBMutant PSA-3 cells were transfected with pSVpssB by the calcium phosphate precipitation method (10), and the resultant transformant, designated as PSA-3/pssB, which was able to grow in the growth medium without exogenous phospholipids, was purified by limited dilution of the transfected cells.
Other MethodsRadioactive labeling, extraction, separation, and quantitation of phospholipids were performed as described in Tables III and IV and in the legends to Figs. 5, 6, and 7. [32P]PtdCho was prepared from CHO-K1 cells metabolically labeled with 32Pi as described (2). Protein was measured according to Lowry et al. (11), using bovine serum albumin as a standard.
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To identify cDNA clones encoding PSS II,
the amino acid sequence of PSS I predicted from the cDNA sequence
(4) was compared with the ESTs in DNA data bases using the TBLASTN
search protocol at the National Center for Biotechnology Information. A
human EST (GenBank number F11951) was found to encode a peptide that exhibited 30% sequence identity, in a stretch of 98 amino acids, with
both CHO PSS I and a putative human PSS I (GenBank number D14694). A
cDNA fragment of the CHO counterpart for human EST F11951 was
generated from a CHO cDNA library, using a polymerase chain
reaction and primers corresponding to parts of the EST sequence. The
resulting cDNA fragment was used as a hybridization probe to screen
the CHO cDNA library, after enrichment of hybridizing clones using
a GeneTrapperTM cDNA-positive selection system with a biotinylated
oligonucleotide specific to the cDNA fragment. Sequence analysis of
a hybridizing cDNA clone revealed a large open reading frame
encoding a protein of 474 amino acid residues with a calculated
molecular mass of 55,003 Da (Fig. 1). The gene of this
protein was designated as pssB. The predicted
pssB protein exhibited 32% amino acid sequence identity
with the pssA gene product, PSS I (Fig. 2).
Hydrophobicity analysis of the predicted pssB protein by the
method of Kyte and Doolittle (12) revealed a highly hydrophobic protein
containing several potential membrane-spanning domains, the
hydrophobicity profile of which was very similar to that of PSS I, as
shown in Fig. 3. In addition, the pssB
protein was found to have, at its NH2 terminus, a
Met-Arg-Arg-Ala-Glu sequence that corresponds to an
NH2-terminal double arginine motif known as an ER targeting
signal (13). The presence of the motif implied that the pssB
protein is localized to ER. It is noteworthy that a strict positional
requirement for the two arginines of the double arginine motif has been
shown: namely, efficient targeting of recombinant reporter proteins to
ER is only accomplished if two adjacent arginines or two arginines
separated by one residue are located in the four residues following the
initiator methionine (13). Thus, given that the double arginine motif
of the pssB protein actually functions as an ER targeting
signal (see "Discussion"), the putative initiator methionine codon
appeared to be the true initiation codon, although the absence of a
5-termination codon raised the possibility that the isolated
pssB cDNA was a partial clone.
Phospholipid Base Exchange Activities in pssB-transfected Cells
To determine whether the pssB gene product is relevant to serine base exchange for PtdSer formation, the pssB cDNA was placed downstream of the mammalian expression promoter of a plasmid, pSV-SPORT1, and the resulting construct, pSVpssB, was introduced into CHO-K1 cells. The transient transfectant with pSVpssB exhibited a 6-fold higher specific activity of serine base exchange for PtdSer formation than CHO-K1 cells transfected with the control vector (Table I). The ethanolamine base exchange activity also increased 10-fold upon transfection with the pssB cDNA (Table I). On the other hand, there was no significant difference in the choline base exchange activity between the transfectant with pSVpssB and the control CHO-K1 cells. In contrast to pSVpssB, a plasmid pcDPSSA (4) encoding CHO PSS I was capable of increasing the choline base exchange activity, as well as the serine and ethanolamine base exchange activities, upon transient transfection (Table I). These results, together with the sequence similarity of the pssB gene product to PSS I, suggested that pssB encoded an enzyme catalyzing both the serine and ethanolamine base exchange but not the choline base exchange in cell extracts.
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To obtain further
evidence that pssB encodes an enzyme catalyzing both the
serine and ethanolamine base exchange, we used heterologous expression
of the pssB gene in insect cells. The pssB
cDNA was placed within the genome of baculovirus under control of
the polyhedrin promoter and expressed by viral infection of Sf9 cells.
The serine and ethanolamine base exchange activities in a homogenate of
Sf9 cells infected with the pssB-containing baculovirus
were, respectively, 5.3- and 7.5-fold higher than those of Sf9 cells
infected with a control virus (Fig. 4). On the other
hand, the choline base exchange activity was not elevated by the
pssB virus infection (Fig. 4). Thus, in this heterologous system the pssB cDNA was capable of increasing both the
serine and ethanolamine base exchange activities, suggesting that the cDNA encodes an enzyme catalyzing these two different base exchange reactions.
pssB cDNA Complements the Growth Defect of PSS I-lacking Mutant PSA-3
A PSS I-lacking mutant of CHO-K1 cells, PSA-3, requires the addition of either PtdSer or PtdEtn to the medium for cell growth (1, 2). To determine whether or not the pssB cDNA compensates for the lack of PSS I activity, the PSA-3 mutant cells were transfected with pSVpssB and then cultured in the absence of exogenous phospholipids. The transfection efficiently yielded transformants that were able to grow in the absence of exogenous phospholipids. The growth rate of the resulting transformant, PSA-3/pssB, was almost the same as that of CHO-K1 in the medium without exogenous phospholipids (Fig. 5). The extract of transformant PSA-3/pssB exhibited strikingly higher serine and ethanolamine base exchange activities than those of the CHO-K1 and mutant PSA-3 cells but remained defective in the choline base exchange activity (Table II). These results indicated that the pssB cDNA, which induced the stable overexpression of the serine and ethanolamine base exchange activities, was able to complement the growth defect of the PSS I-lacking mutant, PSA-3.
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The phospholipid composition and content of cells grown without exogenous phospholipids for 2 days were determined. Transformant PSA-3/pssB exhibited a phospholipid composition and content very similar to those of CHO-K1 cells, whereas the contents of PtdSer and PtdEtn of mutant PSA-3 were 21% and 51% of those of CHO-K1 cells, respectively (Table III). To determine the rate of biosynthesis of PtdSer, cells were pulse labeled with L-[U-14C]serine. Transformant PSA-3/pssB, but not mutant PSA-3, was able to incorporate the label into PtdSer at a rate similar to that of CHO-K1 cells, as shown in Fig. 6. These results showed that the pssB cDNA was able to complement the PtdSer biosynthetic defect of the PSS I-lacking mutant, PSA-3.
The pssB-transformed PSA-3 Mutant Remains Defective in Conversion of PtdCho to PtdSerMutant PSA-3 is defective in the conversion of exogenous [32P]PtdCho to [32P]PtdSer because of a lack of PSS I activity (2). To determine whether or not the pssB cDNA complements this defect, cells were metabolically labeled with [32P]PtdCho. CHO-K1 cells incorporated the radioactivity into PtdSer, in an amount comprising 3.6% of the radioactivity of cellular PtdCho (Fig. 7). In contrast to CHO-K1 cells, both transformant PSA-3/pssB and mutant PSA-3 were incapable of incorporating the radioactivity into PtdSer in significant amounts, although the level of cellular [32P]PtdCho in the transformant and mutant was almost the same as that in CHO-K1 cells (Fig. 7). These results indicated that the transformant PSA-3/pssB remained defective in conversion of PtdCho to PtdSer.
Transformant PSA-3/pssB Cultivated in the Medium with Dialyzed Newborn Calf Serum Exhibits a Normal PtdSer BiosynthesisMutant
PSA-3 incorporates exogenous ethanolamine into PtdEtn which functions
as a precursor phospholipid of PtdSer in the mutant (1, 2). We examined
if exogenous ethanolamine derived from newborn calf serum is involved
in the restoration of PtdSer biosynthesis in transformant
PSA-3/pssB, by using dialyzed newborn calf serum.
Transformant PSA-3/pssB and CHO-K1 cells, but not mutant
PSA-3, grew exponentially in the medium supplemented with 10% dialyzed
newborn calf serum (Fig. 8). When ethanolamine was added
to the medium at a concentration of 10 µM, mutant PSA-3 was able to grow normally for 5 days (Fig. 8). The addition of ethanolamine did not affect the cell growth of transformant
PSA-3/pssB and CHO-K1 cells (Fig. 8). A labeling experiment
with 32Pi for 48 h revealed that the
PtdSer level of transformant PSA-3/pssB grown in the medium
with the dialyzed serum was similar to that of CHO-K1 cells (Table
IV). Although mutant PSA-3 cultivated in the medium with
the dialyzed serum was defective in PtdSer biosynthesis, the addition
of ethanolamine to the medium restored a normal level of PtdSer in the
mutant (Table IV). Upon cultivation with ethanolamine, there was no
significant difference in phospholipid composition among all three
strains (Table IV). These results indicated that the restoration of
PtdSer biosynthesis in transformant PSA-3/pssB occurred
without ethanolamine supplementation to the medium containing the
dialyzed serum and that the addition of ethanolamine to the medium
complemented the PtdSer biosynthetic defect of mutant PSA-3.
We have shown the presence of two different enzymes catalyzing the serine base exchange for PtdSer formation in CHO-K1 cells (1, 2). A CHO pssA cDNA encoding one of the enzymes, PSS I, has been isolated by means of genetic complementation with a PSS I-lacking mutant, PSA-3 (4, 5). Introduction of the pssA cDNA into the mutant leads to increases in choline and ethanolamine base exchange activities, in addition to an elevation of the serine base exchange activity (4). In this study, we have tried to isolate the cDNA of the second PSS, on the assumption that the second PSS is similar in sequence to PSS I. A CHO pssB cDNA isolated here encodes a protein showing a high (32%) amino acid sequence identity with the pssA-encoding PSS I. Transient transfection of CHO-K1 cells with the pssB cDNA results in a 6-fold increase in the serine base exchange activity. The pssB-transfected cells also exhibit a 10-fold elevated ethanolamine base exchange activity. However, the pssB cDNA is incapable of elevating the choline base exchange activity, in contrast to the pssA cDNA. The expression of the pssB gene in Sf9 insect cells also results in striking increases in both serine and ethanolamine base exchange activities. When the pssB cDNA is introduced into the mutant PSA-3, the mutant recovers a normal level of PtdSer biosynthesis. These results suggest that the pssB cDNA encodes the second PtdSer synthase PSS II, which catalyzes the serine and ethanolamine base exchange but not the choline base exchange.
Both the pssB and pssA proteins deduced from the cDNA sequences have several potential membrane-spanning domains, consistent with the fact that the serine and choline base exchange enzymes are firmly embedded in membranes (14). In addition, the pssB protein has a Met-Arg-Arg-Ala-Glu sequence, which corresponds to an NH2-terminal double arginine motif known as an ER targeting signal (13). Although the pssA protein does not have the double arginine motif, it has a unique Gly-Val-Gly-Lys-Lys sequence at its COOH terminus, which is similar to another ER targeting motif, a COOH-terminal double lysine motif (Lys-X-Lys-X-X or X-X-Lys-Lys-X-X) (15, 16). Consistent with the presence of these sequences, the serine and choline base exchange activities are recovered in a microsomal fraction containing the bulk of ER and in a mitochondria-associated membrane fraction containing a subfraction of ER (5, 17, 18).
PtdSer can be synthesized by the exchange of L-serine with the base moiety of both PtdCho and PtdEtn in CHO-K1 cells (2). Because mutant PSA-3 defective in the conversion of PtdCho to PtdSer is incapable of synthesizing a normal amount of PtdSer (1, 2), the exchange of the choline moiety of PtdCho with L-serine appears to be a major route for PtdSer formation in CHO-K1 cells. Although the pssB-transformed PSA-3 mutant remains defective in the conversion of PtdCho to PtdSer, the transformant produces a normal amount of PtdSer. The transformant shows striking increases in both activities of serine base exchange and ethanolamine base exchange, which is supposed to be the reverse reaction of PtdSer formation from PtdEtn. These results suggest that the majority of PtdSer in the pssB-transformed PSA-3 mutant is produced through the exchange of the ethanolamine moiety of PtdEtn with L-serine.
PtdEtn can be synthesized by three pathways. First, in the CDP-ethanolamine pathway, ethanolamine is phosphorylated and converted to CDP-ethanolamine, and then the phosphoethanolamine moiety is transferred to diacylglycerol for PtdEtn formation (19). The second pathway is the decarboxylation of PtdSer. The third is the ethanolamine base exchange. PtdEtn formation through the decarboxylation and ethanolamine base exchange requires PtdSer. Thus, the exchange of L-serine for the ethanolamine moiety of PtdEtn made by the decarboxylation and ethanolamine base exchange does not yield a net increase in cellular PtdSer content. On the other hand, the ethanolamine-serine exchange using PtdEtn produced through the CDP-ethanolamine pathway results in a net increase in cellular PtdSer content. It is therefore likely that PtdEtn produced through the CDP-ethanolamine pathway contributes to the restoration of PtdSer biosynthesis in the pssB-transformed PSA-3 mutant. Because the restoration of PtdSer biosynthesis in the transformant occurs without ethanolamine supplementation to the medium containing dialyzed newborn calf serum, the level of endogenous ethanolamine appears to be sufficient for PtdSer biosynthesis in the transformant. In contrast, in the PSA-3 mutant cultivated without ethanolamine supplementation, the level of endogenous ethanolamine appears to be insufficient for PtdSer biosynthesis because the mutant is defective in PtdSer biosynthesis unless the growth medium is supplemented with either ethanolamine or PtdEtn (2). Why is the pssB-transformed PSA-3 mutant capable of synthesizing normal amounts of PtdEtn and PtdSer in the absence of ethanolamine supplementation? If the ethanolamine-serine exchange reaction is written together with the reutilization of ethanolamine for PtdEtn synthesis via CDP-ethanolamine pathway,
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The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB004109.
We thank Dr. Yoshiharu Matsuura for help in the expression of the pssB gene in Sf9 insect cells, Dr. Yoshimasa Sakakibara for helpful comments during preparation of the manuscript, and Suma Noura for the technical assistance.