Isolation of a Chinese Hamster Ovary (CHO) cDNA Encoding Phosphatidylglycerophosphate (PGP) Synthase, Expression of Which Corrects the Mitochondrial Abnormalities of a PGP Synthase-defective Mutant of CHO-K1 Cells*

Kiyoshi KawasakiDagger §, Osamu KugeDagger , Shao-Chun Chang§, Philip N. Heacock§, Minseok Rho§, Kenji SuzukiDagger , Masahiro NishijimaDagger , and William Dowhan§parallel

From the Dagger  Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan and the § Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77225

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Phosphatidylglycerophosphate (PGP) synthase catalyzes the first step in the cardiolipin (CL) branch of phospholipid biosynthesis in mammalian cells. In this study, we isolated a Chinese hamster ovary (CHO) cDNA encoding a putative protein similar in sequence to the yeast PGS1 gene product, PGP synthase. The gene for the isolated CHO cDNA was named PGS1. Expression of the CHO PGS1 cDNA in CHO-K1 cells and production of a recombinant CHO PGS1 protein with a N-terminal extension in Escherichia coli resulted in 15-fold and 90-fold increases of PGP synthase specific activity, respectively, establishing that CHO PGS1 encodes PGP synthase. A PGP synthase-defective CHO mutant, PGS-S, isolated previously (Ohtsuka, T., Nishijima, M., and Akamatsu, Y. (1993) J. Biol. Chem. 268, 22908-22913) exhibits striking reductions in biosynthetic rate and cellular content of phosphatidylglycerol (PG) and CL and shows mitochondrial morphological and functional abnormalities. The CHO PGS-S mutant transfected with the CHO PGS1 cDNA exhibited 620-fold and 7-fold higher PGP synthase activity than mutant PGS-S and wild type CHO-K1 cells, respectively, and had a normal cellular content and rate of biosynthesis of PG and CL. In contrast to mutant PGS-S, the transfectant had morphologically normal mitochondria. When the transfectant and mutant PGS-S cells were cultivated in a glucose-depleted medium, in which cellular energy production mainly depends on mitochondrial function, the transformant but not mutant PGS-S was capable of growth. These results demonstrated that the morphological and functional defects displayed by the PGS-S mutant are due directly to the reduced ability to make normal levels of PG and/or CL.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

In animal tissue, the anionic phospholipids phosphatidylglycerol (PG)1 and cardiolipin (CL) are thought to be necessary for many cellular functions. PG represents approximately 1% of total lipid phosphorous in mammalian tissues except for lung and is found in many intracellular locations, such as mitochondrial, nuclear, and microsomal membranes. In lung, PG represents approximately 5% of total phospholipid; it is found there predominantly in lamella body membranes (reviewed in Ref. 1) and is also one of the main components of lung surfactant (1, 2). Recent biochemical analysis suggests that PG is a potential activator of the protein kinase C family, including protein kinase C-theta (3) and nuclear protein kinase C-beta II (4). CL represents from 0.2 to 15% of total lipid phosphorous in various animal tissues and is located primarily in the inner mitochondrial membrane (1). Biochemical analysis suggests that CL is required for many enzymatic activities, such as cytochrome c oxidase (5) and carnitine acylcarnitine translocase (6), and is involved in cellular functions, such as mitochondrial protein import (7-10) and binding of matrix Ca2+ (11).

In yeast cells, most of the requirement for CL for mitochondrial functions appears to be substituted by an increase in PG content (12). Disruption of the CRD1 gene, which encodes CL synthase, resulted in no detectable CL and 5-fold elevation of PG content in yeast cells. The yeast crd1-null strain can grow on both fermentable and nonfermentable carbon sources (12-14), although with reduced efficiency on the latter. On the other hand, disruption of the yeast PGS1 gene (also known as PEL1), which encodes phosphatidylglycerophosphate (PGP) synthase resulted in no detectable CL or PG (15) and the inability to grow on nonfermentable carbon sources (15-17).

Early enzymological studies revealed the biosynthetic pathway for PG and CL in animal cells as shown in Fig. 1. PGP synthase catalyzes the committed step in CL biosynthesis with the displacement of the CMP moiety of CDP-diacylglycerol by sn-glycerol 3-phosphate to produce PGP (18). PGP is rapidly dephosphorlylated to generate PG that is utilized as a substrate along with CDP-diacylglycerol for CL synthesis. PGP synthase activity is predominantly localized in mitochondria, but measurable activity is also detected in endoplasmic reticulum (19). Solubilization and partial purification of PGP synthase from pig liver mitochondria was reported (20, 21), but neither complete purification nor isolation of cDNA for PGP synthase from somatic cells has been reported.


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Fig. 1.   Biosynthetic pathway for PG and CL in animal cells.

Chinese hamster ovary (CHO) cell mutants are a good model for studying mammalian phospholipid biosynthesis and functions (22). Previously, we isolated a temperature-sensitive CHO mutant (PGS-S) that is defective in PGP synthase activity. The PGP synthase activity in the mutant was 1% of that in wild type CHO-K1 cells, and biosynthetic rate and cellular content of PG and CL were also markedly reduced in the mutant (23). Moreover, this mutant displays mitochondrial morphological and functional abnormality (24). These previous results implied that PG and/or CL is essential for mitochondrial morphology and function in CHO-K1 cells.

In this study, we report the first isolation of a somatic cell cDNA from CHO cells that encodes a PGP synthase (cPGS1). Using this cDNA, we also demonstrate that the mitochondrial morphological defects and inability to carry out oxidative phosphorylation to support growth on glucose-depleted medium displayed by mutant PGS-S are directly due to the reduced ability to make normal levels of the anionic phospholipids PG and/or CL.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
Discussion
References

Materials-- All chemicals were reagent grade or better. Restriction endonucleases and DNA modifying enzymes were from Promega Corp. and New England Biolabs. Thin layer chromatography Silica Gel 60 plates were from EM Science. Oligonucleotides were prepared commercially by Genosis Biotechnologies, Inc. QIAEXTM gel extraction kit was from Qiagen. sn-[U-14C]Glycerol 3-phosphate was from ICN Radiochemicals. [32P]Orthophosphate and [alpha -32P]dCTP were from Amersham Pharmacia Biotech. CDP-diacylglycerol was from Serdary Research Laboratory. sn-Glycerol 3-phosphate and trypsin were from Sigma. Ham's F-12 medium, newborn calf serum, Geneticin (G418), penicillin G, and streptomycin sulfate were from Life Technologies, Inc. All media for bacterial growth were obtained from BIO 101 or Difco.

Molecular Cloning of CHO PGS1 cDNA-- Oligonucleotides corresponding to parts of human expressed sequence tags (ESTs) (The Institute for Genome Research, gene identification numbers THC122139 and T12593) were used to amplify a CHO PGS1 cDNA fragment from a CHO cDNA library (25) by means of a two-stage polymerase chain reaction. The primers used for the first round of amplification were 5'-ATGGCATCCCTTTACCTGGG-3' (sense) and 5'-ACGTAGCGGATCTGTCGGTT-3' (antisense). Primers, 5'-TCTGAATTCGAGCAGGAACTGGTGGATTG-3' (sense) and 5'-TCTCTCGAGATCTGTCGGTTGGTGAAGTA-3' (antisense) containing an EcoRI site and XhoI site, respectively, were used for the second round of amplification. The amplification reactions were performed for 40 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 2 min, and elongation at 72 °C for 2 min, with Taq polymerase (Perkin-Elmer) according to the manufacturer's instruction. For the second round of amplification, the first round reaction mixture was diluted 20-fold and used as template. The 0.35-kb product of the second round reaction was subcloned into the EcoRI/XhoI sites of pBluescriptII SK+ (Stratagene) and sequenced. Based on the sequence of the cloned 0.35-kb DNA, an oligonucleotide (5'-CTCTGGAGAAGTCACTACAGTCG-3' (sense)) was synthesized, biotinylated, and used for the enrichment of corresponding cDNA clones from a CHO cDNA library (25) by employing the GeneTrapperTM cDNA positive selection system (Life Technologies, Inc.) according to the manufacturer's instructions. The single stranded cDNA clones captured with the biotinylated oligonucleotide were electroporated into ElectroMAX DH10B (Life Technologies, Inc.), and ampicillin-resistant transformants were selected. Colonies of the resultant transformant were subjected to a polymerase chain reaction screening for colonies containing CHO PGS1 cDNA using the same unbiotinylated sense oligonucleotide and 5'-AGTCACTCAGGTTTGCACC-3' (antisense, which corresponds to the sequence of human EST cDNA, The Institute for Genome Research number THC122139) as polymerase chain reaction primers.

DNA Manipulations and Sequence-- Methods for plasmid preparation, restriction enzyme digestion, DNA ligation, and Escherichia coli transformation were preformed as described previously (26). Plasmid DNA to be sequenced and to be transfected into CHO cells was prepared with Wizard DNA purification kits (Promega Corp.). DNA sequencing reactions were performed by the Taq Dye-deoxy Terminator (Applied Biosystems) method with walking primers, and the reaction products were run on an Applied Biosystems sequencer. Both strands of two CHO PGS1 cDNA clones carrying a putative full-length open reading frame (ORF) were determined.

Cell Culture-- Strain CHO-K1 was obtained from the American Type Culture Collection. Mutant PGS-P (parent strain CHO-K1) and mutant PGS-S (parent strain PGS-P) were previously described (23). Unless otherwise indicated all strains were grown in Ham's F-12 medium (27) supplemented with 10% (v/v) newborn calf serum, penicillin G (100 units/ml), and streptomycin sulfate (100 µg/ml), under 5% CO2 atmosphere at 100% humidity at either 40, 37, or 33 °C. Ham's F-12 medium containing galactose (1.8 mg/ml) instead of glucose as the carbon source was prepared with reagent grade chemicals and supplemented with 10% (v/v) dialyzed newborn calf serum, penicillin G (100 units/ml), and streptomycin sulfate (100 µg/ml).

Transient Transfection of CHO-K1 Cells with CHO PGS1 cDNA-- A plasmid (pSPORT1-cPGS1), carrying a putative CHO PGS1 cDNA (see Fig. 2), was cleaved at the SalI and NotI sites flanking the cDNA insert. The resulting fragment was inserted into the same restriction enzyme sites of mammalian expression vector, pSV-SPORT1 (Life Technologies, Inc.), under control of the SV40 promoter. The resulting construct, pSV-cPGS1, and pSV-SPORT1 were introduced into CHO-K1 cells using LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's instructions.

Production of Recombinant CHO PGS1 Gene Product in E. coli-- A plasmid (pSPORT-cPGS1') potentially expressing a chimeric derivative of the CHO PGS1 cDNA ORF under lacOP control was introduced into E. coli strain XL1-Blue. The cDNA insert in pSPORT-PGS1' is identical to that shown in Fig. 2 except for an additional GG at the 5'-end. Because the cDNA library used to make this plasmid employed a SalI linker (5'-TCGACCCACGCGTCCG-3') at the 5'-end, this plasmid should express a chimeric protein with following sequence fused to the N-terminal MET of the putative ORF encoded by the PGS1 cDNA: MTMITPS-SNTTHYRESWYACRYRSGIPGS-THAS-GRVS (the hyphens divide the sequence derived from beta -galactosidase, the vector, the linker, and the region 5' of the ORF, respectively). E. coli strain XL1-Blue transformed with pSPORT-cPGS1' was cultivated in LB-medium containing 100 µg/ml of ampicillin at 30 °C. Protein production was induced by the addition of isopropyl beta -D-thiogalactoside to the medium at a final concentration of 1 mM. After cultivation for 4 h in the presence of isopropyl beta -D-thiogalactoside, cells were collected, suspended in a buffer (50 mM Tris-HCl (pH 7.8), 1 mM EDTA, 100 mM NaCl, and 0.1 mM phenylmethylsulfonyl fluoride), and sonically disrupted for enzyme assay.


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Fig. 2.   Nucleotide and predicted amino acid sequences of CHO PGS1 cDNA. The DNA sequence is that determined for the insert in pSPORT1-cPGS1. The putative initiation codon and stop codon are underlined

Purification of Stable Transformant PGS-S/cPGS1-- A mammalian expression vector (pSV-OKneo) containing the G418 resistant determinant (28) was cleaved at the SalI and NotI sites and ligated with the CHO PGS1 cDNA generated by the digestion of pSPORT1-cPGS1 with same restriction enzymes. The resulting construct (pSVneo-cPGS1) was introduced into mutant PGS-S cells (23) using LipofectAMINE reagent, and then the G418-resistant transformants were selected in medium containing 400 µg/ml of G418 at 37 °C. Stable transformant colonies were isolated with cloning cups, and finally purified by limited dilution to yield only one colony per well.

Enzyme Assay-- PGP synthase activity was measured as described previously (23) with some modification. The assay was performed at 37 °C for 30 min in the presence of 50 mM Tris-HCl (pH 7.4), 0.25 mM CDP-diacylglycerol, 0.1 mM sn-[U-14C]glycerol 3-phosphate (20 mCi/mmol), 0.25% Triton X-100, and 0.4 mg/ml of CHO cell extract protein in a total volume of 100 µl or 0.5 mg/ml of E. coli cell extract protein in a total volume of 200 µl. After incubation, the lipids were extracted by the sequential addition of 600 µl of chloroform/methanol (1:2, v/v), 200 µl of chloroform, and 200 µl of phosphate-buffered saline. The lipid-containing chloroform phase was washed twice with 400 µl of chloroform/methanol/0.1 M KCl (3:47:48, v/v), and the radioactivity in the chloroform phase was quantified as described previously (15).

Extraction and Separation of Phospholipids-- Lipid extraction from 32Pi-labeled cells was performed according to Bligh and Dyer (29). Analysis of lipid composition and separation of lipids were performed by two-dimensional thin layer chromatography (30). Alternatively, one-dimensional thin layer chromatography was performed for the separation of PG or CL from other lipids. For the separation of PG, chloroform/methanol/acetic acid (65:25:10, v/v) was used as solvent. For the separation of CL, chloroform/methanol/water/ammonium hydroxide (130:75:6:2, v/v) was used with boric acid-impregnated Silica gel 60 thin layer plates (31).

Protein Determination-- Protein concentrations were determined by BCA protein assay reagent (Pierce) using bovine serum albumin as a standard.

Northern Hybridization Analysis-- mRNA was isolated using the FastTrackTM 2.0 Kit (Invitrogen) according to the manufacturer's instructions from CHO-K1 and mutant PGS-S cells grown at 40 °C for 4 days. mRNA (4 µg) was separated on 1% agarose gels containing formaldehyde and transferred to a positively charged HybondTM N+ Nylon membrane (Amersham Pharmacia Biotech) by capillary action using 20× SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) at room temperature. RNA was cross-linked to the membrane by baking at 80 °C for 2 h. The NotI/SalI CHO PGS1 cDNA fragment from pSV-cPGS1 and a 2.0-kb fragment of human beta -actin cDNA were 32P-labeled with the Rediprime DNA labeling system (Amersham Pharmacia Biotech). Hybridization reaction was performed in Rapid-Hyb buffer (Amersham Pharmacia Biotech). A mouse Multiple Tissue Northern (MTNTM) Blot (CLONTECH) was hybridized with 32P-labeled CHO PGS1 cDNA and human beta -actin cDNA according to the manufacturer's instructions. The results were visualized by exposure to x-ray film, and quantitative analysis was preformed with a Packard Instant Imager (Packard Instruments Inc.).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Molecular Cloning of a CHO cDNA Encoding a Protein Similar to yPGS1-- In order to identify a mammalian cDNA clone encoding PGP synthase activity, we searched within the The Institute for Genome Research human gene index data base (http://www.tigr.org/) (32) for human ESTs that encode a similar protein to the yeast PGS1 gene product (yPGS1) (15). Two human ESTs (The Institute for Genome Research gene identification number, THC122139 and T12593) were found to encode peptides that exhibited 41.6% identity with yPGS1 in a stretch of 178 amino acids and 43.4% identity in a stretch of 79 amino acids, respectively. A cDNA fragment that has similarity to yPGS1 in sequence was amplified from a CHO cDNA library using polymerase chain reaction with primers corresponding to the sequence of human ESTs. In order to isolate a full-length cDNA clone, an oligonucleotide derived from the sequence of the amplified CHO cDNA fragment was used for screening against a CHO cDNA library as described under "Experimental Procedures." Eight positive plasmid clones were identified that have cDNA inserts of approximately 2.2 kb. Two of the clones, named pSPORT1-cPGS1 and pSPORT1-cPGS1', were used for further analysis, and the gene corresponding to the isolated CHO cDNA was named PGS1. The CHO PGS1 cDNA contained a large ORF encoding a protein of 553 amino acid residues with a calculated molecular mass of 62,329 Da (Fig. 2). Fifteen bases upstream of the poly(A) tail, there is a sequence (TATAAA) similar to the consensus poly(A) attachment signal (AATAAA) that is also functional as a poly(A) attachment signal (33). Because the size of PGS1 mRNA was estimated at about 2.4 kb by Northern blot analysis (see below) and poly(A) length is usually from 150 to 200 bases (34), the 2.2-kb CHO PGS1 cDNA lacking the poly(A) tail appears to be the complete cDNA.

The amino acid sequence of cPGS1 deduced from the cDNA sequence exhibited 30% amino acid sequence identity with yPGS1, and the identical amino acids were scattered throughout the sequence (Fig. 3A). In Fig. 3A, the underlined sequence indicates a region of high homology (79%) between cPGS1 and yPGS1. Within this region is the HKD motif (HXKXXXXDXXXXXXG). This motif (Fig. 3B) is found in several hydrolases and phosphotransferases (35) involved in phospholipid metabolism such as E. coli phosphatidylserine synthase, bacterial CL synthases, eukaryotic phospholipase D, and yPGS1. The presence of the HKD motif is consistent with the CHO PGS1 gene product encoding a PGP synthase (see below).


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Fig. 3.   Comparison of amino acid sequence of cPGS1 with yPGS1 and other phospholipid metabolizing enzymes. A, the amino acid sequence of cPGS1 is aligned with that of yPGS1. Identical amino acids are indicated with an asterisk (*). The highly conserved region listed in B as a phospholipid synthesis and hydrolysis consensus motif is underlined under the asterisks. B, regions of similarity in the amino acid sequences of cPGS1, yPGS1 (15), E. coli PS synthase (ecPSS) (36), yeast CL synthase (yCLS) (12), Bacillus firmus CL synthase (bfCLS) (37), human phospholipase D1 (hPLD1) (38), and yeast phospholipase D (yPLD) (SPO14 gene product) (39). The amino acids identical with cPGS1 are boxed.

Expression of PGP Synthase Activity by CHO PGS1 cDNA-- To examine whether the CHO PGS1 gene encodes PGP synthase activity, the CHO PGS1 cDNA was cloned into mammalian expression vector, pSV-SPORT1, and the resultant plasmid, designated as pSV-cPGS1, and pSV-SPORT1 were introduced into CHO-K1 cells. The cell extract derived from the transient transfectant with pSV-cPGS1 exhibited a 15-fold higher specific activity (3550 pmol/min/mg of protein) of PGP synthase than that from CHO-K1 cells transfected with pSV-SPORT1 (230 pmol/min/mg of protein); the average of duplicate assays varied by less than 10%. This result suggested that the CHO PGS1 gene encodes PGP synthase activity.

To obtain further evidence that CHO PGS1 encodes PGP synthase, E. coli strain XL1-Blue was transformed with pSPORT1-cPGS1'. The resultant transformant was cultured, and production of recombinant cPGS1 was induced by the addition of isopropyl beta -D-thiogalactoside. PGP synthase activity in cell extracts in which recombinant protein production was induced (781 pmol/min/mg of protein) was 90-fold higher than that of cell extracts in which recombinant protein production was not induced (8.68 pmol/min/mg of protein); the average of duplicate assays varied by less than 10%. This result provides definitive evidence that CHO PGS1 encodes cPGS1.

Restoration of PG and CL Biosynthesis in Mutant PGS-S Transfected with the CHO PGS1 cDNA-- PGS-S is a temperature-sensitive CHO mutant strain in which PGP synthase activity is 1% of that in wild type CHO-K1 at the restrictive temperature of 40 °C (Ref. 23 and Table I). The mutant was isolated by introducing a second mutation into mutant PGS-P, in which PGP synthase activity is 50% of that in the parental strain CHO-K1 (23). The biosynthetic rate and content of PG in mutant PGS-S are both reduced to approximately 10% or less of that in CHO-K1. The biosynthetic rate and content of CL in mutant PGS-S are also reduced (23). To examine whether the CHO PGS1 cDNA complements the defect in PG and CL biosynthesis of mutant PGS-S, the CHO PGS1 cDNA was cloned into pSV-OKneo, a mammalian expression vector with a G418-resistant determinant, and introduced into PGS-S cells. We analyzed five independent stable G418-resistant transformant cells for PGP synthase activity and found that PGP synthase activity of all transformants analyzed was higher than that of CHO-K1 cells. This result demonstrated that the CHO PGS1 cDNA complements the defect in PGP synthase activity in mutant PGS-S. A stable G418-resistant transformant, designated PGS-S/cPGS1, was purified and used for further characterization. The cell extract of transformant PGS-S/cPGS1 cultivated at 40 °C exhibited a 620-fold and 7.3-fold higher PGP synthase specific activity than that of mutant PGS-S and wild type CHO-K1, respectively (Table I).

                              
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Table I
PGP synthase activity of stable transformant PGS-S/cPGS1 cells
Mutant PGS-S/cPGS1 cells with a temperature sensitive defect in PGP synthase were transfected with pSVneo-cPGS1, and a stable neo-resistant transformant (PGS-S/cPGS1) was isolated. Cells were grown at 40 °C for 4 days, and the cell extracts were assayed for PGP synthase activity. The data shown are the mean value ± S.D. of triplicate assays.

To investigate the rate of PG biosynthesis in transformant PGS-S/cPGS1, cells were labeled with 32Pi for 2, 4, and 8 h at 40 °C, and the metabolically labeled lipids were analyzed. As shown in Fig. 4, PG biosynthetic rate in PGS-S/cPGS1 cells was approximately 13-fold and approximately 2-fold higher than that in PGS-S and CHO-K1 cells, respectively. The rate of CL biosynthesis in transformant PGS-S/cPGS1 was also higher than that in mutant PGS-S and similar to that in CHO-K1. Phospholipid composition of transformant PGS-S/cPGS1 was determined by long term labeling of intact cells with 32Pi. When the cells were cultivated with 32Pi for 4 days at 40 °C, the PG level in transformant PGS-S/cPGS1 was more than 30-fold higher than that in mutant PGS-S and about 2.5-fold higher than that in wild type CHO-K1. The CL level in transformant PGS-S/cPGS1 was 2.3-fold higher than that in mutant PGS-S cells and similar to that in CHO-K1 (Table II). Under our experimental conditions, the radioactivity incorporated into phospholipid per cell was virtually identical among the transformant, the mutant, and the wild type cells (data not shown). These results indicated that the expression of CHO PGS1 cDNA complements the defect of PG and CL biosynthesis in mutant PGS-S.


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Fig. 4.   Incorporation of 32Pi into PG and CL by transformant PGS-S/cPGS1 cells. Transformant PGS-S/cPGS1 (bullet ), CHO-K1 (open circle ), and PGS-S (black-square) cells were seeded in 6-cm diameter dishes and cultured for 2 days at 40 °C to subconfluence. At time 0, cells were metabolically labeled with 32Pi (2.5 µCi/ml) as described previously. At the time indicated, one dish of each strain was removed, and cellular phospholipids were extracted and separated as described under "Experimental Procedures." The radioactivity of each phospholipid or total phospholipids was determined using a FUJIX BAS2000 IMAGER (Fuji Film). The number of cells at time 0 was determined as described in the legend to Fig. 7 and used to standardize the results.

                              
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Table II
Phospholipid composition of transformant PGS-S/cPGS1
Cells were cultured in the medium containing 32Pi (1 µCi/ml) for 4 days at 40 °C. The cellular phospholipids were extracted and analyzed as described under "Experimental Procedures." Data shown are the mean ± S.D. for three independent experiments.

Restoration of Normal Mitochondrial Morphology in Transformant PGS-S/cPGS1-- When mutant PGS-S is cultivated under conditions where the content of PG and CL is reduced, its mitochondria are enlarged and swollen, and the electron density of the mitochondrial matrix is reduced (Ref. 24 and Fig. 5). To examine whether the expression of the CHO PGS1 cDNA in mutant PGS-S corrects the abnormal mitochondrial morphology in the mutant cells, the structure of mitochondria in transformant PGS-S/cPGS1 was examined by electron microscopy (Fig. 5) and found to be normal with respect to both size and matrix electron density. This result indicated that the correction of the low level of PGP synthase activity in mutant PGS-S is sufficient to correct the abnormal mitochondrial morphology in mutant PGS-S.


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Fig. 5.   Electron micrographs of transformant PGS-S/cPGS1 cells. CHO-K1 (A), PGS-S (B), and PGS-S/cPGS1 (C) cells grown at 40 °C for 4 days were analyzed by electron microscopy as described previously (24). All micrographs are the same magnification.

Complementation of the Defect in Growth of Mutant PGS-S by Expression of CHO PGS1-- As previously demonstrated, the growth rate of mutant PGS-S is reduced under conditions in which the content of PG and CL is reduced (23). To examine whether expression of the CHO PGS1 cDNA complements the growth phenotype of mutant PGS-S, the growth of PGS-S/cPGS1 cells was compared with that of CHO-K1 and mutant PGS-S cells. As shown in Fig. 6, clonal growth from single cells of transformant PGS-S/cPGS1 was faster than that of mutant PGS-S. The colony size of transformant PGS-S/cPGS1 was almost the same as or a little smaller than that of CHO-K1.


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Fig. 6.   Colony formation of transformant PGS-S/cPGS1 cells. CHO-K1, PGS-S, and PGS-S/cPGS1 cells were seeded at 1000 cells/10-cm diameter dish containing 10 ml of cell culture medium and cultivated for 8 days at 40 °C. Colonies were stained with 0.05% Coomassie Blue G in methanol/water/acetic acid (45:45:10, v/v).

We previously showed that mutant PGS-S is defective in mitochondrial oxidative phosphorylation (24). Probably due to this defect, PGS-S cells are incapable of growth in a medium containing galactose instead of glucose because the majority of cellular energy appears to be provided by oxidative phosphorylation under conditions of glucose deprivation (40). In contrast to the mutant, PGS-S/cPGS1 cells are capable of growth in the glucose-depleted medium (Fig. 7). In this medium, the cell growth rate of transformant PGS-S/cPGS1 was slower than that of wild type CHO-K1 but almost the same as that of mutant PGS-P. The latter mutant was initially generated by a first-step mutagenesis of CHO-K1 and was subsequently used as the parent strain for the second-step mutagenesis to produce mutant PGS-S. This result indicates that expression of the CHO PGS1 cDNA complements the defect in mitochondrial oxidative phosphorylation of mutant PGS-S that results in the inability to grow on galactose and that mutants PGS-S and PGS-P contain additional mutation(s) unrelated to PG and CL biosynthesis that affect growth.


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Fig. 7.   Cell growth of transformant PGS-S/cPGS1 cells in medium containing galactose or glucose. Cells were initially grown in standard growth medium at 33 °C. A, PGS-S/cPGS1 (bullet ), CHO-K1 (open circle ), PGS-P (), and PGS-S (black-square) were seeded at 5 × 103 cells/6-cm diameter dish and then grown at 40 °C in cell culture medium containing glucose (1.8 mg/ml). B, the above cells were seeded at 5 × 104 cells/dish and then grown at 40 °C in cell culture medium containing galactose (1.8 mg/ml) instead of glucose. Both cell culture media were supplemented with 10% (v/v) dialyzed newborn calf serum instead of nondialyzed serum. At the times indicated, cells were harvested, treated with trypsin, and counted with a Coulter model ZBI counter.

Expression of PGS1 mRNA in CHO Cells and Mouse Tissues-- Poly(A)+ RNAs from CHO K1 and mutant PGS-S were subjected to Northern blot analysis with CHO PGS1 cDNA as probe. As shown in Fig. 8A, a 2.4-kb PGS1-specific mRNA was detected in the RNAs from both strains. The relative intensity of 2.4-kb PGS1 mRNA in PGS-S cells was 88% of that in CHO-K1 cells after normalization by actin mRNA. This result suggests that the mutation in mutant PGS-S does not affect either PGS1 mRNA expression or stability.


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Fig. 8.   Northern blot analysis of PGS1 mRNA in CHO cells and in various tissues of mouse. 4 µg of poly(A)+ RNA isolated from CHO-K1 and PGS-S cells grown at 40 °C for 4 days (A) and mouse multiple tissue Northern blot filter containing about 2 µg/lane of poly(A)+ RNA isolated from various mouse tissues (B) were hybridized with 32P-labeled CHO PGS1 cDNA. The relative intensity (RI) of the 2.4-kb PGS1 mRNA band compared with total poly(A)+ RNA is indicated below each lane. The RI values were also normalized to the amount of 2.0-kb beta -actin mRNA present per lane. UD, undetectable. The size of bands was estimated by comparison with ribosomal RNA or RNA molecular weight standards with sizes indicated at the left of the figure.

Northern blot analysis of poly(A)+ RNAs isolated from various mouse tissues with CHO PGS1 as a probe revealed that a 2.4-kb mRNA, the putative mouse homologue of CHO PGS1 mRNA, exists in various mouse tissues (Fig. 8B). The highest expression level of PGS1 mRNA (normalized to actin mRNA) was found in testis, followed by liver and brain. The expression levels were low in spleen and undetectable in kidney. Intermediate levels were found in skeletal muscle, heart, and lung. This result indicates that a CHO PGS1 homologue is responsible for PGP synthase activity in various mouse tissues.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Initial characterization of a PGP synthase-defective mutant PGS-S suggested that PG and/or CL formation is critical for mitochondrial function and morphology (23, 24). Unresolved was whether the defect in this mutant was in the gene encoding PGP synthase and whether the phenotype displayed could be attributed directly to the reduced levels of PG and CL. In the current study, we report the isolation of a cDNA encoding PGP synthase from CHO cells initially based on the assumption that CHO PGP synthase is similar in sequence to yPGS1. The CHO PGS1 cDNA encodes a putative protein showing a high (30%) amino acid sequence identity with the yPGS1. The calculated molecular mass (62,329 Da) of the putative cPGS1 deduced from the cDNA sequence is similar to the relative molecular mass (62 kDa) of PGP synthase purified from CHO-K1 cells.2 cPGS1 contains a HKD motif that has been found in a family of lipid hydrolases and phosphotransferases. Transient transfection of CHO-K1 cells with the CHO PGS1 cDNA and production of a recombinant cPGS1 in E. coli cells resulted in highly elevated PGP synthase activity. Moreover, when the CHO PGS1 cDNA was introduced into mutant PGS-S, the mutant recovered normal biosynthesis and cellular content of PG and CL. These results definitively establish that CHO PGS1 cDNA encodes PGP synthase.

The CHO PGS1 cDNA was able to complement the mitochondrial morphological defect, as well as the PG and CL biosynthetic defects, of mutant PGS-S. Furthermore, in contrast to PGS-S cells, the CHO PGS1-transfected PGS-S cells were capable of growth in a glucose-depleted medium, in which mitochondrial oxidative phosphorylation is considered to be critical for cell growth. These results demonstrate that cPGS1 is required for normal mitochondrial morphology and function in CHO cells and that the most dramatic phenotypes associated with mitochondrial morphology and cell growth are directly a result of the reduced ability to make normal levels of PG and/or CL. These results parallel in many ways the properties of pgs1-null mutants of yeast (15).

PGS1 mRNA level in mutant PGS-S is similar to that in CHO-K1 cells. Thus, the mutation in PGS-S appears not to affect the expression level of the PGS1 gene or the stability of PGS1 mRNA. Because introduction of CHO PGS1 cDNA into mutant PGS-S substantially increases PGP synthase activity, the defect in PGS-S also appears not to affect the stability of the wild type enzyme. Considering these results together with the fact that PGP synthase activity in PGS-S cell extract is sensitive to heat (23), it is highly unlikely that the defect in mutant PGS-S is in the regulation of PGS1 gene expression or the posttranscriptional modification of enzyme activity. Short of molecular cloning of PGS1 cDNA from mutant PGS-S and demonstrating a mutation affecting enzymatic activity, the results reported here strongly suggest that the mutation in PGS-S is in the structural gene for cPGS1.

Overexpression of CHO PGS1 cDNA in mutant PGS-S resulted in 2-fold higher PG biosynthetic rate and 2.5-fold higher cellular PG content than that of wild type CHO-K1 cells. However, CL biosynthetic rate and cellular CL content in the transformant did not increase compared with those of wild type cells. Therefore, the expression level of PGS1 may determine the rate of de novo PG synthesis, thereby establishing cellular PG content, but the level of CL appears to be under separate and independent control.

Northern blot analysis of mRNA isolated from mouse tissues revealed that a 2.4-kb PGS1-derived mRNA was highly expressed in testis. This result is consistent with our finding that the specific activity of PGP synthase in the crude mitochondria fraction from pig testis is 7.6-fold and 2.6-fold higher than that from pig heart and thymus, respectively.2 In rabbit sperm, PG content is relatively high, being 6.8% of total phospholipid (41). High level expression of PGP synthase mRNA may be required for sperm production in testis. On the other hand, the level of the PGS1-derived mRNA in lung, where PG content is high in lamellar bodies, is similar to that in other tissues such as skeletal muscle and liver, where PG content is not high. This result raises the possibility that posttranscriptional regulation of PGP synthase or another type of PGP synthase exists in lung.

The enzymes that use CDP-diacylglycerol as a substrate can be classified structurally into two groups. One has the HKD motif as shown in Fig. 3, and the other has a consensus motif, DXXDGXXARXXXXXXXXGXXLDXXXD (42), that is also found in E. coli PGP synthase, yeast phosphatidylinositol synthase, yeast phosphatidylserine synthase, and yeast CL synthase. This structural difference may reflect a difference in the reaction mechanism between these two groups of enzymes (43). The reaction mechanism of E. coli phosphatidylserine synthase (HKD motif), which shares homology with both yPGS1 and cPGS1, has been proposed to proceed via a ping-pong reaction mechanism involving a phosphatidyl-enzyme intermediate. On the other hand, yeast phosphatidylserine synthase (alternate motif), which shares homologous with the E. coli PGP synthase, appears to proceed by a single displacement sequential Bi-Bi reaction mechanism (43). Based on these sequence homologies, the eukaryotic PGP synthases most likely utilize a ping-pong reaction mechanism, in contrast to the highly homologous prokaryotic PGP synthases (44) that employ a Bi-Bi reaction mechanism. Because the PGP synthase is an essential enzyme in bacteria (45), this difference in reaction mechanism between eukaryotic and prokaryotic PGP synthases might represent a target for antibacterial agents.

    FOOTNOTES

* These studies were supported in part by the Social Insurance Agency Contract Fund of the Japan Health Science Foundation (to K. K.), by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (to K. K. and M. N.), by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan (to M. N.), and by the United States National Institutes of General Medical Sciences Grants GM56389 and GM20478 (to W. D.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB016930.

To whom requests for biological material should be sent. Tel.: 011-81-3-5285-1111; Fax: 011-81-3-5285-1157; E-mail: nishim{at}nih.go.jp.

parallel To whom correspondence and reprint requests should be addressed. Tel.: 713-500-6051; Fax: 713-500-0652; E-mail: wdowhan{at}bmb.med.uth.tmc.edu.

The abbreviations used are: PG, phosphatidylglycerol; CL, cardiolipin; PGP, phosphatidylglycerophosphate; CHO, Chinese hamster ovary; EST, expressed sequence tag; kb, kilobase pairs(s); ORF, open reading frame; cPGS1, CHO PGS1 gene product; yPGS1, yeast PGS1 gene product.

2 Kiyoshi Kawasaki, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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