Cloning of a Human cDNA for CTP-Phosphoethanolamine Cytidylyltransferase by Complementation in Vivo of a Yeast Mutant*

(Received for publication, January 21, 1997)

Asae Nakashima Dagger , Kohei Hosaka § and Jun-ichi Nikawa Dagger

From the Dagger  Department of Biochemical Engineering and Science, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, Iizuka, Fukuoka 820 and the § Department of Biochemistry, Gunma University School of Medicine, Maebashi, Gunma 371, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

CTP-phosphoethanolamine cytidylyltransferase (ET) is the enzyme that catalyzes the formation of CDP-ethanolamine in the phosphatidylethanolamine biosynthetic pathway from ethanolamine. We constructed a Saccharomyces cerevisiae mutant of which the ECT1 gene, putatively encoding ET, was disrupted. This mutant showed a growth defect on ethanolamine-containing medium and a decrease of ET activity. A cDNA clone was isolated from a human glioblastoma cDNA expression library by complementation of the yeast mutant. Introduction of this cDNA into the yeast mutant clearly restored the formation of CDP-ethanolamine and phosphatidylethanolamine in cells. ET activity in transformants was higher than that in wild-type cells. The deduced protein sequence exhibited homology with the yeast, rat, and human CTP-phosphocholine cytidylyltransferases, as well as yeast ET. The cDNA gene product was expressed as a fusion with glutathione S-transferase in Escherichia coli and shown to have ET activity. These results clearly indicate that the cDNA obtained here encodes human ET.


INTRODUCTION

Phosphatidylethanolamine (PtdEtn)1 is a major membrane constituent of both prokaryotic and eukaryotic cells. In mammalian cells, this phospholipid can be synthesized de novo via the CDP-ethanolamine pathway, by decarboxylation of phosphatidylserine, and by the calcium-dependent exchange of ethanolamine with the base moiety of pre-existing phospholipids (1). Although the exact contributions of each of these pathways remain to be established, the CDP-ethanolamine pathway probably plays an important role in the synthesis of PtdEtn (2). CTP-phosphoethanolamine cytidylyltransferase (ET; EC 2.7.7.14) catalyzes the synthesis of CDP-ethanolamine from CTP and phosphoethanolamine, and is considered to be an important regulatory enzyme in the CDP-ethanolamine pathway through analogy with the CDP-choline pathway. However, little is known about the regulation of its activity.

CTP-phosphocholine cytidylyltransferase (CT; EC 2.7.7.15) is a key regulatory enzyme in the CDP-choline pathway. The properties of CT have been extensively studied, and the role of CT in the regulation of phosphatidylcholine (PtdCho) biosynthesis has been well established (3). The sequences have been determined for several mammalian CT cDNAs (4-7) and the gene from Saccharomyces cerevisiae (8). CT exists in an inactive soluble form and an active membrane-bound form. Regulation of the translocation process by changes in the lipid composition of cellular membranes seems to be one of the major mechanisms for the control of CT activity. Phosphorylation and dephosphorylation of the enzyme are also correlated to the translocation of CT to membranes; the soluble form is highly phosphorylated, and translocation of CT to membranes is accompanied by extensive dephosphorylation. Multiple phosphorylation sites at the C terminus have been identified (9, 10). CT is thought to have a bipartite structure composed of a globular N-terminal catalytic domain and an extended C-terminal domain. Between the catalytic domain and the phosphorylation region is a sequence that is predicted to form amphipathic alpha -helices and to interact with the membrane bilayer of activating phospholipids (11, 12).

Unlike for CT, the protein structure and regulatory properties of ET have been studied much less extensively. ET has been purified 1,000-fold from a post-microsomal fraction of rat liver by Sundler (13). van Golde et al. recently purified ET to homogeneity from rat liver (14). The purified protein has a molecular weight of about 50,000 and appears to be a dimer. ET has long been tacitly assumed to be regulated like CT. However, there is accumulating evidence that the control mechanisms for the ET and CT activities are different (15).

Only sequence information for the yeast S. cerevisiae gene putatively encoding ET is available. Yeast cells possess phospholipid-synthetic pathways quite similar to those found in other eukaryotic cells. Yeast gene ECT1,2 originally known as MUQ1 (16), encodes a protein similar to CTs of yeast and other species and, therefore, is thought to encode ET. Here, we report the isolation and characterization of a human cDNA encoding ET by genetic complementation of a yeast mutant defective in ECT1.


EXPERIMENTAL PROCEDURES

Strain, Media, and Growth Conditions

Saccharomyces cerevisiae DS15-3 (MATalpha ise leu2 his3) was used as the wild-type strain and as the recipient for ECT1 disruption. DS15-3 is a segregant of the diploid strain, DS15 (17). The ise mutant is a conditional ethanolamine auxotroph whose growth is inhibited by high concentrations of inositol, and the growth defect is suppressed by the addition of ethanolamine to the culture medium (18). Yeast cells were cultured in either YPD or synthetic minimal medium at 30 °C. The compositions of the YPD and inositol-free minimal media were reported previously (18). The composition of nitrogen-free minimal medium (M-N) is given in Ref. 19. Inositol, L-leucine and L-histidine were each added to the culture media at the concentration of 20 µg/ml. Ethanolamine was added to the culture media at the concentration of 10 µg/ml.

Amplification of ECT1

The yeast ECT1 gene was amplified from chromosomal DNA by the polymerase chain reaction (PCR). The PCR primers used were 5'-TCTAGATGACGGTAAACTTAGATC-3' (forward primer) and 5'-ATGTAATACCAGTTTTCTTA-3' (reverse primer). The complementary oligonucleotides contained additional sequences (underlined), so there was a BamHI restriction site at the 5' end and a SalI site at the 3' end of the PCR products. An about 1-kbp PCR product was digested with BamHI and SalI, purified by gel electrophoresis, and then inserted between the BamHI and SalI sites of pUC18 to yield pUC-ECT. By comparing the restriction maps of the isolated clone and the reported one, and partial sequencing of the cloned fragment, plasmid pUC-ECT was confirmed to contain the yeast ECT1 gene.

Construction of an ECT1-disrupted Strain

The 0.4-kbp EcoRV/XhoI fragment of pUC-ECT located within the ECT1 coding frame was replaced with a 1.3-kbp HincII/XhoI fragment containing yeast HIS3 to yield pUC-ect::HIS3. The 1.9-kbp SphI/SalI fragment of this plasmid was used for the transformation of DS15-3. His+ colonies were selected. One disruptant strain, of which the gene disruption was confirmed by Southern blot analysis, was designated as NA9 and used for the isolation of human cDNA.

cDNA Cloning---A human cDNA library made from glioblastoma cells was constructed on a yeast multicopy vector, pADANS, which contains the yeast ADH1 promoter, the following small part of the coding region, and the terminator (20). Inserted cDNAs are designed to be expressed in yeast cells as a fusion protein with ADH1 N-terminal amino acids. NA9 was transformed with the cDNA library, and the colonies that grew on a minimal medium supplemented with inositol and ethanolamine were selected. Colonies that grew on a medium supplemented with inositol alone were excluded. Plasmids were recovered from the transformants and used for Escherichia coli transformation.

DNA Sequencing and Sequence Analysis

DNA was sequenced manually using the dideoxynucleotide chain termination method (21) with Klenow fragment (Takara Shuzo) and [32P]dCTP (Amersham Corp.) on the M13 phage vector. The primers were either universal or a series of oligonucleotides synthesized for the partial cDNA sequence. Amino acid sequence similarities were found using the BLAST program, and the translation of nucleotide sequences in the GenBank and EMBL data bases. Alignments were further improved by eye. Only amino acid identities were considered.

Transcript Analysis

An adult human multitissue Northern blot (Clontech) was hybridized with a random primed (Takara Shuzo) 32P-labeled entire cDNA fragment as a probe. Hybridization was carried out at 42 °C overnight in a hybridization solution containing 50% formamide, 50 mM sodium phosphate (pH 6.5), 5 × SSC (1 × SSC: 0.15 M NaCl, 15 mM sodium citrate, pH7.0), 1 × Denhardt's (0.1% each of bovine serum albumin, polyvinylpyrrolidone, and Ficoll), and 0.1% SDS. The filter was washed twice at room temperature with 2 × SSC containing 0.1% SDS for 10 min, and then twice with 0.1 × SSC containing 0.1% SDS at 50 °C for 15 min.

Metabolite Analysis of Radiolabeled Ethanolamine

Exponentially growing yeast cells were harvested by centrifugation, washed with M-N medium, and then resuspended in the same medium. To 1 ml of culture of each strain was added 7.4 kBq of [1,2-14C]ethanolamine and 0.4 µmol of ethanolamine, and then the mixture was incubated at 30 °C. At the indicated times, 5 ml of 10% trichloroacetic acid was added to the culture. The mixture was then filtered through GF/C glass-microfiber paper (Whatman). The filter paper was washed three times with 5 ml of 10% trichloroacetic acid and dried, and then radioactivity was counted after treatment with Scintilamine-OH (Dojindo) as described previously (21). The uptake of the labeled precursors into cells during the above incubation was determined by counting the radioactivity in cells collected by filtration through GF/C glass-microfiber paper as described previously (22).

For the lipid analysis, cells were cultured under the specified conditions, collected by centrifugation, and then suspended in M-N medium. To 2 ml of cell suspension was added [1,2-14C]ethanolamine or [methyl-14C]choline (37 kBq each). After the incubation at 30 °C for 60 min, cells were collected by centrifugation, suspended in 100 µl of methanol, and then sonicated. To the sonicated sample was added 200 µl of chloroform to extract the lipids. The extract was washed once with 300 µl of saline, twice with 300 µl of methanol-saline (1:1, v/v), and then evaporated. Lipids were dissolved in 50 µl of chloroform and chromatographed on a Silica Gel 60 plate (Merk) with chloroform-methanol-acetic acid-formic acid-water (35:15:6:2:1, by volume) as a developing solvent. Radioactive materials were located by autoradiography.

For the analysis of water-soluble metabolites, 1 ml of a culture of cells in M-N medium was incubated with 37 kBq of [1,2-14C]ethanolamine for 60 min, and then the cells were collected by centrifugation and suspended in 50 µl of 10% trichloroacetic acid. After standing on ice for 20 min, the insoluble materials were removed by centrifugation and the supernatant was subjected to analysis by thin-layer chromatography on a Silica Gel 60 plate with ethanol and 2% ammonia (1:1, v/v). Radioactive materials were located by autoradiography.

Construction of a GST Fusion Protein and Expression in E. coli

A SmaI/EcoRI fragment excised from the cDNA clone was ligated between the SmaI and EcoRI sites of pGEX-2T (Pharmacia Biotech Inc.). The plasmid, pGEX-hECT, thus obtained was used for expression in E. coli. Briefly, overnight cultures of E. coli JM103 harboring the pGEX-hECT plasmid were diluted in fresh medium and then grown for another 2 h at 37 °C. Protein expression was induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside (IPTG), and after a further 3 h of growth, the cells were pelleted and resuspended in 10 mM Tris-Cl buffer (pH 7.5) containing 1 mM EDTA and 20% glycerol. The cells were then lysed by sonication and centrifuged at 8,000 × g for 10 min. The supernatant was used as a crude cell extract for determination of the enzyme activity. A part of the IPTG-treated and untreated cells, respectively, was used for protein analysis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of total E. coli proteins was performed in 10% polyacrylamide mini-slab gels, essentially as described by Laemmli (23). Molecular weight references were purchased from Sigma. Proteins were stained with Coomassie Brilliant Blue using a Quick-CBB kit (Wako).

ET activity of fusion protein was determined in a reaction mixture comprising 20 mM Tris-Cl (pH 7.5), 10 mM MgCl2, 2 mM CTP, 5 mM dithiothreitol, [1,2-14C]phosphoethanolamine, and E. coli cell extract, in a total volume of 25 µl. After 30 min of incubation at 30 °C, the reaction was stopped by heating at 100 °C for 5 min. The reaction product was separated by thin-layer chromatography on a Silica Gel 60 plate as described above. Protein determination was carried out with BCA protein assay reagent (Pierce).

ET Activity

Yeast cells were cultured in a minimal medium on a Bio-Shaker BR-30 (Taitec) to the early stationary phase at 30 °C, harvested, and disrupted by vortexing vigorously with glass beads (diameter, 0.3 mm) in 10 mM Tris-Cl containing 1 mM EDTA and 20% glycerol for 5 min. Disrupted cells were centrifuged at 4,000 × g for 10 min, and the supernatant fraction was used for the determination of ET and CT activities as described previously (22) except that the total volume was reduced to 50 µl. All enzyme reactions were carried out at 25 °C for 10 min or 20 min.

Materials

Restriction endonucleases and other nucleic acid-modifying enzymes were purchased from Takara Shuzo. Taq DNA polymerase was obtained from Boehringer Mannheim. [alpha -32P]dCTP (110 TBq/mmol), [methyl-14C]choline (2.04 GBq/mmol), and [methyl-14C]phosphocholine (2.07 GBq/mmol) were obtained from Amersham. [1,2-14C]Ethanolamine (170 MBq/mmol) was from DuPont NEN. CTP was from Yamasa. Choline chloride, ethanolamine, phosphocholine, and phosphoethanolamine were from Tokyo Kasei. Other chemicals were from Wako Chemicals. [1,2-14C]Phosphoethanolamine was prepared from [1,2-14C]ethanolamine through the action of yeast choline kinase (24) and purified as described by Sundler (25).


RESULTS

Isolation of Human cDNAs by Complementation in Vivo of a Yeast Mutant

Based on the structural similarity, the S. cerevisiae ECT1 gene was thought to encode ET (16). To confirm this, we constructed an yeast strain of which the ECT1 gene was disrupted. Although mutants carrying the ect1 mutation alone could exhibit no growth phenotype, the combination of the ect1 and ise mutants allowed us to detect the phenotype of the ect1 mutation. The ise mutation is a conditional choline- or ethanolamine-auxotrophic mutant of which the growth is inhibited by high concentrations of inositol (18). As was expected, an ect1 ise double mutant, NA9, we constructed did not grow on inositol- and ethanolamine-containing media, although it grew normally on inositol- and choline-containing media or on inositol-free media. The mutant also showed the decreased levels of the synthesis of CDP-ethanolamine and PtdEtn as well as low levels of ET activities (see below). These results strongly indicate that the ECT1 encodes ET.

Using the ect1 ise mutant, we next attempted to clone the human cDNA for ET by the complementation method. Strain NA9 was transformed with a human cDNA expression library, and the colonies that grew on a minimal medium supplemented with inositol and ethanolamine but not on a medium supplemented with inositol alone were selected. Five clones were obtained. Based on their restriction maps, the obtained clones could be classified into two groups: four clones (pH1-pH4) and one clone (pS4a). Fig. 1 shows the growth phenotype of the yeast mutant transformed with the cloned plasmids. Plasmids pH4 and pS4a clearly suppress the growth defect of strain NA9 on inositol- and ethanolamine-containing medium, although the suppression by pH4 is slightly weaker. Partial sequence analysis revealed that plasmid pH4 contains the cDNA for human choline kinase, which has already been isolated (26). On the other hand, plasmid pS4a was found to have a new cDNA. We therefore sequenced the entire region of the new cDNA.


Fig. 1. Suppression of the growth defect of yeast cells by human cDNAs. ise ect1 double mutant NA9 was transformed with the indicated plasmids, and then its growth phenotype was examined on a minimal medium supplemented with or without inositol plus ethanolamine. Plasmid pADANS is the multicopy vector used for the construction of the human cDNA library.
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Nucleotide and Deduced Amino Acid Sequences

A restriction map of the cDNA insert of pS4a is shown in Fig. 2A. The nucleotide sequence of the cDNA was determined on both strands. The cDNA insert consisted of a total of 1,856 bases, with a single long open reading frame (ORF) starting with an ATG codon at position 67 and ending at position 1,236 (Fig. 2B). Although it was not known whether translation started at the first methionine shown in Fig. 2B or at a further upstream one, we predicted that the first methionine shown in Fig. 2B is the likely translation start site, as the sequence flanking it conforms to the consensus sequence for translation initiation proposed by Kozak (27), (A/G)XXATG(A/G)X(C/T). This was supported by sequence comparison with the yeast ET (see below). The ORF encoded a protein of 389 amino acids with a calculated molecular mass of 43.8 kDa. As was expected, the reading frame is in-frame with the ADH1 N-terminal region of the expression vector, pADANS. The consensus site for polyadenylation, 5'-AATAAA-3', is present 579 base pairs after a TAA termination codon.


Fig. 2. Nucleotide and deduced amino acid sequences of the human cDNA. A, restriction map of the cDNA in plasmid pS4a. The hatched box represents the coding region. The restriction sites are: E, EcoRI; H, HindIII; N, NotI; P, PstI; Pv, PvuII; S, SacI; Sm, SmaI; Sp, SphI. EcoRI, HindIII, and NotI are cloning sites of vector pADANS. B, the nucleotide sequence of the cDNA and the deduced amino acid sequence of its product. The largest ORF is translated into an amino acid sequence, which is shown below the nucleotide sequence in the one-letter code. Residue numbers for nucleotides and amino acids are shown on the right. The putative initiation codon is indicated by the underline. The asterisk denotes the termination codon. The double-underline indicates a putative polyadenylation signal.
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Similarity of the Deduced Amino Acid Sequence to Those of Known Cytidylyltransferases

The deduced amino acid sequence for the cDNA coding region was used to search a number of data bases provided by the National Institute of Genetics, Japan. The cDNA gene product was found to be similar to several cytidylyltransferases and found to be most similar (36% identity) to the S. cerevisiae ET (Fig. 3A). The homology extends across the entire lengths of both proteins. It is also noteworthy that both proteins have large internal repeated sequences (Fig. 3B). On the other hand, the sequence similarity between the cDNA gene product and CTs from several species, including from humans, was limited to their N-terminal halves. Limited sequence similarity was also observed at the C termini of human and yeast ET, and yeast CT, but not at the C terminus of human CT (Fig. 3C). These results strongly suggested that the cDNA obtained here is for human ET. We describe below biochemical evidence demonstrating that this is indeed the case.


Fig. 3. Comparison of the deduced amino acid sequences of human and yeast cytidylyltransferases. A, sequences of ET from S. cerevisiae (16), and CTs of S. cerevisiae (8) and humans (7) were aligned in descending order (yET, yCT, and hCT, respectively) with respect to the similarity to the predicted sequence of the human ET (hET). The bold letters show residues identical to the hET sequence. B, sequence alignment of the N-terminal and C-terminal halves of human and yeast ETs together with N-terminal halves of human and yeast CTs. Residues identical among more than four sequences are indicated by bold letters. C, schematic representation of the human and yeast ETs and CTs. The hatched boxes represent the sequences shown in B. The dotted boxes and closed boxes show the sequences of local similarities.
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Northern Analysis

Northern analysis was carried out with a variety of adult human tissues. For this study the entire 2-kbp human cDNA fragment from pS4a was used as a probe. As shown in Fig. 4, a major transcript of approximately 2 kilobases was detected among the RNAs from the several human tissues examined. The size of the transcript is consistent with that of the cDNA obtained. The transcript is very abundant in liver, heart, and skeletal muscle tissues.


Fig. 4. Tissue-specific expression of human ET mRNA revealed by Northern blot analysis. A, a human multitissue blot containing poly(A)+ RNA (Clontech) was probed with a radiolabeled cDNA probe and then autoradiographed. The positions of RNA size markers are indicated on the left. B, the same blot was rehybridized with a manufacturer-supplied beta -actin DNA as a probe.
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Lipid Synthesis via the CDP-ethanolamine Pathway

To confirm that plasmid pS4a contains the cDNA for human ET, we examined whether or not the cDNA could restore the activity of the CDP-ethanolamine pathway in the yeast strain. The flux of ethanolamine through the ET step was analyzed by determining the incorporation of [14C]ethanolamine into PtdEtn. The overall rate of PtdEtn synthesis was determined by measuring the radioactivity in lipids. As shown in Fig. 5, the incorporation of radioactivity in the ect1 mutant was very low. On the other hand, the incorporation of radioactivity into lipids in the mutant strain harboring plasmid pS4a had recovered to 81% (at 60 min) of that in the wild type. It was also shown that the introduction of the human cDNA for choline kinase (plasmid pH4) restored the incorporation of radiolabeled ethanolamine into lipids, although the extent was slightly low (43% of that in the wild type at 60 min).


Fig. 5. Incorporation of [14C]ethanolamine into PtdEtn in the wild-type strain, the ect1 mutant NA9, and the mutants harboring human cDNAs. To 1 ml of a culture of each strain was added 7.4 kBq of [14C]ethanolamine, and then the mixture was incubated at 30 °C. At the indicated times, the incorporation of radioactivity into lipids was determined as described under "Experimental Procedures." A550 refers to the absorbance at 550 nm. square , wild type with pADANS; black-square, mutant with pADANS; open circle , mutant with pH4; bullet , mutant with pS4a. The total radioactivity taken up into cells at 60 min was 3.3 × 104, 3.3 × 104, 5.0 × 104, and 3.5 × 104 dpm, for wild type with pADANS, mutant with pADANS, mutant with pH4, and mutant with pS4a, respectively.
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We next analyzed the radioactive lipids derived from 14C-labeled ethanolamine and choline. Cells were cultured with labeled compounds for 60 min and lipids were extracted and analyzed by thin-layer chromatography. As shown in Fig. 6, the amount of labeled PtdEtn and PtdCho derived from [14C]ethanolamine was very low in the mutant cells (lane 5) when compared with those in wild-type cells (lane 4). Introduction of human cDNA into the yeast mutant clearly restored the synthesis of PtdEtn and PtdCho from [14C]ethanolamine (lane 6). In contrast, there were no significant differences in the amount of labeled PtdCho derived from [14C]choline between these strains (lanes 1-3).


Fig. 6. Analysis of radiolabeled PtdEtn and PtdCho. Yeast cells were incubated with 37 kBq of [14C]choline (lanes 1-3) or 37 kBq of [14C]ethanolamine (lanes 4-6) at 30 °C for 60 min. Lipids were extracted and analyzed by thin-layer chromatography as described under "Experimental Procedures." Lanes 1 and 4, wild type; lanes 2 and 5, mutant with pADANS; lanes 3 and 6, mutant with pS4a.
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We next examined the incorporation of [14C]ethanolamine into the intermediates of the CDP-ethanolamine pathway. The ect1 mutant harboring vector plasmid pADANS and plasmid pS4a, as well as the wild-type strain harboring pADANS were grown at 30 °C in the presence of [14C]ethanolamine for 60 min. Trichloroacetic acid-soluble materials were analyzed by thin-layer chromatography. As shown in Fig. 7, mutant cells accumulated much higher levels of radioactive phosphoethanolamine than wild-type cells. Conversely, the level of radioactive CDP-ethanolamine was very low in the mutant. Introduction of the human cDNA into the yeast mutant clearly restored the level of CDP-ethanolamine. These results strongly indicate that the cDNA product has ET activity. Further evidence supporting this conclusion was obtained by assaying the ET activity of the cDNA product.


Fig. 7. Analysis of the intermediates of the CDP-ethanolamine pathway. Yeast cells were incubated at 30 °C with 37 kBq of [14C]ethanolamine for 60 min. Radiolabeled water-soluble metabolites were analyzed by thin-layer chromatography as described under "Experimental Procedures." Lane 1, mutant with pS4a; lane 2, mutant with pADANS; lane 3, wild type with pADANS.
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ET and CT Activities

ET and CT activities in the transformant were determined and compared with those in the mutant and wild-type cells. As shown in Table I, ect1 mutant strain, NA9 having a vector alone, had a very low ET activity, when compared with wild-type strain, DS15-3. On the other hand, the introduction of human cDNA on a multicopy vector into the mutant cell clearly restored the ET activity. The activity was higher than that in wild-type cells. This might be the results of gene dosage effect. CT activity was measured as a control, and we found no significant differences in the enzyme activities between these strains.

Table I.

ET and CT activities in wild-type, mutant, and transformant cells


Strain Plasmid Specific activitya
ET CT

nmol/min/mg protein
DS15-3 None 1.13  ± 0.26 0.87  ± 0.35
NA9 pADANS 0.07  ± 0.05 1.38  ± 0.03
NA9 pS4a 2.68  ± 0.62 1.65  ± 0.40

a Specific activity data are the averages ± standard deviations from four independent experiments.

Expression of Human ET in E. coli

To determine the ET activity of the cDNA product, we expressed its protein product fused with GST in E. coli. A SmaI/EcoRI cDNA fragment was subcloned between the SmaI and EcoRI sites of expression vector pGEX-2T. The plasmid, pGEX-hECT, thus obtained resulted in an in-frame fusion between the N-terminal GST coding region and codons 18-390 of the ET coding region (including the stop codon). The construct was transformed into E. coli, and expression of the fusion protein was induced with IPTG. The production of the fusion protein was analyzed by SDS-PAGE. The IPTG-dependent induction of a large amount of the fusion protein was detected (data not shown). The molecular mass of the fusion protein appeared to be about 65 kDa, this being consistent with the calculated value.

We next determined the ET activity using [14C]phosphoethanolamine as the substrate and an E. coli cell extract containing the fusion protein as the enzyme source. Reaction products were analyzed by thin-layer chromatography and located by autoradiography. As shown in Fig. 8, the extract prepared from the pGEX-hECT-harboring E. coli cells cultured in the presence of IPTG contained significant ET activity (lane 2). The activity was dependent on CTP (lane 3) and Mg2+ (lane 4). When a boiled extract or an extract prepared from vector-harboring cells was used as the enzyme source, no activity was detected (lanes 6 and 1, respectively). These results clearly show that the cDNA obtained here is of the structural gene for human ET.


Fig. 8. ET activity of the GST-fusion protein expressed in E. coli. Reaction mixtures containing the indicated substrates were incubated at 30 °C for 30 min, and then the products were separated by thin-layer chromatography as described under "Experimental Procedures." The cell extracts were prepared from E. coli cells harboring pGEX-hECT or vector pGEX-2T, and used for the determination of ET activity as the enzyme source (5.0 µg each). a, the cell extracts were boiled for 5 min and used.
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DISCUSSION

We describe here the first isolation and characterization of the cDNA for ET from mammalian cells. The size of the mRNA for ET, as revealed by Northern blot analysis, was almost the same as that of the cDNA. The transcript is very abundant in liver, heart, and skeletal muscle tissues. The high level of expression in liver cells agrees with the fact that liver cells have high ET activity (2). The deduced polypeptide encoded by the cDNA consists of 389 amino acids with a molecular mass of about 43.8 kDa. This value is slightly less than that of ET purified from rat liver, i.e. approximately 49.5 kDa (11, 12).

The predicted amino acid sequence of the putative encoded protein shows a high degree of similarity to the yeast ET throughout the entire region. The putative human protein, however, is longer than the yeast protein in both the N- and C-terminal regions, especially at the C-terminal region. The two proteins are functionally similar. Indeed, the introduction of the human cDNA, even in a single copy, restored the growth defect of the yeast ect1 mutant (data not shown). An additional striking structural feature is that human and yeast ETs have a large repetitive sequence in their N-terminal and C-terminal halves, suggesting that the two halves are generated through a gene duplication event. The regions of greatest identity in the two halves of ETs are also homologous to the sequences in the N-terminal half of CTs. Based on the high degree of homology between the N-terminal domain of rat CT, a similarly located region in yeast CT, and a Bacillus subtilis glycerolphosphate cytidylyltransferase (4, 8, 28), it has been proposed that this region bears the active site of these cytidylyltransferases. This was confirmed by that the catalytic activity of a truncated form of rat CT, comprising residues 1-236, was quite similar to that of the wild-type CT (29). Therefore, human and yeast ETs might have two catalytic domains, and this might reflect the substrate specificity of the enzyme. Further studies involving structural and functional analyses of ETs are required.

In a previous study, we cloned the human cDNA for choline kinase from the same library as used here by complementing the yeast cki1 mutation (26). Choline kinase catalyzes the formation of phosphoethanolamine, which is one of the substrates for ET. As shown in Fig. 1, we again obtained the human choline kinase cDNA as a suppressor for the yeast ect1 mutation. It was also shown that the human choline kinase cDNA restored the defect of PtdEtn synthesis via CDP-ethanolamine when introduced into the yeast ect1 mutant (Fig. 5). These results strongly suggest that the introduction of multicopies of the choline kinase gene might result in overproduction of phosphoethanolamine, and the accumulation of phosphoethanolamine could bypass the defect of ect1 through the action of CT. Indeed, this is the case because we found that multicopies of the yeast CKI1 or CCT1 gene can also suppress the growth defect of the ect1 mutant (data not shown). Although phosphoethanolamine is a poor substrate for CT (22, 30), elevation of the enzyme activity of CT on introducing multicopies of CCT1 into cells could compensate for the defect of ET activity.

Based on the analogy between the CDP-choline and CDP-ethanolamine pathways, it has long been thought that ET would be regulated in a similar manner to CT. However, more recent studies summarized by Tijburg et al. (15) strongly suggest that, at least in liver, the CDP-choline and CDP-ethanolamine routes are subject to independent regulation. Further work on thorough comparison of the two cytidylyltransferases and the significance of structural differences that could be relevant for independent regulation of these enzymes are required. The cDNA and the information on the primary structure for ET obtained here will allow us to develop methods for studying the structural and functional relationship between ET and CT, as well as the regulatory mechanism for ET.


FOOTNOTES

*   This research was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D84307[GenBank].


   To whom correspondence should be addressed. Tel.: 81-948-29-7822; Fax: 81-948-29-7801; E-mail: nikawa{at}bse.kyutech.ac.jp.
1   The abbreviations used are: PtdEtn, phosphatidylethanolamine; CT, CTP-phosphocholine cytidylyltransferase; ET, CTP-phosphoethanolamine cytidylyltransferase; kbp, kilobase pair(s); GST, glutathione S-transferase; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PtdCho, phosphatidylcholine.
2   During the preparation of our manuscript, the characterization of yeast ECT1 gene, which encodes ET, was reported by Min-Seok et al. (31) and shown to be identical to MUQ1 (accession number GenBank D50644[GenBank]). In this paper, we use the name ECT1, which better describes its function.

ACKNOWLEDGEMENT

We thank M. Wigler (Cold Spring Harbor Laboratory) for the cDNA library.


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