From the Department of Food Science, Cook College,
New Jersey Agricultural Experiment Station, Rutgers University,
New Brunswick, New Jersey 08901 and the § Lord and
Taylor Laboratory for Lung Biochemistry and the Anna Perahia Adatto
Clinical Research Center, National Jewish Center for Immunology and
Respiratory Medicine, Denver, Colorado 80206
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ABSTRACT |
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Ethanolamine kinase (ATP:ethanolamine
O-phosphotransferase, EC 2.7.1.82) catalyzes the committed
step of phosphatidylethanolamine synthesis via the CDP-ethanolamine
pathway. The gene encoding ethanolamine kinase
(EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline kinase, which
also exhibits ethanolamine kinase activity. The EKI1 gene
was isolated and used to construct eki1 The major membrane phospholipids in the yeast Saccharomyces
cerevisiae are PC,1 PE,
PI, and PS (1, 2). In addition to being major structural components of
cellular membranes, they play important roles in various cellular
processes including signal transduction (1, 2). Nearly all of the genes
encoding the enzymes responsible for the synthesis of these
phospholipids have been isolated and characterized, and many of the
enzymes have been purified and studied (1, 2). The pathways for the
synthesis of the major phospholipids in S. cerevisiae are
shown in Fig. 1. Two alternative pathways, the CDP-DAG pathway and the CDP-choline pathway (Fig. 1),
synthesize PC, the most abundant membrane phospholipid. Wild-type cells
primarily use the CDP-DAG pathway when they are grown in the absence of
choline (1, 2). The CDP-choline pathway becomes essential for PC
synthesis when the enzymes in the CDP-DAG pathway are defective (1, 2).
Mutants defective in the synthesis of PS (3, 4), PE (5, 6), or PC
(7-10) are auxotrophic for choline. The choline is transported into
these mutant cells and used to synthesize PC via the CDP-choline
pathway. Mutants defective in the synthesis of PS (3, 4) and PE (5, 6) are also auxotrophic for ethanolamine. The ethanolamine is transported into these cells and used to synthesize PE via the CDP-ethanolamine pathway. PE is subsequently methylated to form PC via the CDP-DAG pathway (Fig. 1).
and
eki1
cki1
mutants. A multicopy plasmid
containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1
mutant,
cki1
mutant, and eki1
cki1
double mutant cells. The heterologous expression of the S. cerevisiae EKI1 gene in Sf-9 insect cells resulted in a
165,500-fold overexpression of ethanolamine kinase activity relative to
control insect cells. The EKI1 gene product also exhibited
choline kinase activity. Biochemical analyses of the enzyme expressed
in insect cells, in eki1
mutants, and in cki1
mutants indicated that ethanolamine was the
preferred substrate. The eki1
mutant did not exhibit a
growth phenotype. Biochemical analyses of eki1
,
cki1
, and eki1
cki1
mutants showed that the EKI1 and CKI1 gene
products encoded all of the ethanolamine kinase and choline kinase
activities in S. cerevisiae. In vivo labeling experiments
showed that the EKI1 and CKI1 gene products had overlapping
functions with respect to phospholipid synthesis. Whereas the
EKI1 gene product was primarily responsible for
phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway,
the CKI1 gene product was primarily responsible for
phosphatidylcholine synthesis via the CDP-choline pathway. Unlike
cki1
mutants, eki1
mutants did not
suppress the essential function of Sec14p.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Pathways for the synthesis of the major
phospholipids in S. cerevisiae. The pathways
shown for the synthesis of phospholipids include the relevant steps
discussed in the text. The CDP-ethanolamine, CDP-choline, and CDP-DAG
pathways are indicated. The EKI1-encoded ethanolamine kinase
and CKI1-encoded choline kinase reactions are indicated in
the figure. A more comprehensive description that includes additional
steps in these pathways may be found in Refs. 1 and 2. The
abbreviations used are: Etn, ethanolamine; P-Etn,
phosphoethanolamine; CDP-Etn, CDP-ethanolamine;
Cho, choline; P-Cho, phosphocholine;
CDP-cho, CDP-choline; PME,
phosphatidylmonomethylethanolamine; PDE,
phosphatidyldimethylethanolamine; PG, phosphatidylglycerol;
and CL, cardiolipin.
The prevailing view has been that the CDP-choline pathway is a salvage pathway used by cells when the CDP-DAG pathway is compromised (11). However, recent studies have shown that the CDP-choline pathway contributes to PC synthesis even when wild-type cells are grown in the absence of exogenous choline (12). PC synthesized by the CDP-DAG pathway is constantly metabolized to choline and PA via the action of phospholipase D (13). The choline generated is then incorporated back into PC via the CDP-choline pathway and the PA is recycled back into PC, and other phospholipids (e.g. PI), via the CDP-DAG pathway (13). In fact, proper regulation of the CDP-choline pathway is important to overall lipid synthesis. For example, the activation of the CDP-choline pathway, due to the unregulated synthesis of CTP, results in significant increases in the synthesis of PC and PA and a decrease in the synthesis of PS (14). These changes are accompanied by an increase in total neutral lipid content at the expense of total phospholipids (14). The importance of the CDP-choline pathway to cell physiology is emphasized by the fact that the lethal phenotype of sec14 mutants defective in the PI/PC transfer protein (Sec14p) is suppressed by mutations in the CDP-choline pathway (15, 16).
The role of the CDP-ethanolamine pathway in phospholipid metabolism and
cell physiology has not been studied as extensively as the CDP-choline
pathway. Although the genes encoding the enzymes catalyzing the last
two steps in the CDP-ethanolamine pathway have been isolated and
characterized (17, 18), little is known about the enzyme ethanolamine
kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1.82)
that catalyzes the committed step in this pathway. In this paper we
report the isolation and characterization of the EKI1
(ethanolamine kinase) gene encoding
ethanolamine kinase in S. cerevisiae. Analysis of the
EKI1 gene product expressed in Sf-9 insect cells and the
analysis of cells with deletions in the EKI1 and
CKI1 genes indicated that the EKI1 gene product exhibited both ethanolamine kinase and choline kinase activities. The
EKI1 gene product was primarily responsible for PE synthesis via the CDP-ethanolamine pathway, whereas the CKI1 gene
product was primarily responsible for PC synthesis via the CDP-choline pathway. Unlike cki1 mutants, eki1 mutants did
not suppress the essential function of Sec14p.
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EXPERIMENTAL PROCEDURES |
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Materials
All chemicals were reagent grade. Growth medium supplies were from Difco. Restriction endonucleases, modifying enzymes, and recombinant Vent DNA polymerase were from New England Biolabs. Polymerase chain reaction and sequencing primers were prepared commercially by Genosys Biotechnologies, Inc. DNA sequencing kits were obtained from Applied Biosystems. The PCRScriptTM AMP SK(+) cloning kit was from Stratagene, and the YeastmakerTM yeast transformation system was from CLONTECH. The DNA size ladder used for agarose gel electrophoresis was from Life Technologies, Inc. Radiochemicals and EN3HANCE were from NEN Life Science Products. Scintillation counting supplies were from National Diagnostics. Protein assay reagent was purchased from Bio-Rad. Ethanolamine, phosphoethanolamine, CDP-ethanolamine, choline, phosphocholine, CDP-choline, and bovine serum albumin were from Sigma. Lipids were purchased from Avanti Polar Lipids. High performance thin layer chromatography and Silica Gel 60 thin layer chromatography plates were from EM Science.
Methods
Strains, Plasmids, and Growth Conditions--
The strains and
plasmids used in this work are listed in Tables
I and II,
respectively. Methods for yeast growth and sporulation were performed
as described previously (19, 20). Yeast cultures were grown in YEPD
medium (1% yeast extract, 2% peptone, 2% glucose) or in complete
synthetic medium minus inositol (21) containing 2% glucose at
30 °C. The appropriate amino acid of complete synthetic medium was
omitted for selection purposes. Escherichia coli strain DH5 was grown in LB medium (1% tryptone, 0.5% yeast extract, 1%
NaCl, pH 7.4) at 37 °C. Ampicillin (100 µg/ml) was added to cultures of DH5
-carrying plasmids. Media were supplemented with either 2% (yeast) or 1.5% (E. coli) agar for growth on
plates. Yeast cell numbers in liquid media were determined by
microscopic examination with a hemacytometer or spectrophotometrically
at an absorbance of 600 nm. The inositol excretion phenotype (22) of
yeast strains was examined on complete synthetic medium (minus inositol) by using growth of the inositol auxotrophic indicator strain
MC13 (ino1) (21) as described by McGee et al.
(23).
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DNA Manipulations, Amplification of DNA by PCR, and DNA
Sequencing--
Plasmid and genomic DNA preparation, restriction
enzyme digestion, and DNA ligations were performed by standard
protocols (20). Conditions for the amplification of DNA by PCR were
optimized as described by Innis and Gelfand (24). The annealing
temperature for the PCR reactions was 55 °C, and extension times
were typically between 2 and 3 min at 72 °C. PCR reactions were
routinely run for a total of 30 cycles. DNA sequencing reactions were
performed with the Prism DyeDeoxy Terminator Cycle sequencing kit and
analyzed with an automated DNA Sequencer. Transformations of yeast (25, 26) and E. coli (20) were performed as described previously. Plasmid maintenance and amplifications were performed in E. coli strain DH5. Amplification of the plasmid pKSK1 was
performed in E. coli strain Epicurian
ColiRXL-1.
Isolation of the EKI1 Gene-- A DNA sequence encoding an open reading frame in the Saccharomyces Genome Data Base (locus YDR147W) (GenBankTM accession number Z50046) was named EKI1. A 2.9-kb DNA fragment containing 595 bp of the putative EKI1 promoter, its entire protein coding sequence, and 678 bp of the 3'-flanking sequence was obtained by PCR (primers 5'-TTGTTTACCTTTTTCCTAAACAGG-3' and 5'-TGGTGTTTTTGGTTCATTATACGG-3') using strain W303-1A genomic DNA as a template. The PCR product was ligated into the SrfI site of the pCRScriptTM AMP SK(+) cloning vector resulting in the creation of pKSK1. This plasmid was digested with PstI/SacI. The resulting 2.9-kb fragment containing the open reading frame and 595 bp of the promoter region and 678 bp of the 3'-flanking region was ligated into the PstI/SacI sites of YEp352 (27) to create plasmid pKSK3. This construct was then transformed into W303-1B and the indicated mutants for the overexpression of the EKI1 gene product.
A genomic copy of the EKI1 gene was also isolated. A search of the Saccharomyces Genome Data Base indicated that the EKI1 gene flanked the 3'-end of the KGD2 gene, which was isolated by Repetto and Tzagoloff (28). We obtained the 2-µm plasmid pG104/T1 that was used originally by Repetto and Tzagoloff (28) for isolation of the KGD2 gene. This plasmid contains an insert of yeast genomic DNA of approximately 8 kb. PCR and restriction enzyme analyses indicated that pG104/T1 contained the EKI1 open reading frame and its 5'- and 3'-flanking sequences. A 4.8-kb insert, containing the EKI1 gene with its promoter and 3'-untranslated region, was released from pG104/T1 by digestion with BamHI/SphI. This fragment was ligated into the BamHI/SphI sites of pUC18 to form plasmid pKSK4. Plasmid pKSK4 was used to construct a recombinant viral expression vector of the EKI1 gene in Sf-9 insect cells.
Recombinant Viral Expression of the S. cerevisiae EKI1 Gene in Insect Cells-- Plasmid pKSK4 was digested with BglII/BstZ17I to release the genomic version of the entire EKI1 gene. This DNA fragment was ligated into the BamHI/HincII sites of the pUC18 vector resulting in the formation of plasmid pKSK6. Plasmid pKSK6 was digested with BstYI/PstI to release the EKI1 open reading frame, which was then ligated into the BamHI/PstI sites of the baculovirus vector pVL1393 to form plasmid pKSK7.
Sf-9 cells were maintained and grown as monolayers in TMNFH medium
containing containing 10% heat-inactivated fetal bovine serum (medium
A) (29). Sf-9 cells were co-transfected with pKSK7 and
BaculoGoldTM Autographa californica DNA
(PharMingen) using the CaCl2 method. Sf-9 infection
procedures followed the methods described by O'Reilly et
al. (29). For EKI1 expression, Sf-9 cells (1-2 × 107 cells grown in 75-cm2 tissue culture
flasks) were infected at a viral multiplicity of 10 and grown for
48 h in medium A. The infected cells were collected by gentle
trituration with medium, harvested by centrifugation, and washed twice
with phosphate-buffered saline. The final cell pellet was snap-frozen
over dry ice and stored at 80 °C.
Construction of eki1 and cki1
Mutants--
The plasmid
pKSK1 was digested with BglII/BsaBI to remove the
approximately 70% EKI1 coding region. A 1.8-kb
TRP1 disruption cassette, derived from plasmid pJA52 (30) by
BglII/SmaI digestion, was inserted into the
BglII/BsaBI sites of plasmid pKSK1 to create the
plasmid pKSK2. A linear 4-kb EKI1 disruption cassette was released from the plasmid pKSK2 by digestion with
SacI/XhoI. Strain W303-1B was transformed with
this DNA fragment to disrupt the chromosomal copy of the
EKI1 gene by the one-step gene disruption technique (31).
Transformants were selected for their ability to grow on complete
synthetic medium without tryptophan. Disruption of the chromosomal copy
of the EKI1 gene was confirmed by PCR (32) using the primers
listed above with the extension time increased to 4 min. The template
for the PCRs used to confirm the EKI1 disruption was genomic
DNA isolated from transformed colonies that grew on medium without
tryptophan. One of the eki1
mutants that we isolated was
designated strain KS101.
A similar strategy was used to construct cki1 and
eki1
cki1
double mutants. A linear 4-kb
CKI1 disruption cassette was released from the plasmid
pCTY307 (15) by digestion with ClaI/HpaI. Strain
W303-1A was transformed with this DNA fragment to disrupt the
chromosomal copy of the CKI1 gene (31). Transformants were selected for their ability to grow on complete synthetic medium without
histidine. Similarly, the CKI1 gene was disrupted in the eki1
mutant strain KS101, and transformants were selected
for their ability to grow without tryptophan and histidine. Disruption of the chromosomal copy of the CKI1 gene in these cells was
confirmed by PCR (primers, 5'-TTCGGATTATCTGAAGCAGG-3' and
5'-GGAAGTCAATGATGTAGACG-3') with the extension time increased to 2.5 min. The template for the PCRs used to confirm the CKI1
disruption was genomic DNA isolated from transformed colonies that grew
on medium without histidine and without tryptophan and histidine,
respectively. One of the cki1
mutants and one of the
eki1
cki1
double mutants that we isolated
were designated strains KS105 and KS106, respectively.
The EKI1 and CKI1 deletion cassettes were used to
transform a temperature-sensitive sec14 mutant (strain
CTY5-2D) (31). The appropriate transformants were isolated, and the
disruptions of the chromosomal copies of the EKI1 and
CKI1 genes in the sec14ts background were
confirmed by PCR as described above. The eki1 sec14ts and cki1
sec14ts double mutants were designated KS118 and
KS119, respectively.
Preparation of Cell Extracts-- All steps were performed at 5 °C. Yeast cells were disrupted with glass beads with a Mini-Bead-Beater (Biospec Products) in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM Na2EDTA, 0.3 M sucrose, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethanesulfonyl fluoride, 1 mM benzamide, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin (33). Glass beads and cell debris were removed by centrifugation at 1,500 × g for 5 min. The supernatant was used as the cell extract. Insect cells were disrupted by sonic oscillation in 50 mM Tris-HCl buffer (pH 7.5) containing 0.3 M sucrose, 1 mM Na2EDTA, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethanesulfonyl fluoride, 1 mM benzamide, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin (34). The disrupted cell suspension was centrifuged at 1,500 × g for 5 min to remove unbroken cells and cell debris. The supernatant was used as the cell extract.
Enzyme Assays, Protein Determination, and Analysis of Kinetic Data-- Ethanolamine kinase activity was measured for 20 min at 30 °C by following the phosphorylation of [1,2-14C]ethanolamine (5,000 cpm/nmol) with ATP. The reaction mixture contained 50 mM Tris-HCl buffer (pH 8.5), 5 mM ethanolamine, 10 mM ATP, 10 mM MgSO4, and enzyme protein in a final volume of 50 µl. The product phosphoethanolamine was identified by thin layer chromatography on silica gel plates using the solvent system methanol, 0.6% sodium chloride, ammonium hydroxide (10:10:1) (35). The position of the labeled phosphoethanolamine on chromatograms was determined by fluorography using EN3HANCE and compared with standard phosphoethanolamine. The amount of labeled product was determined by scintillation counting. All assays were performed in triplicate with an average S.D. of ±5%. All assays were linear with time and protein concentration.
Choline kinase activity was measured for 10 min at 30 °C by following the formation of 3H-labeled phosphocholine from [methyl-3H]choline (2,000 cpm/nmol) as described previously (36). The reaction mixture contained 50 mM Tris-HCl buffer (pH 8.5), 5 mM choline, 10 mM ATP, 10 mM MgSO4, and enzyme protein in a final volume of 50 µl. Radiolabeled phosphocholine was separated from the radiolabeled substrate by the precipitation of the substrate as choline reineckate (36). The amount of labeled product in the supernatant was determined by scintillation counting. The product phosphocholine was identified by thin layer chromatography on silica gel plates using the solvent system methanol, 0.5% sodium chloride, ammonium hydroxide (50:50:1) (37). The position of the labeled phosphocholine on chromatograms was determined by fluorography using EN3HANCE and compared with a standard.
A unit of ethanolamine kinase or choline kinase activity was defined as the amount of enzyme that catalyzed the formation of 1 nmol of product/min. Specific activity was defined as units per mg of protein. Protein concentration was determined by the method of Bradford (38) using bovine serum albumin as the standard. Kinetic data were analyzed according to the Michaelis-Menten and Hill equations using the EZ-FIT Enzyme Kinetic Model Fitting Program (39).
Labeling and Analysis of CDP-Ethanolamine and CDP-Choline Pathway Intermediates-- The CDP-ethanolamine and CDP-choline pathway intermediates were labeled with [1,2-14C]ethanolamine and with [methyl-3H]choline, respectively, as described previously (14, 40). The intermediates were isolated from whole cells after lipid extraction (41). The aqueous phase was neutralized and dried in vacuo, and the residue was dissolved in deionized water. Samples were subjected to centrifugation at 12,000 × g for 3 min to remove insoluble material. The CDP-ethanolamine (35) and the CDP-choline (37) pathway intermediates were then separated by thin layer chromatography with Silica gel 60 plates. The positions of the labeled intermediates on chromatograms were determined by fluorography using EN3HANCE and compared with standards. The amount of each labeled compound was determined by liquid scintillation counting.
Labeling and Analysis of Phospholipids--
Labeling of
phospholipids with 32Pi, with
[1,2-14C]ethanolamine, and with
[methyl-3H]choline were performed as described
previously (3, 4, 40, 42). Phospholipids were extracted from labeled
cells by the method of Bligh and Dyer (41) as described previously
(43). Phospholipids were analyzed by two-dimensional thin layer
chromatography on high performance silica gel thin layer chromatography
plates using chloroform/methanol/glacial acetic acid (65:25:10, v/v) as
the solvent for dimension one and chloroform/methanol/88% formic acid
(65:25:10, v/v) as the solvent for dimension two (44). The
32P-labeled phospholipids were visualized by
autoradiography. The 14C-labeled and 3H-labeled
phospholipids were visualized by fluorography using EN3HANCE. The position of the labeled phospholipids on
chromatography plates were compared with standard lipids after exposure
to iodine vapor. The amount of each labeled lipid was determined by
liquid scintillation counting of the corresponding spots on the chromatograms.
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RESULTS |
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Isolation of the S. cerevisiae EKI1 Gene Encoding Ethanolamine
Kinase and the Deduced Primary Structure of Its Encoded
Protein--
The EKI1 gene was identified in the
Saccharomyces Genome Data Base on the basis that its deduced
amino acid sequence showed 40% sequence identity to the C-terminal
amino acid sequence of the CKI1-encoded choline kinase of
S. cerevisiae (45, 46) (Fig.
2). The homologous region of these
proteins contain a phosphotransferase consensus sequence domain (47),
which is also present in the ethanolamine kinase encoded by the
easily shocked (eas) gene of Drosophila
melanogaster (48) (Fig. 2). The overall amino acid sequence
homologies of the EKI1 gene product to the CKI1
and eas gene products are 35 and 17%, respectively. Based
on this information we hypothesized that the EKI1 gene
encoded an ethanolamine kinase. The EKI1 gene is located on
the right arm of chromosome IV (49). The EKI1 gene coding
sequence along with its 5'- and 3'-flanking sequences was isolated by
PCR amplification using genomic DNA from strain W303-1A as the
template. The EKI1 gene and its flanking sequences were also
isolated from plasmid pG104/T1, a multicopy plasmid that contains
EKI1 on a 8-kb insert of genomic DNA. The PCR-derived and
genomic-derived genes were sequenced by automated DNA sequence
analysis. This analysis showed that both versions of the gene were
identical and matched the sequence in the data base.
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Inspection of the EKI1 DNA sequence did not reveal any sequence motifs that would suggest the existence of introns in the gene. The predicted protein product is 534 amino acids in length with a minimum subunit molecular mass of 61.7 kDa. The PSORT computer program2 predicts that the EKI1 gene product is a cytosolic protein. The PROSITE Motif program3 predicts that the EKI1 gene product has two protein kinase A, nine protein kinase C, eight casein kinase II, and one tyrosine kinase phosphorylation target sites.
The DNA sequence upstream (217 to
208) of the EKI1
coding region contains one UASINO element
(5'-CATGTGAAAA-3'). The UASINO element is found in the
promoters of several enzymes, including choline kinase, that are
involved with the synthesis of phospholipids in S. cerevisiae (2, 50). The expression of these enzymes is
coordinately down-regulated by inositol at the level of mRNA abundance (1, 2, 50).
Ethanolamine Kinase and Choline Kinase Activities in S. cerevisiae
Cells and in Sf-9 Insect Cells Overexpressing the EKI1 Gene--
A
multicopy plasmid bearing the EKI1 gene was used to
overexpress the EKI1 gene product in wild-type S. cerevisiae. Cells bearing the multicopy plasmid were grown to the
exponential phase, and cell extracts were prepared and assayed for
ethanolamine kinase activity. The plasmid containing the
EKI1 gene directed a 2.6-fold overexpression of ethanolamine
kinase activity when compared with cells not bearing the plasmid (Fig.
3). The overexpression of ethanolamine
kinase activity in these cells supported the conclusion that the
EKI1 gene encoded the enzyme. However, this experiment did
not rule out the possibility that the EKI1 gene was a
regulatory gene that controlled the expression of ethanolamine kinase
activity in S. cerevisiae. To test further the hypothesis
that the EKI1 gene was the structural gene encoding
ethanolamine kinase, we used heterologous expression of the gene in
Sf-9 insect cells. The EKI1 gene was placed within the
genome of baculovirus under control of the polyhedrin promoter and
expressed by viral infection of Sf-9 cells. Infection of the cells with
the baculovirus containing the EKI1 gene resulted in the
massive overexpression (165,500-fold) of ethanolamine kinase activity
when compared with uninfected cells (Fig. 3). This massive level of
ethanolamine kinase expression was equivalent to a 675-fold
purification over the activity expressed in the cell extract of
wild-type S. cerevisiae (Fig. 3).
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We also examined the ability of the EKI1 gene product to utilize choline as a substrate. Choline was used for this analysis because of the homology that the EKI1 gene product showed to the CKI1-encoded choline kinase (Fig. 2). The choline kinase activity in wild-type cells was 11-fold higher than the ethanolamine kinase activity in wild-type cells. There was only a 1.1-fold increase in choline kinase activity in wild-type cells bearing the EKI1 gene on the multicopy plasmid when compared with the control cells (Fig. 3). Infection of the Sf-9 insect cells with the baculovirus containing the EKI1 gene resulted in the massive overexpression (76,000-fold) of choline kinase activity (Fig. 3). The choline kinase activity expressed in the infected insect cells was 41-fold greater than the choline kinase activity expressed in wild-type S. cerevisiae. The ethanolamine kinase activity was 1.5-fold greater than the choline kinase activity in the insect cells infected with virus containing the yeast EKI1 gene. These data provided strong evidence that the EKI1 gene encoded an enzyme with both ethanolamine kinase and choline kinase activities.
The dependence of the EKI1-encoded kinase on the concentrations of ethanolamine and choline was examined using the cell extract of Sf-9 insect cells expressing the EKI1 gene. The kinase exhibited saturation kinetics with respect to ethanolamine and to choline using a saturating concentration (10 mM) of ATP. Ethanolamine was the preferred substrate for the enzyme based on the relative values for Vmax and Km (Table III). The specificity constant (Vmax/Km) for ethanolamine was 2.2-fold higher than that for choline.
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Deletion of the EKI1 Gene--
The EKI1 gene was
deleted to test further the hypothesis that its gene product encoded an
ethanolamine/choline kinase enzyme. In addition, the availability of an
eki1 mutant would allow us to examine whether the
EKI1 gene was essential for cell growth and to examine the
role this gene plays in phospholipid metabolism. A genomic construct
containing the EKI1 gene was manipulated to delete about
70% of its coding sequence. The EKI1 deletion construct was
introduced into the genome of haploid cells by homologous recombination
as described under "Experimental Procedures." Haploid eki1
mutant cells were viable and exhibited growth
properties similar to wild-type control cells when grown vegetatively
in YEPD medium at 30 °C. However, the eki1
mutant grew
at a slightly slower rate in complete synthetic medium at 30 °C when
compared with the control cells. Microscopic examination of
eki1
mutant cells showed no apparent gross morphological
differences when compared with wild-type cells when grown in either
complete synthetic medium or YEPD medium. In addition, mating and
sporulation were not affected in the eki1
mutant.
Overall, these results indicated that the EKI1 gene was not
essential for cell growth under typical laboratory growth conditions.
The EKI1 gene was also deleted in a cki1
mutant background to construct an eki1
cki1
double mutant. Haploid eki1
cki1
mutant
cells were viable and appeared normal by microscopic examination. Like
the eki1
mutant, the eki1
cki1
double mutant grew slightly slower than the control
cells in complete synthetic medium. The cki1
mutant
exhibited growth properties similar to wild-type control cells when
grown vegetatively in YEPD medium and in complete synthetic medium at
30 °C.
The eki1 mutant and the eki1
cki1
double mutant were examined for an inositol
excretion phenotype (22). This phenotype is the result of the
derepression of the INO1 gene (51) and is a characteristic
trait of mutants defective in the structural genes for several
phospholipid biosynthetic enzymes (50, 51). Growth of the
ino1 mutant was used as an indicator of the phenotype, and
the opi1 mutant, which excretes inositol (22) due to
unregulated derepression of the INO1 gene (50, 51), was used
as a positive control. Neither one of these mutants exhibited the
inositol excretion phenotype. As described previously (12, 52), the
cki1
mutant did not exhibit the inositol excretion phenotype.
Sec14p is a PI/PC transfer protein that is essential for cell viability
and vesicle budding from the Golgi complex (53, 54). It has been
proposed that a function of Sec14p is to down-regulate the synthesis of
PC via the CDP-choline pathway (53). It appears that too much PC
synthesized via the CDP-choline pathway is detrimental to the secretory
process (55). Mutations in the CKI1 gene can suppress
(i.e. bypass) the lethal phenotype of a sec14
mutant (15, 16). For example, a cki1 sec14ts double
mutant is viable at the restrictive temperature (15). Thus, a block in
the CDP-choline pathway removes the need for Sec14p function (15, 53).
Given the fact that the EKI1 gene product exhibited
ethanolamine kinase and choline kinase activities, we examined whether
the eki1 mutation would suppress the essential function
of Sec14p. An eki1
sec14ts double
mutant was constructed as described under "Experimental Procedures." We also constructed a cki1
sec14ts double mutant in the same genetic background
to be used as a positive control. The sec14ts
mutant, the eki1
sec14ts double
mutant, and the cki1
sec14ts double
mutant were grown at 25 °C (permissive temperature) and 37 °C
(restrictive temperature). As described previously (15), the
cki1
sec14ts double mutant grew at the
restrictive temperature. On the other hand, the eki1
sec14ts double mutant was not viable at the
restrictive temperature, whereas the mutant grew normally at the
permissive temperature. Thus, the mutation in the EKI1 gene
did not suppress the essential Sec14p function.
Ethanolamine Kinase and Choline Kinase Activities in the S. cerevisiae eki1 Mutant, cki1
Mutant, and eki1
cki1
Double
Mutant--
The eki1
mutant was grown to exponential
phase, and cell extracts were prepared and assayed for ethanolamine
kinase and choline kinase activities. Ethanolamine kinase activity was
reduced by 40% in the eki1
mutant when compared with the
ethanolamine kinase activity found in the wild-type parent strain (Fig.
4). However, the expression of choline
kinase activity was not affected in the eki1
mutant (Fig.
4). Transformation of the eki1
mutant with the multicopy
plasmid containing the EKI1 gene resulted in an
overexpression of ethanolamine kinase and choline kinase activities of
8- and 1.3-fold, respectively, when compared with these activities in
the eki1
mutant (Fig. 4).
|
To examine the expression of the ethanolamine kinase and choline kinase
activities encoded by the EKI1 gene product in the absence
of the CKI1-encoded choline kinase, we utilized a
cki1 mutant. Ethanolamine kinase and choline kinase
activities were reduced in the cki1
mutant by 82 and
98%, respectively, when compared with the wild-type parent (Fig. 4).
These data were consistent with previous studies showing that the
CKI1 gene product possessed both choline kinase and
ethanolamine kinase activities (45, 56). Transformation of the
cki1
mutant with the EKI1 gene on the
multicopy plasmid resulted in increases in ethanolamine kinase and
choline kinase activities of 16- and 2.8-fold, respectively, when
compared with these activities in the cki1
mutant (Fig. 4).
The eki1 cki1
double mutant was examined
for ethanolamine kinase and choline kinase activities. These activities
were not detectable in the double mutant (Fig. 4). Thus, the
EKI1 and CKI1 genes accounted for all of the
measurable ethanolamine kinase and choline kinase activities in
S. cerevisiae. We expressed the EKI1 gene in the
eki1
cki1
double mutant to explore further the ethanolamine kinase and choline kinase activities encoded by the
EKI1 gene. In this double mutant background, the
ethanolamine kinase activity (2.6 units/mg) encoded by the
EKI1 gene was 3.6-fold greater than the choline kinase
activity (0.72 units/mg) encoded by the gene (Fig. 4). These data
indicated that ethanolamine was preferred over choline as a substrate
and were also consistent with the data using the enzyme derived from
insect cells expressing the EKI1 gene.
Effect of the eki1, cki1
, and eki1
cki1
Mutations on
the Composition of the CDP-Ethanolamine Pathway Intermediates--
The
CDP-ethanolamine pathway intermediates include ethanolamine,
phosphoethanolamine, and CDP-ethanolamine (Fig. 1). Cells were labeled
with [1,2-14C]ethanolamine to steady state to analyze the
composition of the CDP-ethanolamine pathway intermediates. The
eki1
and the cki1
mutants exhibited
alterations in the incorporation of ethanolamine into the total pool of
CDP-ethanolamine pathway intermediates (Fig.
5A). The deletion of the
EKI1 gene resulted in a 1.7-fold increase in the amount of
ethanolamine incorporated into this pool when compared with the
wild-type control. The deletion of the CKI1 gene resulted in
a 70% decrease in the ethanolamine incorporated into the pool of
CDP-ethanolamine pathway intermediates. The incorporation of
ethanolamine into the intermediates of the eki1
cki1
double mutant was similar to that of the
cki1
mutant. The effects of the eki1
and
cki1
mutations on the relative amounts of the
CDP-ethanolamine pathway intermediates are shown in Fig. 5B.
In the control cells, 8.7 and 4% of the label was incorporated into
phosphoethanolamine and CDP-ethanolamine, respectively, whereas most of
the label was found in ethanolamine. The deletion of the
EKI1 gene resulted in dramatic decreases in the relative
amounts of phosphoethanolamine (77%) and CDP-ethanolamine (85%),
respectively. The deletion of the CKI1 gene resulted in a
small decrease in the amount of phosphoethanolamine (12%) and a small
increase in the amount of CDP-ethanolamine (1.5-fold). The only
intermediate found in the eki1
cki1
double
mutant was ethanolamine. Thus, the CDP-ethanolamine pathway was totally
blocked in the double mutant.
|
Effect of the eki1, cki1
, and eki1
cki1
Mutations
on the Composition of the CDP-Choline Pathway Intermediates--
Cells
were labeled with [methyl-3H]choline to steady
state to analyze the composition of the CDP-choline pathway
intermediates, which include choline, phosphocholine, and CDP-choline
(Fig. 1). Data for the incorporation of choline into the total pool of
CDP-choline pathway intermediates is shown in Fig.
6A. The incorporation of choline into the CDP-choline pathway intermediates was about 40-fold greater than the incorporation of ethanolamine into the
CDP-ethanolamine pathway intermediates (note the difference in the
y axes labels between Figs. 5A and
6A). Furthermore, the choline that was transported into the
cells was more readily incorporated into the CDP-choline pathway
intermediates when compared with the incorporation of ethanolamine into
the CDP-ethanolamine pathway intermediates (Figs. 5B and
6B). The deletion of the EKI1 gene did not have a
significant effect on the incorporation of choline into the CDP-choline
pathway intermediates, whereas the deletion of the CKI1 gene
resulted in a 90% decrease in the incorporation of choline into the
intermediates. The amount of choline incorporated into the pool of
intermediates in the eki1
cki1
double
mutant was similar to that of the cki1
mutant. The
effects of the eki1
and cki1
mutations on
the relative amounts of the CDP-choline pathway intermediates are shown
in Fig. 6B. The deletion of the EKI1 gene
resulted in a 2.4-fold increase in the amount of choline and a 50%
decrease in the amount of phosphocholine when compared with the
wild-type control cells. Deletion of the CKI1 had a more
dramatic effect on the relative amounts of choline and phosphocholine
when compared with the deletion of the EKI1 gene. The amount
of choline increased 4.3-fold and the amount of phosphocholine
decreased by 89% when compared with the control cells. The relative
amounts of CDP-choline were not significantly affected in the
eki1
and cki1
mutants. Phosphocholine and
CDP-choline were not detected in the eki1
cki1
double mutant. Thus, the CDP-choline pathway was
totally blocked in the double mutant.
|
Effect of the eki1, cki1
, and eki1
cki1
Mutations
on Phospholipid Composition--
The phospholipid composition was
analyzed in the eki1
mutant, the cki1
mutant, and the eki1
cki1
double mutant.
Cells were grown in complete synthetic medium without inositol,
ethanolamine, and choline to remove the regulatory effects these
precursors have on phospholipid synthesis (1, 50). The composition of phospholipids was examined by labeling cells to steady state with 32Pi, [1,2-14C]ethanolamine, and
[methyl-3H]choline.
32Pi will be incorporated into phospholipids
synthesized by the CDP-ethanolamine, CDP-choline, and CDP-DAG pathways.
The labeled ethanolamine will only be incorporated into PE via the
CDP-ethanolamine pathway, whereas the labeled choline will only be
incorporated into PC synthesized by the CDP-choline pathway (1, 50).
The effects of the eki1
and cki1
mutations
on phospholipid composition are shown in Fig.
7A. These mutations did not
have a significant effect on the incorporation of
32Pi into phospholipids and did not have a
major effect on the overall phospholipid composition when compared with
the wild-type control cells. The eki1
cki1
double mutant showed a 4% decrease in the amount of PI; the
cki1
mutant and the eki1
cki1
double mutant showed a 5% increase in the amount of PE, and the
cki1
mutant showed a 6% decrease in the amount of
PC.
|
Radiolabeled ethanolamine was incorporated into PE and PC during the
labeling experiments (Fig. 7B). This indicated that PE was
synthesized by the CDP-ethanolamine pathway in the eki1
and cki1
mutants as well as in the control cells. The
fact that the label was also incorporated into PC indicated that the PE
synthesized was used for the synthesis of PC via the phospholipid
methyltransferase enzymes that are used in the CDP-DAG pathway (1, 50).
The ethanolamine label was not incorporated into PE and PC in the eki1
cki1
double mutant. This result was
consistent with the labeling experiments of the CDP-ethanolamine
pathway intermediates where the label was only found in ethanolamine
(Fig. 5B). The data shown in Fig. 7B are plotted
as the ratio of the cpm of 14C incorporated into PE + PC to
the cpm of 32P incorporated into these phospholipids. This
allowed us to determine the effects of the eki1
and
cki1
mutations on the pathways by which cells synthesized
PE. The eki1
mutant showed an 80% decrease in the ratio
of PE + PC when compared with the control cells. These results
indicated that the deletion of the EKI1 gene resulted in a
major decrease in the utilization of the CDP-ethanolamine pathway for
PE synthesis. The deletion of the CKI1 gene did not have a
significant effect on the synthesis of PE via the CDP-ethanolamine pathway (Fig. 7B).
Radiolabeled choline was incorporated into PC in the eki1
and cki1
mutants during the labeling experiments (Fig.
7C). This indicated that PC was synthesized by the
CDP-choline pathway in both mutants. The data shown in Fig.
7C are plotted as the ratio of the cpm of 3H
incorporated into PC to the cpm of 32P incorporated into
PC. This allowed us to determine the effects of the eki1
and cki1
mutations on the pathways by which cells synthesized PC (40). The deletion of the EKI1 gene did not
have a significant effect on the ratio of
3H/32Pi when compared with the
control cells. However, the deletion of the CKI1 gene
resulted in an 91% decrease in this ratio (Fig. 7C). These
results indicated that the deletion of the CKI1 gene resulted in a major decrease in the utilization of the CDP-choline pathway for PC synthesis. Labeled choline was not incorporated into PC
in the eki1
cki1
double mutant. This was
consistent with the labeling of the CDP-choline pathway
intermediates where the label was only found in choline (Fig.
6B).
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DISCUSSION |
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There have been a number of conflicting reports as to
whether a single enzyme catalyzes the phosphorylation of ethanolamine and choline in various systems (46, 57). Results of genetic and
biochemical experiments have indicated that nearly all of the choline
kinase activity in S. cerevisiae is encoded by the CKI1 gene (45, 56). The purified CKI1-encoded
choline kinase enzyme also catalyzes the phosphorylation of
ethanolamine, albeit with only 14% of the activity when choline is
used as the substrate (56). Although choline kinase activity is barely
detectable in a cki1 mutant, cell extracts derived from
the mutant exhibit ethanolamine kinase activity (45). These data
suggest that a distinct ethanolamine kinase enzyme exists in S. cerevisiae (46). Indeed, such an enzyme exists, and in this work
we isolated the structural gene that encodes it.
The EKI1 gene was identified on the basis upon a deduced
amino acid sequence that showed homology to the CKI1-encoded
choline kinase (46). The EKI1 gene, which is found on
chromosome IV, was isolated and characterized. A multicopy plasmid
containing the EKI1 gene directed the overexpression of
ethanolamine kinase activity in S. cerevisiae wild-type
cells, in an eki1 mutant, in a cki1
mutant,
and in an eki1
cki1
double mutant.
Moreover, the heterologous expression of the EKI1 gene in
Sf-9 insect cells resulted in a massive overexpression of ethanolamine
kinase activity. The deletion of the EKI1 gene in S. cerevisiae resulted in a 40% reduction in ethanolamine kinase
activity. The remaining ethanolamine kinase activity in the
eki1
mutant was attributed to the ethanolamine kinase
activity of the CKI1-encoded choline kinase. Collectively, these data provided conclusive evidence for the identification of the
EKI1 gene as the structural gene encoding ethanolamine kinase.
The EKI1 gene product also exhibited choline kinase
activity. Based on the specificity constants for ethanolamine and
choline, and the levels of overexpression of the EKI1 gene
product in the eki1 mutant, cki1
mutant,
and the eki1
cki1
double mutant, ethanolamine was the preferred substrate for the enzyme. These analyses
also indicated that together the EKI1 and CKI1
gene products accounted for all of the ethanolamine kinase and choline
kinase activities in S. cerevisiae.
The eki1 mutant, cki1
mutant, and
eki1
cki1
double mutant were used to
examine the contributions of the EKI1 and CKI1 gene products to phospholipid synthesis via the CDP-ethanolamine and
CDP-choline pathways. The results of these studies showed that the
synthesis of phospholipids via the CDP-ethanolamine pathway was
dramatically reduced in the eki1
mutant, whereas the
synthesis of phospholipids via the CDP-choline pathway was dramatically reduced in the cki1
mutant. These studies also showed
that the EKI1 gene product made a small contribution to
phospholipid synthesis via the CDP-choline pathway and that the
CKI1 gene product made a small contribution to phospholipid
synthesis via the CDP-ethanolamine pathway. Thus, the EKI1
and CKI1 gene products exhibited overlapping functions with
respect to phospholipid synthesis. These results agreed with previous
data showing that in the absence of a functional CDP-choline pathway,
enzymes of the CDP-ethanolamine pathway can contribute to the synthesis
of PC (23). Cells did not synthesize phospholipids via the
CDP-ethanolamine and CDP-choline pathways when both the EKI1
and CKI1 genes were deleted from the genome. However, even
in the absence of the EKI1 and CKI1 genes, cells synthesized an essentially normal complement of membrane phospholipids. Whether the CDP-DAG pathway enzymes were regulated in the
eki1
mutant and cki1
mutant backgrounds to
compensate for these mutations will require additional studies.
The deletion of the EKI1 gene in S. cerevisiae
revealed that the EKI1 gene was not essential for growth
under typical laboratory conditions. The eki1 mutant did
not exhibit a significant growth phenotype. The yeast
cki1
mutant also lacks a growth phenotype (45). However,
the cki1
mutant exhibits a strong choline excretion phenotype in a sec14ts genetic background (12). This
phenotype is dependent on the SPO14/PLD1-encoded
phospholipase D-mediated turnover of PC, synthesized by the
CDP-DAG pathway, and the inability of cells to reincorporate the
choline back into PC by the CDP-choline pathway (12, 13). As discussed
above, the cki1
mutation can suppress the
sec14ts phenotype (15). However, the
cki1
mutation does not suppress the
sec14ts phenotype if the SPO14 gene is
also deleted (13). This situation holds for other CDP-choline pathway
mutations (i.e. cct1 and cpt1) (12,
13), which can also suppress the sec14ts phenotype
(15). Thus, the CDP-choline pathway and phospholipase D are both
important for Sec14p-mediated secretion and viability in S. cerevisiae (55). Although the EKI1-encoded ethanolamine kinase possessed choline kinase activity, the deletion of the EKI1 gene did not suppress the sec14ts
phenotype. The EPT1-encoded ethanolamine phosphotransferase, which catalyzes the final step in the CDP-ethanolamine pathway (Fig.
1), possesses choline phosphotransferase activity (18). Like the
eki1
mutation, the ept1 mutation does not
suppress the sec14ts phenotype (15). Thus, the
CDP-choline and CDP-ethanolamine pathways do not appear to have
overlapping functions with respect to Sec14p function.
Utilization of the CDP-ethanolamine and CDP-choline pathways by
S. cerevisiae requires the transport of ethanolamine and
choline, respectively, into the cell (58). Ethanolamine and choline are both transported by a single transporter encoded by the CTR
gene (58, 59). The in vivo labeling experiments showed that
the incorporation of both ethanolamine and choline into cells was very
reduced in the cki1 mutant and the eki1
cki1
double mutant when compared with wild-type cells. On
the other hand, the incorporation of ethanolamine and choline into the
eki1
mutant was actually greater than that of wild-type
cells. These results suggested that the deletion of the EKI1
and CKI1 genes, and/or the defect in PE and PC synthesis via
the CDP-ethanolamine and CDP-choline pathways, affected either the
expression and/or function of the CTR-encoded transporter.
These studies also showed that the incorporation of choline into
wild-type and mutant cells was much greater than that of ethanolamine.
Moreover, the utilization of choline for phospholipid synthesis via the
CDP-choline pathway was greater than that of ethanolamine for
phospholipid synthesis via the CDP-ethanolamine pathway. The
availability of the mutants described in this study will permit
additional studies to address the regulation of the choline/ethanolamine transporter as well as the utilization of choline
and ethanolamine for phospholipid synthesis.
The EKI1-encoded ethanolamine kinase is a novel enzyme not
described previously. It differed from the ethanolamine kinase encoded
by the eas gene of D. melanogaster. The
ethanolamine kinase from Drosophila is highly specific for
ethanolamine (48), which is in sharp contrast with the yeast
EKI1-encoded ethanolamine kinase which also utilized choline
as a substrate. Pavlidis et al. (48) have shown that a
mutation in the eas gene causes seizure, neuronal failure,
and paralysis. These phenotypes have been attributed to a defect in the
synthesis of PE via the CDP-ethanolamine pathway (48). Further detailed
insights into the physiological role of the EKI1-encoded
ethanolamine kinase and the CDP-ethanolamine pathway are likely to be
revealed through molecular genetic approaches with the yeast system.
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ACKNOWLEDGEMENTS |
---|
We thank Vytas A. Bankaitis for providing us with plasmid pCTY307 and strain CTY5-2D and Alexander Tzagoloff for providing us with plasmid pG104/T1. We thank Darin B. Ostrander for sequencing the EKI1 gene. We also acknowledge helpful discussions with David A. Toke.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service, National Institutes of Health Grants GM-50679 (to G. M. C.) and GM-32453 (to D. R. V.), and the Charles and Johanna Busch Memorial Fund (to G. M. C.). This is New Jersey Agricultural Experiment Station Publication D-10581-1-99.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901 Tel.: 732-932-9611 (ext. 217); Fax: 732-932-6776; E-mail: carman{at}aesop.rutgers.edu.
2 Information available on-line at the following address: http://psort.nibb.ac.jp/form.html.
3 Information available on-line at the following address: http://www.genome.ad.jp/sit/motif.html.
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ABBREVIATIONS |
---|
The abbreviations used are: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; CDP-DAG, CDP-diacylglycerol; PA, phosphatidate; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair.
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REFERENCES |
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