CDP-diacylglycerol is an important branch point intermediate in
glycerophosphate-based phospholipid biosynthesis in both prokaryotic
and eukaryotic
organisms(1, 2, 3, 4) . In
eukaryotic cells, phosphatidic acid is converted either to
CDP-diacylglycerol by the CDP-diacylglycerol synthase or to
diacylglycerol by a phosphatase. In mammalian cells, CDP-diacylglycerol
is the precursor to phosphatidylinositol (and its polyphosphorylated
derivatives), phosphatidylglycerol, and cardiolipin; in yeast, it is
also the precursor in the endoplasmic reticulum to the de novo synthesis of phosphatidylserine, phosphatidylethanolamine, and
phosphatidylcholine. Diacylglycerol is the precursor to
triacylglycerol, phosphatidylethanolamine, and phosphatidylcholine in
all eukaryotic cells. Therefore, the partitioning of phosphatidic acid
between CDP-diacylglycerol and diacylglycerol must be an important
regulatory point in eukaryotic phospholipid metabolism. In all
eukaryotic cells, CDP-diacylglycerol is required in the mitochondria
for phosphatidylglycerol and cardiolipin synthesis and in the
endoplasmic reticulum and possibly other organelles for the synthesis
of phosphatidylinositol.
A cDNA (derived from the CDS gene)
encoding a photoreceptor cell-specific isoform of CDP-diacylglycerol
synthase has been isolated from Drosophila(5) . The
gene product shares sequence identity with CDP-diacylglycerol synthase
from Escherichia coli(6, 7) , suggesting that
this enzyme has been highly conserved during evolution. This particular
isoform is an important regulator of the reutilization of phosphatidic
acid for the formation of phosphatidylinositol 4,5-bisphosphate, which
is the substrate for a phospholipase C-mediated signal cascade linked
to a G-protein-initiated signal. Overexpression of the CDS gene increases the amplitude of the light response of
photoreceptor cells, and cds mutant cells undergo
light-dependent retinal degeneration dependent on phospholipase C
function. However, the mutant flies develop normally, indicating that
the synthase isoform responsible for bulk phospholipid synthesis is
unaffected by this mutation. These results are also consistent with
there being multiple synthase activities in higher eukaryotic cells
derived from either a single gene or multiple genes, which is in
contrast to E. coli, which encodes a single
synthase(6, 7) .
CDP-diacylglycerol synthase has
been purified from the yeast Saccharomyces cerevisiae(8) and appears to be composed of two identical 56-kDa
subunits(9) , although some preparations also contain variable
amounts of a 54-kDa species(8) . The enzyme has been localized
to the endoplasmic reticulum, the cytoplasmic side of the outer
mitochondrial membrane, and the inner mitochondrial membrane (10) . This enzymatic activity is also enriched in the plasma
membrane over total membranes(11) , consistent with the finding
that the activity is also enriched in post-Golgi secretory
vesicles(12) . Its product, CDP-diacylglycerol, may play an
important role as both a precursor to phosphoinositide biosynthesis in
the plasma membrane and as a negative effector of phosphatidylinositol
4-kinase activity, thereby exerting an effect on cell proliferation via
a lipid-dependent signal transduction cascade(13) . The
multiple locations of this enzyme in yeast mirrors the results seen in Drosophila.
In order to gain more in-depth understanding of
the cellular distribution, function, and regulation of
CDP-diacylglycerol synthase in eukaryotic cells, we report in this
paper the isolation of the CDS1 gene from S.
cerevisiae. The gene product was verified by overexpression of
CDP-diacylglycerol synthase in yeast transformants. By gene
interruption, we demonstrate that CDS1 is an essential gene
for cell growth and encodes the majority, if not all, of the synthase
activity in yeast.
EXPERIMENTAL PROCEDURES
Materials
All chemicals were of reagent grade or
better. Radiochemicals, Hybond N nylon membranes, and CTP were obtained
from Amersham Corp. Liquiscint(TM) was purchased from National
Diagnostics. Restriction endonucleases were from Promega Corp., New
England Biolabs, Stratagene, and Boehringer Mannheim. The Gene Amp PCR (
)reagent kit was from Perkin Elmer Cetus. The Genius(TM)
1 kit (DNA Labeling and Detection Kit, Nonradioactive),
digoxigenin-labeled DNA molecular weight markers, positively-charged
nylon membranes, and Lumi-Phos(TM) 530 were purchased from
Boehringer Mannheim. Oligonucleotides were prepared commercially by
Genosys Biotechnologies. Geneclean II kit, YEP broth, and synthetic
media for yeast growth and selection were from BIO 101. Yeast nitrogen
base without amino acids was from Difco. The BCA kit was from Pierce. L-
-phosphatidic acid was from Sigma.
Strains, Plasmids, and Growth Conditions
A list of
the strains and plasmids used in this work is given in Table 1.
Methods of yeast growth, sporulation, and tetrad analysis were as
reported previously(14, 15) . YEPD medium consisted of
1% of Bacto-yeast extract, 2% Bacto-peptone, and 2% dextrose. In YEPR
or YEPG medium, 2% of raffinose or galactose (glucose free),
respectively, replaced dextrose as the carbon source. The induction
medium YEPRG or repression medium YEPRD contained 2% glucose-free
galactose or 2% glucose, respectively, in addition to raffinose.
Complete synthetic media (CSM) was constituted as described previously (15) and contained the indicated sugar carbon source (D, G, or
R). Yeast selection media contained the components of CSM except those
noted for selection purposes (i.e. minus tryptophan
(-Trp), minus leucine (-Leu), or minus uracil
(-Ura)). Yeast strains were grown at 30 °C. E. coli strain DH5
was grown in LB medium (1% Bacto-tryptone, 0.5%
Bacto-yeast extract, 1% NaCl, pH 7.4) at 37 °C. Ampicillin (200
µg/ml) was added to cultures of DH5
carrying plasmids. All the
above media were supplemented with 2% agar (yeast) or 1.5% agar (E.
coli) for preparation of agar plates.
DNA Manipulations
Methods for plasmid and genomic
DNA preparation, restriction enzyme digestion, and DNA ligation have
been described previously(16, 17) . Prior to
transformation, E. coli cells were washed with cold, sterile
water and concentrated in 10% glycerol as described in the
Biotechnologies and Experimental Research, Inc. protocol for E.
coli transformation. Concentrated cells were transformed by
electroporation using a BTX electroporation system at 1.0 kV/mm and
resistance of 129
. Yeast strains were transformed using a
modified method based on the protocol for E. coli transformation above. Yeast cells were grown to stationary phase,
washed with ice-cold water, and resuspended in sterile 10% glycerol.
Cells were transformed by electroporation at 1.0 kV/mm and 186
.
Amplification of DNA by Polymerase Chain
Reaction
For both analytical and preparative purposes, PCR was
performed after optimizing conditions as described by Innis and
Gelfand(18) . Amplification of the CDS1 gene from a
YES yeast cDNA library(19) , from a YEp13 yeast genomic
library(20) , or from yeast chromosomal DNA employed the
following primers: primer 1 (5`-CCATATCTCGAGAATGTCTGACAACCCTGAG-3`) and
primer 2 (5`-CCGGTCTAGATCAAGAGTGATTGGTCAATG-3`). They were designed
according to the DNA sequence of open reading frame YBR029c (GenBank
number Z35898); the underlined codons in primers 1 and 2 indicate the
start and stop codons, respectively, for this open reading frame.
DNA Labeling and Detection
The Genius 1 kit was
used according to the manufacturer's directions for preparation
and detection of digoxigenin-labeled DNA probes. The kit utilized
random priming of template DNA and incorporation of digoxigenin-dUTP
into the probe. Template DNA was produced by PCR and isolated by
agarose gel electrophoresis. The desired band was excised from the gel
and extracted by using the Geneclean II kit. An antibody against
digoxigenin coupled to alkaline phosphatase, which in the presence of
Lumi-Phos 530 produces a chemiluminescent signal, was used to permit
detection of hybridized probe by x-ray film.
Screening of a Genomic DNA Library
E. coli colonies bearing a yeast genomic DNA library carried on the E.
coli-yeast shuttle vector YEp13 (20) were transferred to
positively charged nylon membranes and screened for hybridization to
the labeled PCR probe generated by using genomic DNA as template.
Transfer of colonies to membranes, hybridization, and development of
blots were carried out using the manufacturer's instructions for
use of positively charged nylon membrane and the Genius 1 kit. SSC
dilutions were prepared from 20
SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0). Hybridization was performed
overnight at 68 °C in hybridization solution (5
SSC, 0.5%
Genius 1 kit blocking reagent, 0.1% N-lauroylsarcosine, 0.02%
SDS) containing the labeled PCR probe (10 ng/ml). Following
hybridization, membranes were washed twice for 5 min in 2
SSC,
0.1% SDS at room temperature and twice for 15 min in 0.1% SSC, 0.1% SDS
at 68 °C. Colonies corresponding to the positive signals on the
blot were picked from the original plates for further screening,
including a second round of hybridization screening and Southern blot
analysis of their DNA.
Southern Analysis of Genomic DNA
DNA samples were
digested with restriction enzymes and separated by agarose gel
electrophoresis. DNA was transferred to positively charged nylon
membranes by capillary transfer using 20
SSC at room
temperature and then cross-linked to the membrane by using a UV
Stratalinker(TM) 1800. The labeled PCR product used for library
screening was also used for hybridization to the Southern blots.
Methods for hybridization and development of blots were the same as
those in library screening. For high stringency hybridization,
membranes were washed twice for 5 min in 2
SSC, 0.1% SDS at
room temperature and twice for 15 min in 0.1% SSC, 0.1% SDS at 68
°C. For low stringency hybridization, membranes were washed twice
for 5 min in 2
SSC, 0.1% SDS at room temperature, and twice for
15 min in the same wash buffer at 68 °C.
Preparation of Cell Fractions and Measurement of
CDP-diacylglycerol Synthase Activity
All cell fractionation
procedures were carried out at 4 °C. For the measurement of
CDP-diacylglycerol synthase activity in the total membrane fraction, S. cerevisiae cells were grown to the exponential phase of
growth, and the cells were collected by centrifugation in tared
containers. Cells were washed in 50 mM Tris-maleate, pH 6.5,
0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride;
centrifuged; and resuspended in the same solution. The cell suspension
was mixed with an equal volume of pre-chilled silicon beads (diameter,
0.3 mm) and disrupted in a Mini-Beadbeater (Biospec Products) by six
15-s bursts with a 2-min pause between bursts. Silicon beads and
unbroken cells were removed by centrifugation at 2,000
g for 1 min. The total membrane fraction was separated from the
cytoplasmic fraction by centrifugation at 100,000
g for 1 h. The membrane pellet was suspended in 50 mM
Tris-maleate, pH 6.5, containing 40 mM MgCl
.
CDP-diacylglycerol synthase activity was measured by the incorporation
of [
H]CTP into chloroform-soluble material
dependent on phosphatidic acid as described previously(9) . For
the measurement of CDP-diacylglycerol synthase activity in the
mitochondria and microsomes, yeast cells were grown to an optical
density (A
) of 1.0. Isolation of yeast
organelles was carried out by the method of Zinser and Daum (21) . Mitochondria were isolated from the cell-free homogenate
(3,000
g supernatant) by centrifugation at 9,000
g for 10 min, while microsomes were collected by
centrifugation of the postmitochondrial supernatant at 100,000
g for 1 h.
Plasmid Shuffling and Overexpression of CDS1 by Galactose
Induction
Plasmids (URA3) carrying the CDS1 gene under the regulation of the GAL1 promoter were
introduced into the cds1 null mutant by ``plasmid
shuffling'' as follows. Briefly, strain YSD90A/YEp30 was
transformed with a P
-CDS1 plasmid. A
transformant carrying both plasmids was grown in YEPG medium for 2
overnights before plating for single colonies on CSMG-URA plates to
select cells still containing the P
-CDS1 plasmid; resulting colonies were screened for lack of growth on
CSMD-LEU and CSMG-LEU plates to verify the absence of plasmid YEp30.
Induction of the expression of CDS1 from the GAL1 promoter was carried out as follows. Cell transformants growing
exponentially in either CSMR-URA or YEPR medium were diluted to an A
of 0.1 unit with the same medium. Galactose
(2%) was added to the cell culture when A
reached 0.75 unit, and growth was continued before harvesting at
various times; parallel growth after addition of glucose to 2% was
carried out for those transformants that can grow in the presence of
glucose. After preparation of membrane fractions, samples were diluted
to 0.2-0.8 mg/ml of protein concentration before they were
assayed for CDP-diacylglycerol synthase activity.
Labeling and Analysis of Phospholipids
The CDS1 haploid strain YPH102 and the cds1::TRP1/P
-CDS1 transformant
YSD90A/pSDG1 were grown in YEPR medium to the exponential phase of
growth (A
about 1.0-1.2). For steady state
labeling, 5-ml aliquots of YEPRD and YEPRG medium were inoculated with
the above strains to an A
of 0.05. Following the
addition of 50 µCi of [
P]orthophosphate,
cells were grown for 16 h (six generations) to assure uniform labeling
before harvesting by centrifugation at 1,500
g. The
cell pellets were resuspended in 1 ml of 80% ethanol and incubated at
80 °C for 15 min. After centrifugation at 1,500
g,
the pellets were suspended in 0.67 ml of chloroform, methanol, 0.1 N HCl (1:2:0.8 (v/v)). Cells were lysed using glass beads, and
the phospholipids were extracted as described previously(22) .
Isolated radiolabeled phospholipids were applied to boric
acid-impregnated silica gel plates(8) , which were developed in
one dimension with chloroform/methanol/water/ammonium hydroxide
(60:37.5:3:1) as the solvent system. Labeled phospholipids were
detected and quantified directly from the thin layer plate using a
Betascope (Betagen Corp.). For pulse labeling of phospholipids, 250
µCi of [
P]orthophosphate was added to each
5-ml cell culture (A
= 1.0) in either
YEPRD or YEPRG medium. Cells were grown for 30 min before harvesting.
Phospholipid extraction and analysis were by the same procedure as
described above.
Assessment of Inositol-Excretion Phenotype
The
inositol excretion capacity of yeast strains was tested on CSMD-URA or
CSMG-URA plates lacking inositol, choline, and ethanolamine
(I
C
E
) as reported
previously (23) using growth of the inositol auxotrophic
reporter strain MC13 (ino1). The yeast strain to be tested was
patched onto the
I
C
E
tester plate
and permitted to grow for 24 h. The inositol auxotrophic reporter
strain was then streaked away from the patch as described
previously(24) , and cross-feeding was scored after an
additional 48-h incubation.
RESULTS AND DISCUSSION
Screening of the Genomic Library
Amino acid
sequences of CDP-diacylglycerol synthases both from Drosophila(5) and from E. coli(6) were compared
with DNA data bases by using the TBLAST search protocol (25) at
the National Center for Biotechnology Information (Bethesda, MD). An
unassigned open reading frame on chromosome II from S. cerevisiae corresponding to the open reading frame denoted as YBR0313 (26) or YBR029c (GenBank number Z35898) encodes a protein with
high homology to the above two enzymes. PCR primers were designed
according to the DNA sequence of the above open reading frame to
generate a DNA fragment encoding the putative yeast CDP-diacylglycerol
synthase. Only one PCR product with a length of about 1.4 kb was
generated either from a
YES yeast cDNA library (19) or
from a YEp13 yeast genomic library(20) . Digoxigenin-labeled
PCR product was used for Southern blot analysis and for genomic library
screening. Southern blot analysis of strain YPH102 genomic DNA digested
either with EcoRI or HindIII restriction enzyme
revealed only one hybridization-positive DNA fragment of either 4.9 or
3.7 kb, respectively, under both low- or high-stringency hybridization
conditions (data not shown). This result is consistent with the
restriction digestion map of the open reading frame YBR0313 (26) and its surrounding region on chromosome II. Screening of
the YEp13 yeast genomic DNA library carried in E. coli strain
DH5
was performed as described under ``Experimental
Procedures.'' Out of 70,000 colonies screened, two positive
colonies containing plasmids YEp2 and YEp30 were identified as
potential candidates carrying the putative yeast CDS1 gene.
Restriction mapping of these two clones revealed that they were
identical (data not shown).
Overexpression of CDP-diacylglycerol Synthase in
Yeast
Total membrane fractions from exponential phase cultures
of the strain YPH102 transformed with either plasmid YEp2 or YEp30 and
grown on CSMD-LEU medium were examined for CDP-diacylglycerol synthase
activity. Enzyme activity in strain YPH102 was increased about 10-fold
when carrying either plasmid YEp2 or plasmid YEp30 as shown in Table 2. Since both plasmids exhibited identical restriction
patterns and brought about a similar overproduction of enzyme activity,
they appeared to carry the same CDS1 structural gene. A 1.7-kb SspI fragment from the genomic clone was subcloned into the
plasmid pYES2 downstream of P
to yield plasmid pSDG1,
which was introduced into the wild-type yeast strain YPH102; this
construct carries 108 bp of CDS1 gene upstream sequence after
the P
. Induction of the CDS1 gene in CSMG-URA
medium brought about an 10-fold overexpression of CDP-diacylglycerol
synthase activity (Table 2) when compared with either strain
YPH102 alone or strain YPH102/pSDG1 (not shown) grown on CSMD-URA
medium (uninduced).
CDS1 Gene and CDP-diacylglycerol
Synthase
Restriction digestion mapping and DNA sequencing from
both ends (total of approximately 800 bp) of the gene (1371 bp)
confirmed that the CDS1 gene is identical with open reading
frame YBR029c (GenBank number Z35898). Inspection of the DNA sequence
did not reveal any sequence motifs(27) , suggesting the
existence of introns in or near the CDS1 gene, which minimizes
the possibility of multiple synthases being derived from alternate
splicing of a common RNA transcript. In the 3`-region, two potential
transcriptional termination sequences (28, 29) (1416-TAG

T-rich
region

TAG

TATGT

AT-rich
region

TTT-1502 and 1465-TAGNNTATGTA-1475) and a
polyadenylation site (30) (1548-AATAAA-1553) were found. A
sequence (ATGTGAAAA) homologous to the upstream activation sequence UAS
(1, 31, 32) was
found beginning 161 bp 5` to the gene. No recognizable TATA promoter
element was observed in this region. UAS
has
been found 5` to several genes (INO1, CHO1, PEM1, PEM2, PIS, and PSD1) related to phospholipid
metabolism(1, 33) . This sequence appears to be
related to the coordinate transcriptional depression of the expression
of phospholipid biosynthetic enzymes via the products of the INO2 and INO4 genes (31) when cells are grown in the
absence of inositol and either choline or ethanolamine. Its presence
upstream of the CDS1 gene may explain the decreased level of
CDP-diacylglycerol synthase when cells are grown in the presence of
inositol, choline, and ethanolamine(34) . Although multiple
copies of this regulatory sequence exist in the promoter region of many
of the genes reported above, only one conserved UAS
is located 5` to the CDS1 gene. This may explain the
smaller response in synthase activity (about a 2-fold range) to these
water-soluble precursors to phospholipids (34) when compared
with the response of other enzymes of phospholipid metabolism
(3-4-fold range).The predicted open reading frame encodes a
protein of 457 amino acids with a molecular mass of 51,789 Da. A
comparison of the deduced yeast CDP-diacylglycerol synthase amino acid
sequence with the sequences of the CDP-diacylglycerol synthases from E. coli(6) and Drosophila(5) revealed a high degree of homology as shown in Fig. 1. The amino acid sequence from S. cerevisiae shares 37% identity and 60% similarity with the Drosophila enzyme, and 29% identity and 56% similarity with the E. coli enzyme. In regions that were highly conserved among the three
enzymes, identities of approximately 90% were observed. Hydrophobicity
analysis of the yeast enzyme by the method of Kyte and Doolittle (35) showed a highly hydrophobic protein containing several
potential membrane spanning domains and a hydrophilic N terminus, which
is absent in the E. coli CDP-diacylglycerol synthase. The
profile of the yeast and Drosophila enzymes are remarkably
similar. Analysis of the sequence by the PSORT program (36) showed no potential mitochondrial targeting sequence
within the N-terminal hydrophilic region or any potential endoplasmic
retention sequence (KKXX or HDEL) at the C
terminus(37) , although this enzyme activity has been localized
primarily to the mitochondria and the endoplasmic
reticulum(10) . The lack in E. coli of the N-terminal
and C-terminal extensions found in the eukaryotic enzymes may indicate
that these sequences are required for organelle targeting. The PSORT
program did predict possible plasma membrane and Golgi body
localization for this sequence, which is consistent with finding the
activity in secretory vesicles and the plasma
membrane(11, 12) .
Figure 1:
Comparison of predicted amino acid
sequences of CDP-diacylglycerol synthases. Identical amino acids are
shaded in gray and boxed. The putative yeast (Sc) amino acid sequence and the Drosophila (Dm) (5) and E. coli (Ec) (6) amino acid sequences are numbered in the margins. These
sequences are aligned using the Pileup program in GCG(56) . A dash represents a gap placed by the computer
program.
The CDP-diacylglycerol synthase
isolated from the total membranes of yeast (minus the nuclear fraction)
was reported to have a molecular weight of 56,000(9) . The
discrepancy with the predicted molecular weight could be explained by
the inaccuracy of SDS gel electrophoresis methods with
membrane-associated proteins. However, any additional posttranslational
processing normally associated with organelle targeting of proteins
would only make this discrepancy greater. Since the CDS1 gene
encodes most if not all of the synthase activity in the cell (see
below) and there are no sequence motifs consistent with alternate
splicing of the primary transcript, how is this activity directed to
multiple sites in the cell? Are there additional synthase isoforms that
have not been yet identified? There is precedence in yeast for the use
of alternative AUG start sites on a common RNA transcipt for the
synthesis of tRNA modification enzymes, which are localized to the
mitochondria, cytoplasm, and nucleus (38, 39, 40) . The protein products from a
common transcript have different N termini, which appears to account
for their different locations in the cell. In contrast to the
CDP-diacylglycerol synthase, the N termini of the complete open reading
frames of these modifying enzymes contain predicted mitochondrial
targeting sequences. Phosphatidylserine synthase of yeast is found
associated with both the mitochondria (most likely the outer membrane)
and the endoplasmic reticulum(41) . Both forms have the same
molecular weight of 30,804 and are derived from the CHO1 gene;
whether this dual localization occurs in vivo and how it may
happen has not been resolved. Removal of the first AUG of the open
reading frame of the CHO1 gene results in localization of the
majority of the protein (molecular weight of 22,400) to the cytoplasm
in an inactive form but with sufficient membrane-associated active
protein of reduced size to complement a cho1 mutant(42) . This N terminus region cannot localize a
soluble marker protein to either membranes or specific organelles, yet
it appears to be important in localizing the CHO1 gene
product. Translation beginning at the second methionine of the
CDP-diacylglycerol synthase would only reduce the size of the protein
by 600 mass units, which would be indistinguishable from the complete
open reading frame by SDS gel electrophoresis methods but may
inactivate an unrecognized mitochondrial targeting sequence. The third
AUG lies 72 codons from the start of the open reading frame, which
would encode a protein much too small to be in agreement with the size
of major protein thus far isolated; however, a smaller minor isoform of
the synthase may have been missed in the earlier work. Such a smaller
product might still be active and membrane associated (with the plasma
membrane for example) because it would still retain the regions
homologous to the E. coli enzyme.
Disruption of the CDS1 Gene
The CDS1 gene
was disrupted in vitro and introduced into the genome by
homologous recombination as described below. A 3-kb EcoRI-BamHI fragment from plasmid YEp30 containing
the CDS1 gene was subcloned into pUC19. The NruI-MscI region (853 bp in length) internal to the CDS1 gene was replaced by a 2 kb PstI-BspHI
fragment carrying the TRP1 gene from plasmid
pRS304(43) . The disrupted CDS1 gene (cds1::TRP1) was excised by EcoRI-BamHI
digestion and used to transform the trp1 homozygous diploid
YPH501(43) . Tryptophan prototrophy (growth on CSMD-TRP plates)
was used to select for replacement of one of the CDS1 genes by
homologous recombination with the cds1::TRP1 fragment. PCR
reactions using genomic DNA of the interrupted diploid as template
confirmed that the disrupted CDS1 gene had integrated at the CDS1 locus of one of the two chromosomes (Fig. 2).
Figure 2:
Interruption of the CDS1 gene as
confirmed by PCR analysis. Genomic DNA was prepared from control
(YPH501) and interrupted strains (tryptophan prototrophs derived from
strain YPH501) and subjected to PCR amplification as described under
``Experimental Procedures.'' Presence of the wild-type CDS1 gene in the chromosome is indicated by a 1.4-kb fragment,
and the interrupted allele is indicated by a 2.6-kb fragment. Unlabeled lane, 1-kb DNA ladder; a, YPH501; b-e, interrupted strains.
The CDS1/cds1::TRP1 heterozygous diploid strain YSD3 was
sporulated and subjected to tetrad analysis. Each of the 10 tetrads
dissected gave rise to only two viable spores, all of which were
tryptophan auxotrophs; no spores with a TRP1 phenotype
survived. Inspection of the nonviable spores by microscopy showed that
none of them had undergone germination, single cell division, or
budding, indicating that the residual amount of CDP-diacylglycerol
synthase in the spore was not sufficient for germination. The 2:2 ratio
of viable to nonviable spores, together with the segregation of
tryptophan auxotrophy and the CDS1 gene with the viable
spores, indicates that the CDS1 gene is essential for cell
growth.
In order to rescue the nonviable spores, plasmid YEp30 (CDS1), which also carries a LEU2 marker, was
transformed into the heterozygous diploid YSD3 (CDS1/cds1::TRP1) prior to sporulation. Each of the four
tetrads dissected gave rise to four viable spores. These spores were
tested for growth in the absence of leucine and tryptophan. Among the
four spores within each tetrad, two were tryptophan auxotrophs (YSD90B,
YSD90D) and the other two were prototrophic for tryptophan (YSD90A,
YSD90C). All spores were leucine prototrophs, indicating that plasmid
YEp30 segregated efficiently during meiosis and that a plasmid-borne
copy of the CDS1 gene had rescued the nonviable spores. These
spores were grown in YEPD medium for one or two overnights, and the
cell cultures were sampled. After 24 h of growth, 90% of the tryptophan
auxotrophs (CDS1 wild type) were leucine auxotrophs,
indicating that the wild-type cells lost the YEp30 plasmid rapidly. The
tryptophan prototrophs (cds1::TRP1) remained prototrophic for
leucine even after 48 h of growth. Supplementation of the liquid growth
medium and the selection plates with choline and ethanolamine did not
result in the loss of the covering plasmid YEp30 from the null mutants.
Therefore, lack of dependence on CDP-diacylglycerol for
phosphatidylethanolamine and phosphatidylcholine biosynthesis by
utilization of the diacylglycerol-dependent pathway does not suppress
the need for the CDS1 gene.
Regulated Expression of the CDS1 Gene
To study the
cellular response to different CDP-diacylglycerol synthase levels,
plasmid pSDG1 (P
-CDS1, multicopy) was introduced into
the null mutant YSD90A (cds1::TRP1) by ``plasmid
shuffling'' as described under ``Experimental
Procedures.'' This transformant also showed an 10-fold increase of
the CDP-diacylglycerol synthase activity above the wild-type yeast
background, which was dependent on growth in CSMG-URA induction medium (Table 2). The increase in CDP-diacylglycerol synthase specific
activity relative to a wild-type control was the same (9-fold) in the
mitochondrial- and the endoplasmic reticulum-enriched fractions
dependent on galactose induction of the only CDS1 gene in a
haploid cds1 null background. One-third of the total enzyme
activity was in the mitochondria, and the remainder was in the
microsomal fraction as was also observed in wild-type cells lacking any
plasmids. Although the reported distribution of activity between these
two organelles varies (8, 10, 44) , both
fractions were proportionately enriched in synthase activity when the CDS1 gene was overexpressed, indicating that the CDS1 gene product is associated with both subcellular fractions. When
strain YSD90A/pSDG1 was grown in either CSMD-URA or CSMR-URA medium
(noninducing conditions), only 10% of the wild-type CDP-diacylglycerol
synthase activity was detected (Table 2), which was sufficient to
support robust cell growth on agar plates and in liquid medium.
Introduction of the low copy number plasmid pSDG2
(P
-CDS1), which carries a LEU2 marker
into strain YSD90A, also supported growth and overproduction of
synthase activity (10-fold) in CSMG-LEU medium; this plasmid contains
no DNA derived from the 5` upstream region of the CDS1 gene.
However, unlike the high copy number plasmid pSDG1, the latter plasmid
could not complement the null allele when grown on CSMD-LEU agar
plates. Plating cultures for single colonies resulted in the appearance
after 4 days incubation of very small colonies, which when restreaked
to CSMD-LEU plates did not form single colonies. Therefore, the
original colonies appear to have resulted from utilization of residual
synthase activity produced under induction conditions followed by cell
arrest on glucose-containing media once insufficient synthase activity
was present to sustain growth. Similarly, liquid cultures of this
transformant growing in CSMG-LEU arrested several generations after
switching to CSMD-LEU media. These results are consistent with the
earlier conclusion that the CDS1 gene is essential and encodes
the majority of the synthase activity. The ability of plasmid pSDG1 and
not plasmid pSDG2 to complement the null allele under repressed
conditions is most likely due to leak through transcription, which
would result in higher levels of transcript from the multicopy plasmid
(about 10-20 copies/cell) than the low copy number plasmid (about
1-2 copies/cell)(45) .
Inositol Excretion Phenotype
The cdg1 mutant of yeast (46) exhibits about a 75% reduction in the
derepressed level of CDP-diacylglycerol synthase activity. The synthase
activity in this mutant also no longer responds to regulation by
inositol and choline, and the mutant excretes inositol into the growth
medium. The product of the CDG1 gene has not been established.
In order to determine whether inositol excretion could be caused by
simply reducing the steady state level of synthase activity, strain
YSD90A/pSDG1 was streaked as patches to
I
C
E
plates
containing glucose (uninduced) or galactose (induced) and grown for 24
h to test for inositol excretion. The inositol auxotrophic strain MC13 (ino1) showed detectable and robust growth 24-48 h after
being streaked next to the patches of strain YSD90A/pSDG1 grown under
uninduced conditions but not next to the patches grown under induced
conditions; strain YPH102 did not exhibit an inositol excretion
phenotype under either growth condition. Therefore, an
inositol-excretion phenotype is exhibited by yeast with depressed
levels of CDP-diacylglycerol synthase similar to that observed in the cdg1 mutant.
CDS1 Expression and Phospholipid Metabolism
Strain
YSD90A/pSDG1 was pulse-labeled with
[
P]orthophosphate in both YEPG and YEPD media to
examine the initial rate of phospholipid synthesis as a function of the
capacity to make CDP-diacylglycerol (Fig. 3A) versus the wild-type strain YPH102 grown under similar
conditions; the results for the latter strain were independent of the
carbon source. Changes in the relative percent incorporation of label
into the various phospholipid classes during a pulse labeling
experiment should be related to changes in the initial rate of
synthesis of each phospholipid. The most significant difference brought
about by overproduction of the synthase (induced) is a marked increase
in the rate of synthesis of phosphatidylinositol and a decrease in the
rate of synthesis of phosphatidylserine and its downstream metabolic
products. A 90% reduction in the level of the synthase over wild-type
levels (uninduced) resulted in a significant increase in
phosphatidylserine labeling with a reduction in phosphatidylinositol
labeling. The increase in phosphatidic acid from 0.2% for wild-type and
induced cells to 0.8% for uninduced synthase was significant and
reproducible consistent with this synthase being involved in the major
phospholipid biosynthetic pathways of the cell. To analyze phospholipid
composition, both strain YSD90A/pSDG1 and strain YPH102 were labeled to
steady state with [
P]orthophosphate in the above
media as described under ``Experimental
Procedures''(Fig. 3B). Strain YPH102 grown in YEPG
medium (data not shown) gave the same results as cells grown in YEPD
medium. Except possibly for phosphatidylethanolamine, the differences
in labeling patterns among the strains reflected the pulse labeling
results. Relative to wild-type cells, overproduction of the synthase
increased the proportion of phosphatidylinositol while underexpression
of the synthase reduced the proportion of phosphatidylinositol.
Although the levels were low, phosphatidic acid appears to be elevated
under uninduced conditions and cardiolipin levels were highest under
induced conditions. These results are consistent with variations in
either the steady state level of CDP-diacylglycerol or its rate of
synthesis affecting the relative rate of synthesis of phospholipid at
this branch point in metabolism. Accumulation of phosphatidic acid and
increases in cardiolipin are also consistent with low and high levels,
respectively, of synthase activity.
Figure 3:
Dependence of phospholipid metabolism on
the level of CDP-diacylglycerol synthase activity. A, the
relative initial rate of biosynthesis of individual phospholipids was
estimated by the percent distribution of
[
P]orthophosphate into the indicated
phospholipids in 30 min. Individual determinations varied by less than
4% of the respective mean of each duplicate determination. B,
steady state phospholipid composition was estimated by the percent
distribution of [
P]orthophosphate incorporated
into the indicated phospholipids after long term labeling as described
under ``Experimental Procedures.'' Individual determinations
varied by less than 7% of the respective mean of each duplicate
determination. Wild type strain YPH102 was grown in YEPD (
) and
the cds1::TRP1 mutant strain YSD90A transformed with the
overexpression plasmid pSDG1 was grown in either YEPG (&cjs2108;
induced) or YEPD (
, uninduced). Phospholipids were extracted
and separated as described under ``Experimental
Procedures.''
The fact that only 10% of the
wild type level of the synthase can support normal growth and near
normal lipid composition is consistent with similar results for other
phospholipid biosynthetic enzymes seen in E.
coli(47) , which indicates that the catalytic capacities
of these biosynthetic enzymes are in large excess and that their
activities, as has been demonstrated in yeast(1) , are highly
regulated in response to growth conditions. However, changes over a
100-fold range in the level of synthase activity, which are presumably
somewhat reflected in the steady state level of CDP-diacylglycerol,
should have significant effects on the partitioning of product at this
branch point in metabolism. The excretion of inositol under repressed
expression of the P
-CDS1 gene in the null
background is consistent with the level of CDP-diacylglycerol being
limiting for phosphatidylinositol synthesis. The phosphatidylinositol
synthase would appear to be more sensitive to changes in the in
vivo concentration of CDP-diacylglycerol than the
phosphatidylserine synthase, even though the affinity for
CDP-diacylglycerol by these two enzymes measured in vitro at
saturation for their second substrates is the same (48, 49, 50) . With two substrate enzymes,
the apparent K
of one substrate is inversely
related to the concentration of the second substrate when the latter is
below its saturation concentration. At physiological concentrations of
serine, the phosphatidylserine synthase should be saturated for serine
and operating at its minimum apparent K
for
CDP-diacylglycerol(50) . However, the physiological
concentration of inositol is 9-fold below its K
in
the case of the phosphatidylinositol synthase(50) . Therefore,
the apparent K
of the latter enzyme for the
CDP-diacylglycerol should be much higher than the former enzyme under
physiological conditions, which is reflected in the effects brought
about by overproduction and repression of CDP-diacylglycerol synthase
activity under the control of P
.
Conclusions
Clearly the CDS1 gene encodes
an essential CDP-diacylglycerol synthase activity associated with both
the yeast endoplasmic reticulum and mitochondrial fractions. Results
with complementation of the cds1 null mutant with plasmids
pSDG1 and pSDG2 demonstrate that the CDS1 gene encodes more
than 90% of the synthase activity in the cell and does not encode an
activity that is targeted solely to either the mitochondria or the
endoplasmic reticulum. If a second CDS gene exists, as is the
case with the expression of phosphatidylserine decarboxylase (PSD1 and PSD2)
activity(33, 51, 52) , it would account for
significantly less than 10% of the total activity. Unlike the PSD genes(52, 53) , which can complement each other,
a possible second CDS gene does not support growth in the
absence of the CDS1 gene.There is a clear difference in
the germination phenotype of null mutants of the CDS1 and PIS (encoding phosphatidylinositol synthase) genes. Although
both genes are essential for vegetative growth, null spores derived
from heterozygous null/wild-type diploids of the PIS gene
undergo sporulation and at least one cell division before arresting
with buds(54) , while spores containing the null cds1 gene do not germinate. Therefore, supplying CDP-diacylglycerol for
functions other than bulk phosphatidylinositol biosynthesis may be
crucial to cell viability. One such function might be in supplying
substrate for the plasma membrane-associated signal transduction
pathway responsible for phosphoinositide formation, which has been
linked to regulation of cell growth in yeast(13, 55) .