THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 275, No. 52, Issue of December 29, pp. 40887–40896, 2000
Printed in U.S.A.
Regulation of the DPP1-encoded Diacylglycerol Pyrophosphate
(DGPP) Phosphatase by Inositol and Growth Phase
INHIBITION OF DGPP PHOSPHATASE ACTIVITY BY CDP-DIACYLGLYCEROL AND ACTIVATION OF
PHOSPHATIDYLSERINE SYNTHASE ACTIVITY BY DGPP*
Received for publication, September 6, 2000, and in revised form, September 18, 2000
Published, JBC Papers in Press, October 2, 2000, DOI 10.1074/jbc.M008144200
June Oshiro, Shanthi Rangaswamy, Xiaoming Chen, Gil-Soo Han, Jeannette E. Quinn, and
George M. Carman‡
From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University,
New Brunswick, New Jersey 08901
The regulation of the Saccharomyces cerevisiae DPP1encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase
was examined. Addition of inositol to the growth medium resulted in a dose-dependent increase in the level
of DGPP phosphatase activity in both exponential and
stationary phase cells. Activity was greater in stationary
phase cells when compared with exponential phase
cells, and the inositol- and growth phase-dependent regulations of DGPP phosphatase were additive. Analyses
of DGPP phosphatase mRNA and protein levels, and
expression of ␤-galactosidase activity driven by a PDPP1lacZ reporter gene, indicated that a transcriptional
mechanism was responsible for this regulation. Regulation of DGPP phosphatase by inositol and growth phase
occurred in a manner that was opposite that of many
phospholipid biosynthetic enzymes. Regulation of
DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in opi1⌬, ino2⌬,
and ino4⌬ phospholipid synthesis regulatory mutants.
CDP-diacylglycerol, a phospholipid pathway intermediate used for the synthesis of phosphatidylserine and
phosphatidylinositol, inhibited DGPP phosphatase activity by a mixed mechanism that caused an increase in
Km and a decrease in Vmax. DGPP stimulated the activity
of pure phosphatidylserine synthase by a mechanism
that increased the affinity of the enzyme for its substrate CDP-diacylglycerol. Phospholipid composition
analysis of a dpp1⌬ mutant showed that DGPP phosphatase played a role in the regulation of phospholipid metabolism by inositol, as well as regulating the cellular
levels of phosphatidylinositol.
The yeast Saccharomyces cerevisiae serves as a model eukaryote where the regulation of phospholipid synthesis can be
studied (1– 4). The major phospholipids found in the membranes of S. cerevisiae include PC,1 PE, PI, and PS (1– 4).
* This work was supported in part by United States Public Health
Service Grant GM-28140 from the National Institutes of Health. The
costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
‡ To whom correspondence and reprint requests 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:
[email protected].
1
The abbreviations used are: PC, phosphatidylcholine; PE, phosThis paper is available on line at http://www.jbc.org
Mitochondrial membranes also contain phosphatidylglycerol
and cardiolipin (1– 4). The synthesis of these phospholipids is a
complex process that contains a number of branch points (Fig.
1). PS, PE, and PC are synthesized from PA by the CDP-DG
pathway (Fig. 1). CDP-DG is also used for the synthesis of PI
and cardiolipin. PE and PC are also synthesized by the CDPethanolamine and CDP-choline pathways, respectively (Fig. 1).
The CDP-DG pathway is primarily used by wild-type cells for
the synthesis of PE and PC when they are grown in the absence
of ethanolamine or choline (1, 2, 4 – 6). The CDP-ethanolamine
and CDP-choline pathways assume a critical role in phospholipid synthesis when enzymes in the CDP-DG pathway are
defective (1, 2, 4, 7). Mutants in the CDP-DG pathway require
choline for growth and synthesize PC by way of CDP-choline
(8 –15). Mutants defective in the synthesis of PS (8, 9) or PE
(10, 11) can also synthesize PC if they are supplemented with
ethanolamine. The ethanolamine is used for PE synthesis by
way of CDP-ethanolamine. The PE is subsequently methylated
to form PC (Fig. 1).
The CDP-ethanolamine and CDP-choline pathways were
once viewed as auxiliary or salvage pathways used by cells
when the CDP-DG pathway was compromised (1, 2). However,
it is now known that these pathways contribute to the synthesis of PE and PC even when wild-type cells are grown in the
absence of ethanolamine and choline, respectively (16 –18). For
example, the PC synthesized via the CDP-DG pathway is constantly hydrolyzed to free choline and PA by phospholipase D
(18, 19). Free choline is incorporated back into PC via the
CDP-choline pathway, and PA is incorporated into phospholipids via reactions utilizing CDP-DG and DG (2– 4) (Fig. 1).
Genetic, molecular, and biochemical studies have shown that
the regulation of phospholipid synthesis is a complex and
highly coordinated process (2– 4). The mechanisms that govern
this regulation control the mRNA and protein levels of the
biosynthetic enzymes, as well as their activity (1– 4). The factors that regulate phospholipid synthesis in S. cerevisiae include water-soluble phospholipid precursors, nucleotides, lipids, and growth phase (1, 3, 4, 7, 17).
DGPP is a minor phospholipid recently identified in S. cerevisiae (20). It contains a pyrophosphate group attached to DG
(21). DGPP is derived from PA via the reaction catalyzed by PA
kinase (20). DGPP phosphatase catalyzes the removal of the
␤-phosphate from DGPP to yield PA and then removes the
phatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidate; CDP-DG, CDP-diacylglycerol; DGPP, diacylglycerol pyrophosphate; DG, diacylglycerol; PCR, polymerase chain
reaction; kb, kilobase(s); bp, base pair.
40887
40888
Regulation of DPP1-encoded DGPP Phosphatase
FIG. 1. Pathways for the synthesis of
the major phospholipids in S. cerevisiae. The pathways shown for phospholipid synthesis include the relevant steps
discussed in the text. The four major phospholipids (PC, PE, PI, and PS) are indicated by boxes. The CDP-DG, CDP-choline,
and CDP-ethanolamine pathways are indicated. DGPP is indicated by the ellipse. The
DPP1-encoded DGPP phosphatase, CHO1encoded PS synthase, and INO1-encoded
inositol-1-P synthase reactions are indicated in the figure. A more comprehensive
description that includes the synthesis of
PA and additional steps in these pathways
can be found elsewhere (2, 17). The abbreviations used are: PME, phosphatidylmonomethylethanolamine; PDE, phosphatidyldimethylethanolamine;
TG,
triacylglycerol; PGP, phosphatidylglycerophosphate; CL, cardiolipin.
phosphate from PA to generate DG (20). The function of DGPP
has not been established in S. cerevisiae. However, phospholipid composition analysis of a dpp1⌬ mutant devoid of DGPP
phosphatase activity (22) has revealed that the DPP1 gene
product plays a role in the regulation of phospholipid metabolism (23). The dpp1⌬ mutant exhibits a reduction in the cellular level of PI and an elevation in the levels of PA and DGPP
(23). PA plays a central role in phospholipid synthesis as the
precursor of all phospholipids synthesized via the CDP-DG,
CDP-ethanolamine, and CDP-choline pathways (Fig. 1). Moreover, of all the major phospholipids in S. cerevisiae, PI is the
only one that is essential (1, 2, 24, 25).
Because inositol (precursor of PI (Fig. 1)) has regulatory
effects on the expression of many phospholipid biosynthetic
enzymes (1, 2, 4, 7, 17), we examined the effect of inositol on
expression of the DPP1-encoded DGPP phosphatase. We also
examined the influence of growth phase on DGPP phosphatase,
since it has a major influence on the expression of many phospholipid biosynthetic enzymes (1, 2, 4, 7, 17). We discovered
that DGPP phosphatase expression was regulated by inositol
and growth phase. However, this regulation occurred in an
opposite manner to that of most phospholipid biosynthetic enzymes. We also discovered that the activity of DGPP phosphatase was inhibited by CDP-DG and that the activity of PS
synthase was stimulated by DGPP. The work reported here
increases the understanding of the long and short term regulation of DGPP phosphatase and the influence of the enzyme on
phospholipid metabolism.
EXPERIMENTAL PROCEDURES
Materials—All chemicals were reagent grade. Growth medium supplies were purchased from Difco. Radiochemicals were from
PerkinElmer Life Sciences. Scintillation counting supplies were from
National Diagnostics. Triton X-100, bovine serum albumin, aprotinin,
benzamidine, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, diethyl pyrocarbonate, inositol, choline, CDP, and O-nitrophenyl ␤-Dgalactopyranoside were purchased from Sigma. Lipids were purchased
from Avanti Polar Lipids. DE52 (DEAE-cellulose) was from Whatman.
Protein assay reagent, Zeta ProbeTM membranes, electrophoresis reagents, and immunochemical reagents were purchased from Bio-Rad.
The NEBlot kit, restriction endonucleases, modifying enzymes, and
recombinant Vent DNA polymerase with 5⬘- and 3⬘-exonuclease activity
were purchased from New England Biolabs. Primers for polymerase
chain reaction were prepared commercially by Genosys Biotechnologies, Inc. Nitrocellulose membranes were purchased from Schleicher &
Schuell. ProbeQuant G-50 columns, polyvinylidene difluoride membranes, protein A-Sepharose, and the ECF Western blotting chemifluorescent detection kit were purchased from Amersham Pharmacia Biotech. Silica Gel 60 thin layer chromatography plates were from EM
Science.
Strains, Plasmids, and Growth Conditions—The strains and plasmids used in this work are listed in Table I. Methods for yeast growth
were performed as described previously (26, 27). Yeast cultures were
grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose)
containing adenine (40 mg/liter) or in complete synthetic medium minus inositol (28) 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 ␮g/ml)
was added to cultures of DH5␣-carrying plasmids. Media were supplemented with 2% agar for growth on plates. Yeast cell numbers in liquid
media were determined spectrophotometrically at an absorbance of 600
nm. Exponential phase corresponded to a cell density of 1 ⫻ 107 cells/
ml, whereas stationary phase was 1 ⫻ 108 cells/ml. Stationary phase
cultures were harvested 24 h after an initial inoculation density of 1 ⫻
106 cells/ml. For the overexpression of CHO1-encoded PS synthase, cells
were grown to the exponential phase in complete synthetic medium
containing 2% raffinose. Galactose (2%) was then added to the growth
medium to induce the expression of PS synthase. Maximum induction
(60-fold) was achieved after 2 h.
DNA Manipulations and Amplification of DNA by PCR—Plasmid
and genomic DNA preparation, restriction enzyme digestion, and DNA
ligations were performed by standard methods (27). Transformation of
yeast (29) and E. coli (27) were performed as described previously.
Conditions for the amplification of DNA by PCR were optimized as
described previously (30). Plasmid maintenance and amplifications
were performed in E. coli strain DH5␣.
Construction of Plasmids—Plasmid pJO2 contains the putative promoter of the DPP1 gene fused to the lacZ gene of E. coli. The plasmid
was constructed by replacing the EcoRI fragment of plasmid pSD90
with the DPP1 promoter, which was obtained by PCR (primers, 5⬘GTGAAGAAGCAGGAATTCATAAAGGGACAACACGG-3⬘ and 5⬘GTTTTAATAAACGAAACTGAATTCATTTTGGTCG-3⬘) using strain
W303-1A genomic DNA as a template. The PCR primer used in the
forward direction corresponds to ⫺1156 bp to the start codon, and the
primer used in the reverse direction corresponds to ⫹25 bp to the start
codon. Plasmid pSD90, derived from pMA109, contains the CRD1 promoter. Plasmid pMA109, derived from plasmid YEp357R, contains the
PIS1 promoter (31). The correct orientation of the DPP1 promoter in
plasmid pJO2 was checked by digestion with PstI and by measurement
of ␤-galactosidase activity. The PDPP1-lacZ construct does not contain
any sequences of the DPP1 open reading frame. The pJO2 plasmid was
introduced into the indicated strains to examine the expression of the
DPP1 gene by measuring ␤-galactosidase activity.
Regulation of DPP1-encoded DGPP Phosphatase
40889
TABLE I
Strains and plasmids used in this work
Strain or plasmid
S. cerevisiae
W303–1A
DTY1
SH304
SH303
SH307
E. coli
DH5␣
Plasmids
pDT1-DPP1
pSD90
pJO2
pJ699Z
pRS425
YCpGPSS
YEpGPSS
Genotype or relevant characteristics
MATa leu2-3 112 trp1-1 can1-100 ura3-1 ade2-1 his2-11,15
dpp1⌬⬋TRP1/Kanr derivative of W303–1A
MATa trp1⌬63 his3⌬200 ura3–52 leu2⌬1 opi1⌬⬋LEU2
MATa trp1⌬63 his3⌬200 ura3–52 leu2⌬1 ino2⌬⬋TRP1
MAT␣ trp1⌬63, his3⌬200 ura3–52, leu2⌬1 ino4⌬⬋LEU2
F⫺ ␾80dlacZ⌬M15 ⌬(lacZYA-argF)U169 deoR, recA1 endA1 hsdR17(rk⫺ mk⫹) phoA, supE44
␭⫺thi-1 gyrA96 relA1
DPP1 gene ligated into the SrfI site of pCRScript AMP SK(⫹)
PCRD1-lacZ reporter construct on a multicopy plasmid containing URA3, derivative of pMA109
PDPP1-lacZ reporter construct on a multicopy plasmid containing URA3, derivative of pSD90
PINO1-lacZ reporter construct on a multicopy plasmid containing LEU2, derivative of pJH359
Multicopy E. coli/yeast shuttle plasmid containing LEU2
PGAL7-CHO1 on a centromere plasmid containing TRP1
PGAL7-CHO1 on a multicopy plasmid containing LEU2, derivative of YCpGPSS
A 1.8-kb insert, containing the CHO1 gene fused to the GAL7 promoter, was released from plasmid YCpGPSS (32) by digestion with
SalI/BamHI. This DNA fragment was ligated into the SalI/BamHI sites
of pRS425, a multicopy E. coli/yeast shuttle vector containing the LEU2
gene (33) to form plasmid YEpGPSS. This construct was transformed
into W303-1A for the overexpression of the CHO1-encoded PS synthase.
RNA Isolation and Northern Blot Analysis—Total yeast RNA was
isolated using the methods of Schmitt et al. (34) and Herrick et al. (35).
Equal amounts (25 ␮g) of total RNA from each sample were resolved on
a 1.1% formaldehyde gel for 2.5 h at 100 V (36). The RNA samples were
then transferred to a Zeta ProbeTM membrane by vacuum blotting.
Pre-hybridization, hybridization with a specific probe, and washes to
remove unbound probe were carried out according to the manufacturer’s instructions. The DPP1 probe was a 0.87-kb fragment isolated from
pDT1-DPP1 by MfeI/BamHI digestion. A TCM1 probe was used as a
constitutive standard and a loading control. This probe was generated
from an PCR-amplified (primers, 5⬘-CTCACAGAAAGTACGAAGCACC-3⬘ and 5⬘-CAAGTCCTTCTTCAAAGTACC-3⬘) region of genomic
DNA. The DPP1 and TCM1 probes were labeled with [␣-32P]dATP
using the NEBlot random primer labeling kit. Unincorporated nucleotides were removed using ProbeQuant G-50 columns. Images of radiolabeled species were acquired by PhosphorImaging analysis.
Preparation of Anti-DGPP Phosphatase Antibodies and Immunoblotting—The peptide sequence SDVTLEEAVTHQRIPDE (residues 263–
279 at the C-terminal end of the deduced protein sequence of DPP1) was
synthesized and conjugated to carrier protein at Bio-Synthesis, Inc.
(Lewisville, TX). Antibodies were raised against the peptide in New
Zealand White rabbits by standard procedures (37) at Bio-Synthesis,
Inc. The IgG fraction was isolated from antisera by protein A-Sepharose
chromatography (37). SDS-polyacrylamide gel electrophoresis (38) using 12% slab gels and immunoblotting (39) using polyvinylidene difluoride membranes were performed as described previously. The antiDGPP phosphatase antibodies were used at a dilution of 1:1000, and the
DGPP phosphatase protein was detected using the ECF Western blotting chemifluorescent detection kit as described by the manufacturer.
The DGPP phosphatase protein on immunoblots was acquired by FluoroImaging analysis. The relative density of the protein was analyzed
using ImageQuant software. Immunoblot signals were in the linear
range of detectability.
Preparations of Enzymes—Cells from all strains were disrupted with
glass beads (40) in 50 mM Tris maleate buffer, pH 7.0, containing 1 mM
Na2EDTA, 0.3 M sucrose, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 5 ␮g/ml each of aprotinin, leupeptin, and pepstatin. Glass beads and unbroken cells were
removed by centrifugation at 1,500 ⫻ g for 10 min. The supernatant
(cell extract) was used for enzyme assays and immunoblot analysis.
DPP1-encoded DGPP phosphatase (20) and CHO1-encoded PS synthase
(41) were purified to near homogeneity as described previously. The
specific activities of DGPP phosphatase and PS synthase were 60 and 2
␮mol/min/mg, respectively.
Preparation of Substrates—DGPP (42) and CDP-diacylglycerol (43)
were synthesized as described previously. [␤-32P]DGPP was synthesized enzymatically using purified Catharanthus roseus phosphatidate
kinase as described by Wu et al. (20).
Source or
Ref.
95
22
S. A. Henry
S. A. Henry
S. A. Henry
27
22
W. Dowhan
This work
S. A. Henry
64
33
32
This work
Preparation of Triton X-100/Phospholipid Mixed Micelles—Phospholipids in chloroform were transferred to a test tube, and solvent was
removed in vacuo for 40 min. The surface concentration of phospholipids in Triton X-100/phospholipid mixed micelles was varied by the
addition of various amounts of an 80 mM solution of Triton X-100 to the
dried phospholipids. The total phospholipid concentration in the mixed
micelles did not exceed 15 mol % to ensure that the structure of the
micelles was similar to that of pure Triton X-100 (44, 45). The mol % of
a phospholipid in a mixed micelle was calculated with the following
formula: mol %phospholipid ⫽ ([phospholipid (mM)]/[phospholipid (mM)] ⫹
[Triton X-100 (mM)]) ⫻ 100.
Enzyme Assays, Protein Determination, and Analysis of Kinetic
Data—DGPP phosphatase activity was measured by following the release of water-soluble 32Pi from chloroform-soluble [␤-32P]DGPP
(10,000 –15,000 cpm/nmol) as described by Wu et al. (20). The reaction
mixture contained 50 mM citrate buffer, pH 5.0, 2 mM Triton X-100, 0.1
mM DGPP, and enzyme protein in a total volume of 0.1 ml. ␤-Galactosidase activity was determined by measuring the conversion of Onitrophenyl ␤-D-galactopyranoside to O-nitrophenol (molar extinction
coefficient of 3,500 M⫺1 cm⫺1) by following the increase in absorbance at
410 nm on a recording spectrophotometer (46). The reaction mixture
contained 100 mM sodium phosphate buffer, pH 7.0, 3 mM O-nitrophenyl ␤-D-galactopyranoside, 1 mM MgCl2, 100 mM 2-mercaptoethanol,
and enzyme protein in a total volume of 0.1 ml. PS synthase activity
was measured by following the incorporation of water-soluble
[3-3H]serine (10,000 – 40,000 cpm/nmol) into chloroform-soluble PS as
described by Bae-Lee and Carman (41). The reaction mixture contained
50 mM Tris-HCl buffer, pH 8.0, 10 mM MgCl2, 3.2 mM Triton X-100, 0.2
mM CDP-diacylglycerol, 0.5 mM serine, and enzyme protein in a total
volume of 0.1 ml. MgCl2 was used instead of MnCl2 (the alternative
cofactor (41)) because DGPP chelates manganese ions. DGPP phosphatase and ␤-galactosidase assays were conducted at 30 and 25 °C, respectively. The average standard deviation of the enzyme assays (performed in triplicate) was ⫾ 5%. The enzyme reactions were linear with
time and protein concentration. A unit of enzymatic activity was defined as the amount of enzyme that catalyzed the formation of 1 ␮mol
of product/min unless otherwise indicated. Specific activity was defined
as units/mg protein. Protein concentration was determined by the
method of Bradford (47) using bovine serum albumin as the standard.
Kinetic data were analyzed with the EZ-FIT enzyme kinetic modelfitting program according to the Michaelis-Menten and Hill equations.
EZ-FIT uses the Nelder-Mead Simplex and Marquardt/Nash nonlinear
regression algorithms sequentially and tests for the best fit of the data
among different kinetic models (48). IC50 values were calculated from
plots of the log of activity versus the inhibitor concentration.
Labeling and Analysis of Phospholipids—Labeling of phospholipids
with 32Pi was performed as described previously (8, 9, 49). Lipids were
extracted from labeled cells by the method of Bligh and Dyer (50) as
described previously (51). Phospholipids were separated by DEAEcellulose chromatography followed by one-dimensional thin layer chromatography on silica gel plates (52).2 DEAE-cellulose (acetate form)
2
This method of phospholipid analysis obviated the frustrations en-
40890
Regulation of DPP1-encoded DGPP Phosphatase
FIG. 2. Effect of inositol supplementation on the level of DGPP phosphatase activity in the exponential and
stationary phases of growth. Wildtype cells were grown in complete synthetic media with the indicated concentrations of inositol. Cultures were
harvested in the exponential (A) and stationary (B) phases of growth. Cell extracts
were prepared and assayed for DGPP
phosphatase activity. Each data point
represents the average of triplicate enzyme determinations from a minimum of
two independent experiments ⫾ S.D.
was packed (0.5-ml bed volume) and equilibrated in disposable Pasteur
pipettes with the solvent system chloroform/methanol/water (2:3:1, v/v).
Samples, in the same solvent system, were applied to columns under
the flow of gravity. The columns were washed with 3 ml of chloroform/
methanol/water (2:3:1, v/v) (fraction 1), 3 ml of chloroform/methanol/80
mM ammonium acetate (2:3:1, v/v) (fraction 2), and 3 ml of chloroform/
methanol/120 mM ammonium acetate (2:3:1, v/v) (fraction 3). PC and
PE emerged in fraction 1; PI, PS, and PA emerged in fraction 2, and
DGPP and CDP-DG emerged in fraction 3. Chloroform and water were
added to each fraction so that the final ratio of chloroform/methanol/
aqueous solvent was 1:1:1 (v/v). The system was mixed; the phases were
separated, and the chloroform phase was dried in vacuo. Samples from
each fraction were dissolved in chloroform/methanol (9:1) and subjected
to one-dimensional thin layer chromatography on silica gel plates using
the solvent system chloroform/pyridine/88% formic acid/methanol/water (60:35:10:5:2, v/v). Prior to the application of the samples, 20 ␮l of a
3% solution of butylated hydroxyanisol in methanol was applied to the
origin to protect samples against oxidation. The 32P-labeled phospholipids were visualized and quantified by PhosphorImaging analysis.
The positions of the labeled phospholipids on chromatography plates
were compared with standard lipids after exposure to iodine vapor.
RESULTS
Effects of Inositol and Growth Phase on the Level of DGPP
Phosphatase Activity—We examined the effect of inositol on the
level of DGPP phosphatase activity. Wild-type cells were grown
in complete synthetic medium in the absence and presence of
various concentrations of inositol. Cultures were harvested in
the exponential phase of growth. Cell extracts were prepared
and assayed for DGPP phosphatase activity. The addition of
inositol to the growth medium resulted in a dose-dependent
increase in DGPP phosphatase activity (Fig. 2A). Maximum
DGPP phosphatase activity was found in cells grown with
40 – 60 ␮M inositol. This level of activity was 2-fold greater than
that found in cells grown without inositol. Concentrations of
inositol above 60 ␮M led to a dose-dependent decrease in DGPP
phosphatase activity (Fig. 2A). The reason for this effect was
unclear and was not examined further. Studies with the purified DGPP phosphatase enzyme showed that the effect of inositol on the level of DGPP phosphatase activity was not due to
a direct effect of inositol on the enzyme (data not shown).
We examined the effect of growth phase on the level of DGPP
phosphatase activity. The specific activity of DGPP phosphatase in stationary phase cells was elevated (2.2-fold) when
compared with the activity in exponential phase cells (Fig. 2B).
In addition, the inositol-dependent regulation of DGPP phosphatase activity was also observed in stationary phase cells
(Fig. 2B). The expression of activity reached a maximum becountered with the poor resolution of phospholipids using two-dimensional thin layer chromatography due to humid summer days in New
Jersey.
tween 60 and 100 ␮M inositol. The growth phase-dependent and
inositol-dependent regulation of DGPP phosphatase activity
appeared to be additive. The specific activity of DGPP phosphatase in inositol-supplemented stationary phase cells was
5.5-fold greater than the activity of the enzyme in exponential
phase cells grown without inositol (Fig. 2).
Effect of Inositol and Growth Phase on DGPP Phosphatase
mRNA Abundance and Protein Levels—To gain insight into the
mechanism of DGPP phosphatase regulation by inositol and
growth phase, the amount of DPP1 mRNA was examined.
Wild-type cells were grown in complete synthetic medium in
the absence and presence of 50 ␮M inositol. This concentration
of inositol is commonly used for studies on the regulation of
phospholipid synthesis by inositol (1, 2). Total RNA was isolated from exponential and stationary phase cells and used for
Northern blot analysis with a DPP1 probe. The expression of
TCM1 mRNA was also determined and served as a loading
control. The TCM1 gene encodes a ribosomal protein that is not
regulated by inositol supplementation (53, 54). This analysis
showed that the presence of inositol in the growth medium
resulted in an increase in the relative amount of DPP1 mRNA
in both exponential (Fig. 3A) and stationary (Fig. 3C) phase
cells. This analysis also showed that the relative amount of
DPP1 mRNA in stationary phase cells supplemented with inositol was greater than that found in exponential phase cells
grown without inositol. During the course of these experiments, we noted that the total RNA derived from stationary
phase cells was especially subject to degradation during its
isolation. Thus, it was difficult to quantify the levels of mRNA.
The levels of the DGPP phosphatase protein in response to
inositol supplementation and growth phase were examined.
Antibodies were generated against a peptide sequence found at
the C-terminal end of the DGPP phosphatase protein. These
antibodies recognized purified DGPP phosphatase (data not
shown), as well as the enzyme in cell extracts. DGPP phosphatase migrates on SDS-polyacrylamide gels as a doublet with a
molecular mass of ⬃34 kDa (20, 55). Immunoblot analysis
using these antibodies showed that the levels of the DGPP
phosphatase protein were elevated (4- and 3-fold, respectively)
in response to inositol supplementation in both the exponential
(Fig. 3B) and stationary (Fig. 3D) phases of growth. This analysis also showed that in cells grown without inositol, the level
of the DGPP phosphatase protein was elevated (3-fold) in stationary phase when compared with the exponential phase of
growth. The amount of the DGPP phosphatase protein in stationary phase cells supplemented with inositol was 7-fold
greater when compared with that of exponential phase cells
grown without inositol. These results were consistent with the
Regulation of DPP1-encoded DGPP Phosphatase
FIG. 3. Effect of inositol supplementation on the levels of
DGPP phosphatase mRNA and protein in the exponential and
stationary phases of growth. Wild-type cells were grown in complete
synthetic media in the absence and presence of 50 ␮M inositol. Total
RNA was extracted from cells harvested in the exponential (A) and
stationary (C) phases of growth. The abundance of DPP1 mRNA was
determined by Northern blot analysis. 25 ␮g of total RNA was applied
to each lane. Portions of Northern blots are shown, and the positions of
DPP1 mRNA and TCM1 mRNA (loading control) are indicated. Cell
extracts were prepared from cells harvested in the exponential (B) and
stationary (D) phases of growth and subjected to immunoblot analysis
using a 1:1000 dilution of anti-DGPP phosphatase antibodies. 37.5 ␮g of
total protein was applied to each lane. Portions of the immunoblot are
shown, and the position of the 34-kDa DGPP phosphatase protein
(Dpp1p) is indicated. The data shown is representative of two independent experiments.
levels of DGPP phosphatase activity found in these cells
(Fig. 2).
Effect of Inositol and Growth Phase on the Expression of
␤-Galactosidase Activity in Cells Bearing the PDPP1-lacZ Reporter Gene—We utilized a PDPP1-lacZ reporter gene to facilitate further studies on the regulation of DPP1 expression. The
PDPP1-lacZ gene in plasmid pJO2 was constructed by fusing the
DPP1 promoter in frame with the coding sequence of the E. coli
lacZ gene. Thus, the expression of ␤-galactosidase activity was
dependent on transcription driven by the DPP1 promoter. The
␤-galactosidase activity in extracts derived from wild-type cells
bearing plasmid pJO2 was linear with time and with protein
concentration (data not shown). To verify that this reporter
gene could be used to examine the expression of the DPP1 gene,
we examined the regulation of ␤-galactosidase activity in exponential phase cells supplemented with inositol. The addition
of inositol to the growth medium resulted in a dose-dependent
increase in ␤-galactosidase activity (Fig. 4). The level of activity
in cell extracts derived from cells supplemented with 40 –50 ␮M
inositol was 2.6-fold greater than the activity from cells grown
in the absence of inositol (Fig. 4). The addition of inositol to
cells at concentrations greater than 50 ␮M inositol resulted in a
dose-dependent decrease in ␤-galactosidase activity (Fig. 4).
These results were consistent with the data on DGPP phosphatase activity (Fig. 2A).
The ␤-galactosidase activity, driven by the PDPP1-lacZ reporter gene, was also measured in stationary phase cells grown
in the absence and presence of 50 ␮M inositol (Fig. 5C). In the
absence of inositol supplementation, the ␤-galactosidase activity in stationary phase cells was 4.6-fold greater then the
activity found in exponential phase cells (Fig. 5A). The level of
␤-galactosidase activity in inositol-supplemented stationary
phase cells was 1.8-fold greater when compared with stationary
phase cells grown without inositol (Fig. 5C). The specific activity of ␤-galactosidase in stationary phase cells supplemented
with inositol (Fig. 5C) was 8.5-fold greater than that of exponential phase cells grown with inositol (Fig. 5A). These data
further confirmed that a transcriptional mechanism was responsible for the changes in DGPP phosphatase activity (Fig. 5,
40891
FIG. 4. Effect of inositol supplementation on expression of
␤-galactosidase activity in wild-type cells bearing the PDPP1lacZ reporter gene. Wild-type cells bearing the PDPP1-lacZ reporter
plasmid pJO2 were grown to the exponential phase of growth in the
absence and presence of the indicated concentrations of inositol. Cell
extracts were prepared and used for the assay of ␤-galactosidase activity. Each data point represents the average of triplicate determinations
from a minimum of two independent experiments ⫾ S.D.
B and D) that occurred in response to inositol supplementation
and growth phase.
For many phospholipid biosynthetic enzymes, the repressive
effect of inositol is enhanced by the inclusion of 1 mM choline to
the growth medium (1, 2, 4, 7, 17). We examined whether
choline alone or in combination with inositol affected the expression of the DPP1 gene and DGPP phosphatase activity.
This analysis showed that choline supplementation had no
effect on the regulation of DGPP phosphatase (data not shown).
Regulation of DGPP Phosphatase by Inositol and Growth
Phase in Mutants Defective in the OPI1, INO2, and INO4
Regulatory Genes—We examined the regulation of DGPP phosphatase in mutants defective in the OPI1, INO2, and INO4
regulatory genes. Opi1p, Ino2p, and Ino4p are transcriptional
regulators of phospholipid biosynthetic enzymes that are repressed by inositol (1, 2, 4, 7). For example, Opi1p represses the
expression of INO1-encoded inositol-1-P synthase and CHO1encoded PS synthase, whereas Ino2p and Ino4p induce the
expression of these enzymes (40, 54, 56 – 65). An opi1 mutant
exhibits elevated expression of phospholipid biosynthetic enzymes, and the expression of these enzymes does not respond to
inositol supplementation (1, 7, 17). Owing to the constitutive
low expression of the INO1 gene (53), ino2 and ino4 mutants
are inositol auxotrophs (28). These mutants also exhibit repressed levels of the phospholipid biosynthetic enzymes that
are repressed by inositol (1, 7, 17, 66, 67). Moreover, the expression of the inositol-regulated enzymes in ino2 and ino4
mutants is not affected by inositol supplementation (1, 7, 17,
66, 67). For cells grown to exponential phase without inositol,
the expression of PDPP1-lacZ driven ␤-galactosidase activity
was 2-fold lower in opi1⌬ mutant cells when compared with
wild-type cells (Fig. 5A). In contrast to wild-type cells, the
addition of inositol to the growth medium of opi1⌬ cells did not
result in an increase in DPP1 expression. Instead, inositol
supplementation caused a small decrease in DPP1 expression.
We next examined the expression of DGPP phosphatase activity in exponential phase opi1⌬ cells (Fig. 5B). The DGPP phosphatase activity in cells grown in the absence of inositol was
slightly higher than that found in wild-type cells. This level of
DGPP phosphatase activity did not correlate with the reduced
level of ␤-galactosidase activity in opi1⌬ cells when compared
with the wild-type control (Fig. 5A). Whether this difference
was due to transcriptional control versus translational control
will require additional studies. The addition of inositol to opi1⌬
cells resulted in a small reduction in DGPP phosphatase activ-
40892
Regulation of DPP1-encoded DGPP Phosphatase
FIG. 5. Effects of the opi1⌬, ino2⌬,
and ino4⌬ mutations on the regulation of DGPP phosphatase by inositol
and growth phase. Wild-type, opi1⌬,
ino2⌬, and ino4⌬ cells bearing the PDPP1lacZ reporter plasmid pJO2 were grown
in the absence and presence of 50 ␮M inositol. Cultures were harvested in the exponential and stationary phases of
growth. Cell extracts were prepared and
assayed for ␤-galactosidase activity (A
and C) and for DGPP phosphatase activity (B and D). Each data point represents
the average of triplicate enzyme determinations from a minimum of two independent experiments ⫾ S.D. WT, wild-type.
ity (Fig. 5B), which correlated with the small effect of inositol
on the expression of ␤-galactosidase activity (Fig. 5A).
The growth phase-dependent regulation of DGPP phosphatase was examined in opi1⌬ mutant cells (Fig. 5, C and D). As
in wild-type cells, the expressions of ␤-galactosidase and DGPP
phosphatase activities were elevated in stationary phase opi1⌬
cells when compared with these activities in exponential phase
cells. However, in contrast to the lower activity found in the
exponential phase, the level of expression of ␤-galactosidase
activity in stationary phase of opi1⌬ cells was similar to the
␤-galactosidase expression found in wild-type stationary phase
cells. Like exponential phase opi1⌬ cells, the expression of
␤-galactosidase and DGPP phosphatase activities did not increase in response to inositol supplementation in stationary
phase (Fig. 5, C and D).
The regulation of DGPP phosphatase was examined in ino2⌬
and ino4⌬ mutant cells grown in the presence of 10 and 60 ␮M
inositol. The 10 ␮M concentration of inositol was required to
support the growth of these inositol auxotrophs (68). This
growth condition was considered analogous to the growth condition of wild-type cells grown in the absence of inositol (53).
The expression of ␤-galactosidase activity in ino2⌬ mutant cells
was reduced in the exponential phase of growth when compared with expression of ␤-galactosidase activity in wild-type
cells (Fig. 5A). However, ino4⌬ mutant cells showed ␤-galactosidase activity that was slightly higher than that found in
wild-type cells (Fig. 5A). The DGPP phosphatase activity in
exponential phase ino2⌬ and ino4⌬ mutant cells was similar to
that of wild-type cells (Fig. 5B). The addition of 60 ␮M inositol
to the growth medium had small effects on the expression of
both ␤-galactosidase (Fig. 5A) and DGPP phosphatase (Fig. 5B)
activities in the ino2⌬ and ino4⌬ mutant cells.
The effects of the ino2⌬ and ino4⌬ mutations on DGPP
phosphatase regulation by growth phase were examined. The
␤-galactosidase (Fig. 5C) and DGPP phosphatase (Fig. 5D)
activities in stationary phase ino2⌬ and ino4⌬ mutant cells
were elevated when compared with these activities from exponential phase ino2⌬ and ino4⌬ mutant cells. However, both
␤-galactosidase and DGPP phosphatase activities were lower
(2.7- and 1.6-fold, respectively) in ino2⌬ mutant cells when
compared with wild-type cells. The ␤-galactosidase and DGPP
phosphatase activities in the ino4⌬ mutant were similar to the
levels of these activities found in wild-type cells (Fig. 5, C and
D). The expression of ␤-galactosidase and DGPP phosphatase
activities in stationary phase ino2⌬ and ino4⌬ mutant cells
supplemented with 60 ␮M inositol were elevated but not to the
levels observed in wild-type cell supplemented with inositol
(Fig. 5, C and D).
Effect of the dpp1⌬ Mutation on the Regulation of the INO1
Gene by Inositol and Growth Phase—We questioned whether
the dpp1⌬ mutation affected the expression of genes encoding
phospholipid biosynthetic enzymes. Of these genes, INO1 is the
most highly regulated by inositol supplementation (1, 2), and it
was used as a representative gene for this study. Expression of
INO1 in the dpp1⌬ mutant was examined by using a PINO1lacZ reporter gene in plasmid pJ699Z. The expression of ␤-galactosidase activity in wild-type and dpp1⌬ mutant cells was
the same in both exponential and stationary phase cells (data
not shown). As described previously (53, 56, 57, 64, 69, 70), the
INO1 gene was repressed by supplementation with 50 ␮M inositol. The dpp1⌬ mutation did not have a major effect on this
regulation (data not shown).
Inhibition of DGPP Phosphatase Activity by CDP-DG—We
initiated studies to examine the regulation of DGPP phosphatase activity on a biochemical level. Being a membrane-associated enzyme (20, 22), DGPP phosphatase has a distinct relationship with its neighboring phospholipids. Thus, we
examined whether phospholipids played a role in the biochemical regulation of the enzyme. For these studies, we used purified DGPP phosphatase and Triton X-100/phospholipid mixed
micelles so the effects of phospholipids on activity could be
examined under well defined conditions. The nonionic detergent Triton X-100 is required to elicit a maximum turnover for
DGPP phosphatase activity in vitro (20). The function of Triton
X-100 in the assay system for DGPP phosphatase is to form a
uniform mixed micelle with the substrate DGPP (20). The
Triton X-100 micelle serves as a catalytically inert matrix in
which the DGPP is dispersed, preventing a high local concentration of substrate at the active site (71). In addition, this
micelle system permitted the analysis of DGPP phosphatase
Regulation of DPP1-encoded DGPP Phosphatase
FIG. 6. Effect of phospholipids on DGPP phosphatase activity.
A, DGPP phosphatase activity was measured under standard assay
condition with 0.3 mol % DGPP in the absence and presence of various
surface concentrations of the indicated phospholipids. B, DGPP phosphatase activity was measured under standard assay conditions with
0.3 mol % DGPP in the absence and presence of the indicated concentrations of CDP. C, DGPP phosphatase activity was measured as a
function of the surface concentration (mol %) of DGPP in the absence
and presence of 6 mol % of CDP-DG.
activity in an environment that mimics the physiological surface of the membrane (71). In Triton X-100/phospholipid-mixed
micelles, DGPP phosphatase activity follows surface dilution
kinetics (71), where activity is dependent on both the molar and
surface concentrations of DGPP (20). In the experiments reported here, DGPP phosphatase activity was measured such
that activity was only dependent on the surface concentration
of DGPP (20). The concentrations of DGPP and other phospholipids were expressed as a surface concentration (in mol %), as
opposed to a molar concentration, since phospholipids form
uniform mixed micelles with Triton X-100 (44, 45).
DGPP phosphatase activity was assayed in the presence of
various phospholipids. The major yeast phospholipids PC, PE,
PI, and PS inhibited DGPP phosphatase activity at a final
concentration of 10 mol % (Fig. 7A). However, these phospholipids were not considered to be strong inhibitors (30% or less)
and were not pursued further. PA, at concentrations up to 10
mol %, does not affect DGPP phosphatase activity (20). On the
other hand, the addition of CDP-DG to the assay system resulted in a dose-dependent inhibition (IC50 ⫽ 5.3 mol %) of
DGPP phosphatase activity (Fig. 6A). DGPP phosphatase activity was measured in the presence of CDP and DG to examine
which part of the CDP-DG molecule was responsible for the
inhibition. CDP caused a dose-dependent inhibition (IC50 ⫽ 2.6
mM) of activity (Fig. 6B). DG caused a small increase (25%) in
DGPP phosphatase activity at a final concentration of 4 mol %
(Fig. 6A). These data indicated that the inhibition by CDP-DG
may be attributed to the CDP moiety of the molecule. A kinetic
analysis was performed on DGPP phosphatase to explore the
40893
FIG. 7. Effect of DGPP on PS synthase activity. A, PS synthase
activity was measured under standard assay condition with 1 mol %
DGPP in the absence and presence of the indicated surface concentrations of DGPP. B, PS synthase activity was measured as a function of
the surface concentration (mol %) of CDP-DG in the absence and presence of 1 mol % of DGPP. The serine concentration was held constant at
0.5 mM. C, PS synthase activity was measured as a function of the
concentration of serine in the absence and presence of 1 mol % of DGPP.
The CDP-DG concentration was held constant at 2 mol %.
mechanism of inhibition by CDP-DG. The dependence of DGPP
phosphatase activity on DGPP was examined in the absence
and presence of 4 mol % CDP-DG (Fig. 6C). As described
previously (20), DGPP phosphatase exhibited saturation kinetics with respect to the surface concentration of DGPP. CDP-DG
inhibited DGPP phosphatase activity in a dose-dependent
manner at each DGPP concentration. An analysis of the kinetic
data showed that CDP-DG was a mixed type of inhibitor (72)
causing an increase in Km and a decrease in Vmax. The Ki value
for CDP-DG was calculated to be 5 mol %.
Activation of PS Synthase Activity by DGPP—PS synthase is
one of the most highly regulated enzymes of phospholipid metabolism (1, 2, 7, 17), and the biochemical regulation of the
purified enzyme (41) has been extensively studied (3, 4). In
light of the fact that CDP-DG regulated DGPP phosphatase
activity, we examined the effect of DGPP on PS synthase activity. For these studies, we utilized pure enzyme and the
Triton X-100/phospholipid mixed micelle system as discussed
above for DGPP phosphatase. The addition of DGPP to the
assay system resulted in a dose-dependent stimulation of PS
synthase activity (Fig. 7A). The concentration of DGPP that
resulted in half-maximum activation (A0.5) was 0.13 mol %. The
stimulation by DGPP could be attributed to the pyrophosphate
moiety of the molecule since DG does not stimulate PS synthase activity (73). At high concentrations (e.g. 11 mol %), DG
40894
Regulation of DPP1-encoded DGPP Phosphatase
TABLE II
Effect of the dpp1⌬ mutation on phospholipid composition in stationary phase cells grown in the absence and presence of inositol
The indicated S. cerevisiae strains were grown to the stationary (1 ⫻ 108 cells/ml) phase of growth in complete synthetic medium in the absence
and presence of 50 ␮M inositol. The steady-state phospholipid composition was determined by labeling cells for 12 generations with 32Pi (5 ␮Ci/ml).
The incorporation of 32Pi into phospholipids during the labeling was approximately 2400 cpm/108 cells. The phospholipid composition of the cells
was determined as described under “Experimental Procedures.” The percentages shown for phospholipids were normalized to the total 32Pi-labeled
chloroform-soluble fraction that included sphingolipids and other unidentified phospholipids. The values reported are the average of two
independent experiments.
Growth condition
PC
PE
PI
PS
PA
DGPP
CDP-DG
PI:PC
%
Complete synthetic
medium
Wild type
dpp1⌬
⫹ 50 ␮M inositol
Wild type
dpp1⌬
38 ⫾ 1.0
39 ⫾ 1.4
10 ⫾ 3.0
12 ⫾ 0.6
4.0 ⫾ 0.3
3.5 ⫾ 0.2
3.6 ⫾ 0.05
2.4 ⫾ 0.03
1.2 ⫾ 0.3
0.9 ⫾ .08
0.4 ⫾ 0.1
0.4 ⫾ 0.2
0.20 ⫾ 0.1
0.37 ⫾ 0.08
0.11
0.09
35 ⫾ 2.3
39 ⫾ 1.1
12.1 ⫾ 1.2
12.8 ⫾ 1.6
18 ⫾ 1.0
4.8 ⫾ 0.1
3.9 ⫾ 0.6
4.0 ⫾ 0.8
1.2 ⫾ 0.1
1.1 ⫾ 0.04
0.4 ⫾ 0.2
0.3 ⫾ 0.01
0.26 ⫾ 0.1
0.36 ⫾ 0.1
0.51
0.12
inhibits PS synthase activity (73). The dependence of PS synthase activity on the surface concentration of CDP-DG was
examined in the absence and presence of 1 mol % DGPP (Fig.
7B). In the absence of DGPP, PS synthase exhibited positive
cooperative kinetics (n ⫽ 3.2) toward CDP-DG with a Km value
of 1.9 mol %. The enzyme was stimulated by DGPP in a dosedependent manner at each CDP-DG concentration. Moreover,
DGPP caused a decrease in the cooperative (n ⫽ 1.6) kinetic
behavior of the enzyme and a decrease in the Km value (0.8 mol
%) for CDP-DG. Thus, at low CDP-DG concentrations DGPP
had a major stimulatory effect on PS synthase activity. The
Vmax of the reaction was not significantly affected by DGPP.
The effect of DGPP (1 mol %) on the kinetics of the enzyme with
respect to serine was examined (Fig. 7C). The enzyme exhibited
saturation kinetics toward serine in the absence (Km ⫽ 2.7 mM)
and presence (Km ⫽ 2.3 mM) of DGPP. The Vmax of the reaction
in the presence (2.84 units/mg) of DGPP was 2-fold greater
than that in absence (1.4 units/mg) of DGPP. These kinetic
data indicated that DGPP stimulated PS synthase activity by a
mechanism that increased the affinity of the enzyme for
CDP-DG (72).
Effect of a dpp1⌬ Mutation on the Phospholipid Composition
of Stationary Phase Cells Grown in the Presence of Inositol—We
examined the effects of a dpp1⌬ mutation on the phospholipid
composition of stationary phase cells. Stationary phase cells
that were grown in the absence and presence of 50 ␮M inositol
were labeled with 32Pi. Phospholipids were extracted and analyzed as described under “Experimental Procedures.” When
grown without inositol supplementation, the amounts of the
major phospholipids (i.e. PC, PE, PI, and PS) as well as PA and
DGPP were not significantly affected by the dpp1⌬ mutation
(Table II). The amount of CDP-DG in the dpp1⌬ mutant was
nearly 50% greater than that of wild-type cells grown without
inositol (Table II). The presence of inositol in the growth medium of wild-type cells resulted in a 4.5-fold increase in PI
content and a small increase in CDP-DG content (Table II). On
the other hand, the PI content in dpp1⌬ mutant cells increased
by only 1.37-fold, and the CDP-DG content did not change
when cells were supplemented with inositol (Table II). The
PI:PC ratio in the dpp1⌬ mutant grown in the presence of
inositol was 4.2-fold lower than that of wild-type cells (Table
II). The amounts of the other phospholipids in the dpp1⌬ mutant supplemented with inositol were not significantly different
from that of wild-type cells supplemented with inositol.
DISCUSSION
The identification and initial analysis of DPP1-encoded
DGPP phosphatase indicates that this enzyme plays a previously unidentified role in phospholipid metabolism in S. cerevisiae (20, 22). It has been postulated that DGPP may function in
a novel lipid-signaling pathway (4, 74). Studies with plants,
where DGPP was first discovered (21), have shown that this
phospholipid accumulates upon G protein activation (75) or
hyperosmotic stress (76, 77). DGPP accumulation is transient
(77), and it is rapidly converted to PA and then to DG (42). In
S. cerevisiae, DGPP phosphatase may function to regulate cellular levels of DGPP, PA, and DG (3, 4). DGPP has not been
identified in mammalian cells. However, Balboa et al. (78) have
shown that exogenous DGPP activates mouse macrophages for
enhanced secretion of arachidonic acid metabolites, a key event
in the immunoinflammatory response of leukocytes.
To gain insight into the role that DGPP phosphatase plays in
S. cerevisiae, we examined the effects of inositol supplementation and growth phase on the expression of the enzyme. These
two growth conditions have a major impact on the expression of
several phospholipid biosynthetic enzymes (1, 2, 4, 7, 17). Inositol supplementation to wild-type cells resulted in the elevation of DGPP phosphatase activity in both the exponential and
stationary phases of growth. DGPP phosphatase activity was
higher in stationary phase cells when compared with exponential phase cells. Moreover, the inositol- and growth phase-dependent regulation of the enzyme were additive. Analyses of
the DGPP phosphatase mRNA abundance and protein levels,
as well as the expression of ␤-galactosidase activity driven by a
PDPP1-lacZ reporter gene, showed that a transcriptional mechanism was responsible for this regulation. The effects of inositol
and growth phase on DGPP phosphatase expression was opposite that of most phospholipid biosynthetic enzymes. For example, CDP-DG pathway enzymes (i.e. CDP-DG synthase, PS
synthase, PS decarboxylase, and phospholipid N-methyltransferases) are repressed when wild-type cells are supplemented
with inositol and when they enter the stationary phase of
growth (1, 2, 4, 7, 17). The repression of these enzymes by
inositol supplementation or in stationary phase is mediated by
a UASINO cis-acting element (1, 64, 79) present in the promoters of their genes (1, 2, 4, 7, 17). The promoter of the DPP1 gene
does not contain a UASINO element. Additional studies will be
required to identify the promoter element(s) responsible for the
inositol- and growth phase-dependent regulation of the DPP1
gene.
DGPP phosphatase is not the first example of an enzyme
that is regulated by inositol or by growth phase in a manner
that is opposite to the co-regulated phospholipid biosynthetic
enzymes whose genes contain a UASINO element (4). These
enzymes include 45-kDa Mg2⫹-dependent PA phosphatase (51,
80) and inositol-1-P phosphatase (81, 82). The magnitude of the
regulation by inositol supplementation and growth phase observed for DGPP phosphatase was significantly greater than
that of these other enzymes. Expression of cardiolipin synthase
Regulation of DPP1-encoded DGPP Phosphatase
is not regulated by inositol supplementation, but it is derepressed in stationary phase (83). Phosphatidylglycerophosphate synthase, whose promoter contains a UASINO element, is
repressed by inositol supplementation (84, 85) but is derepressed in the stationary phase (86).
The UASINO element contains the binding site for the Ino2pIno4p complex, which is necessary for maximum expression of
the co-regulated UASINO-containing genes (4, 7, 17, 53, 67).
Repression of the co-regulated genes requires the transcription
factor Opi1p (59, 87). Although Opi1p functions via the UASINO
element (63), it does not bind the element directly and does not
interact with Ino2p or Ino4p (88). The DPP1 gene does not have
a UASINO element, yet Opi1p, Ino2p, and Ino4p played a role in
its regulation by inositol. In both exponential and stationary
phase cells, expression of DPP1 was not regulated normally by
inositol in the opi1⌬, ino2⌬, and ino4⌬ mutants. Moreover, the
aberrant regulation observed in the ino2⌬ and ino4⌬ mutants
differed from each other (expression of DPP1 was greater in the
ino4⌬ mutant). This suggests that an Ino2p-Ino4p complex
may be required for proper DPP1 regulation. Differential regulatory effects between the ino2⌬ and ino4⌬ mutants have
been observed for the 45-kDa Mg2⫹-dependent PA phosphatase
(51), PI synthase (89), and for cardiolipin synthase.3 The regulation of DPP1 by growth phase was not affected in the regulatory mutants suggesting that Opi1p, Ino2p, and Ino4p do
not play a role in the growth phase-dependent regulation of
DGPP phosphatase.
Phospholipid synthesis in S. cerevisiae has been described as
existing in two regulatory states designated as “on” and “off”
(4). The system is on when the UASINO-containing genes are
maximally expressed and off when they are repressed. When
the UASINO-containing genes are on, the DPP1 gene is off and
vice versa. Analysis of phenotypes associated with mutations in
the UASINO-containing genes have led to the hypothesis that
PA, or a closely related metabolite, is responsible for generating a signal that causes the derepression of these genes (4, 17).
According to the model, the system is on when PA is produced
more rapidly than it is consumed, and the system is off when
PA is consumed more rapidly than it is produced (4). Since
DGPP phosphatase is involved with the metabolism of PA, we
questioned whether the enzyme was involved in the regulation
of the UASINO-containing genes. The dpp1⌬ mutant did not
have a significant effect on the regulation of the INO1 gene by
inositol. If DGPP phosphatase were involved, other enzymes
(e.g. PA kinase, CDP-DG synthase, and phospholipase D) that
catalyze reactions affecting the metabolism of PA may have
compensated for the dpp1⌬ mutation. A mutation in the LPP1
gene, which encodes a lipid phosphatase that utilizes PA and
DGPP as substrates (23, 90), does not affect the regulation of
the INO1 gene.4
CDP-DG and DGPP, substrates of PS synthase and DGPP
phosphatase, regulated DGPP phosphatase and PS synthase
activities, respectively. CDP-DG was a mixed type inhibitor of
DGPP phosphatase. The inhibitor constant for CDP-DG (Ki ⫽ 5
mol %) was about 12-fold higher than its cellular concentration
in stationary phase cells (Table II). On the other hand, the Ki
value was within the range of the cellular concentration of
CDP-DG in exponential phase cells (2–11 mol %) (91, 92). Thus,
it is unlikely that CDP-DG regulates DGPP phosphatase activity in stationary phase cells, but regulation of the phosphatase
by CDP-DG may be physiologically relevant in exponential
phase cells. DGPP stimulated PS synthase through a mechanism that involved an increase in the affinity of the enzyme for
3
4
W. Dowhan, personal communication.
J. E. Quinn and G. M. Carman, unpublished data.
40895
CDP-DG. The activation constant for DGPP (A0.5 ⫽ 0.13 mol %)
was within the range of its cellular concentration in both exponential (0.2– 0.4 mol %) (20, 22) and stationary (Table II)
phase cells. Thus, regulation of PS synthase activity by DGPP
may occur in vivo during both phases of growth.
Previous studies have shown that the regulation of PS synthase is the major factor that controls the synthesis of PS and
PI from their common precursor CDP-DG (1, 3, 4). Stimulation
of PS synthase by DGPP would favor the synthesis of PS at the
expense of PI. DGPP did not affect the activity of PI synthase
(data not shown). The partitioning of CDP-DG between PS and
PI may be controlled by the activity and/or expression of DGPP
phosphatase. For example, reduced levels of DGPP phosphatase would favor the synthesis of PS over PI. Indeed, the major
impact of the dpp1⌬ mutation in stationary phase cells supplemented with inositol was a decrease in PI content when compared with wild-type cells. Moreover, this caused a major
change in the PI:PC ratio, an index of phospholipid synthesis
regulation (93, 94).
In summary, the work reported here supported the conclusion that the DPP1-encoded DGPP phosphatase was regulated
by genetic and biochemical mechanisms. Moreover, the enzyme
played a role in the regulation of phospholipid synthesis by
inositol. The synthesis of PI is coordinately regulated with the
synthesis of PC by both genetic and biochemical mechanisms
(1–3, 7, 17). Clearly, DGPP phosphatase plays a role in this
complex regulation.
Acknowledgments—We thank William Dowhan for helpful discussions and for plasmid pSD90. David A. Toke is acknowledged for helpful
suggestions with PCR and with the construction of plasmids. We thank
Susan A. Henry for providing the opi1⌬, ino2⌬, and ino4⌬ mutants and
plasmid pJ699Z and Akinori Ohta for plasmid YCpGPSS. We acknowledge Christian R. H. Raetz and Nanette L. S. Que for helpful suggestions in the analysis of phospholipids.
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