From the Department of Food Science, Cook College,
New Jersey Agricultural Experiment Station, Rutgers University,
New Brunswick, New Jersey 08903 and the § Lord and
Taylor Laboratory for Lung Biochemistry and the Anna Perahia Adatto
Clinical Research Center, National Jewish Center for Immunology and
Respiratory Medicine, Denver, Colorado 80206
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ABSTRACT |
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The DPP1-encoded diacylglycerol
pyrophosphate (DGPP) phosphatase enzyme accounts for half of the
Mg2+-independent phosphatidate (PA) phosphatase activity in
Saccharomyces cerevisiae. The LPP1
(lipid phosphate phosphatase)
gene encodes a protein that contains a novel phosphatase sequence motif
found in DGPP phosphatase and in the mouse Mg2+-independent
PA phosphatase. A genomic copy of the S. cerevisiae LPP1
gene was isolated and was used to construct lpp1 and
lpp1
dpp1
mutants. A multicopy plasmid
containing the LPP1 gene directed a 12.9-fold
overexpression of Mg2+-independent PA phosphatase activity
in the S. cerevisiae lpp1
dpp1
double
mutant. The heterologous expression of the S. cerevisiae LPP1 gene in Sf-9 insect cells resulted in a 715-fold
overexpression of Mg2+-independent PA phosphatase activity
relative to control insect cells. The Mg2+-independent PA
phosphatase activity encoded by the LPP1 gene was
associated with the membrane fraction of the cell. The LPP1 gene product also exhibited lyso-PA phosphatase and DGPP phosphatase activities. The order of substrate preference was PA > lyso-PA > DGPP. Like the dpp1
mutant, the
lpp1
mutant and the lpp1
dpp1
double mutant were viable and did not exhibit
obvious growth defects. Biochemical analyses of lpp1
,
dpp1
, and lpp1
dpp1
mutants showed that the LPP1 and DPP1 gene
products encoded nearly all of the Mg2+-independent PA
phosphatase and lyso-PA phosphatase activities and all of the DGPP
phosphatase activity in S. cerevisiae. Moreover, the
analyses of the mutants showed that the LPP1 and
DPP1 gene products played a role in the regulation of
phospholipid metabolism and the cellular levels of phosphatidylinositol
and PA.
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INTRODUCTION |
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PA1 phosphatase in the yeast Saccharomyces cerevisiae catalyzes the dephosphorylation of PA to yield DG and Pi (1). The DG derived from PA is used for the synthesis of phosphatidylethanolamine and phosphatidylcholine via the Kennedy (CDP-ethanolamine- and CDP-choline-based) pathway and is also used for the synthesis of triacylglycerols (2-4). Two types of PA phosphatase have been identified in S. cerevisiae (5). One type of PA phosphatase is Mg2+-dependent and N-ethylmaleimide-sensitive, and the other enzyme type is Mg2+-independent and N-ethylmaleimide-insensitive (5). Two membrane-associated forms (104- and 45-kDa) of the Mg2+-dependent PA phosphatase have been purified and characterized from S. cerevisiae (5-7). These enzymes are regulated by growth phase (7, 8), inositol supplementation (7, 8), phosphorylation via protein kinase A (9), phospholipids (10), sphingoid bases (11), and nucleotides (12). This regulation correlates with changes in the synthesis of phospholipids and triacylglycerols (8, 13, 14).
Much less is known about the Mg2+-independent type of PA
phosphatase and its role in phospholipid metabolism in S. cerevisiae. It was first identified as an activity of a 34-kDa
DGPP phosphatase enzyme (15) encoded by the DPP1 gene (16).
Pure DGPP phosphatase catalyzes the removal of the -phosphate of
DGPP to yield PA and then catalyzes the removal of the phosphate from
PA to yield DG (15). Although the DGPP phosphatase enzyme utilizes PA
as a substrate in the absence of DGPP, the specificity constant for PA
is 10-fold lower than that of DGPP (15). The deletion of the
DPP1 gene in S. cerevisiae results in the loss of
all detectable DGPP phosphatase activity and only a 50% loss in
Mg2+-independent PA phosphatase activity (16). These data
indicate the existence of an additional gene in S. cerevisiae encoding Mg2+-independent PA phosphatase
activity (16). The isolation of the gene encoding this enzyme is
required for defined studies to examine its role in phospholipid
metabolism.
The Mg2+-independent type of PA phosphatase also exists in mammalian cells (17-19). Indeed, the enzyme has been purified and characterized (20-24), and cDNAs encoding the enzyme have been isolated (24, 25). The purified enzyme from rat liver also utilizes LPA, ceramide 1-phosphate, sphingosine 1-phosphate, and DGPP as substrates (26, 27). Given the fact that these substrates and their hydrolysis products mediate a variety of cellular activities (18, 26, 28, 29), it has been postulated that Mg2+-independent PA phosphatase plays a role in lipid signaling pathways (17-19, 30).
In this paper we report the isolation and characterization of the
LPP1 (lipid phosphate
phosphatase) gene in S. cerevisiae. The
expression of the LPP1 gene in S. cerevisiae
cells on a multicopy plasmid and in Sf-9 insect cells by baculovirus
infection resulted in the overexpression of
Mg2+-independent PA phosphatase activity. The overexpressed
gene product also exhibited LPA phosphatase and DGPP phosphatase
activities. Biochemical analyses of an lpp1 mutant, a
dpp1
mutant, and an lpp1
dpp1
double mutant showed that the LPP1 and DPP1 gene
products encoded nearly all of the Mg2+-independent PA
phosphatase and LPA phosphatase activities and all of the DGPP
phosphatase activity in S. cerevisiae. Moreover, the
analyses of the mutants showed that the LPP1 and
DPP1 gene products played a role in the regulation of
phospholipid metabolism in S. cerevisiae and in particular
the cellular contents of phosphatidylinositol and PA.
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EXPERIMENTAL PROCEDURES |
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Materials
Growth medium supplies were purchased from Difco. Protein assay reagents were purchased from Bio-Rad. Restriction endonucleases, modifying enzymes, and recombinant Vent DNA polymerase with 5'-and 3'-exonuclease activity were purchased from New England Biolabs. Polymerase chain reaction (PCR) and sequencing primers were prepared commercially by Genosys Biotechnologies, Inc. The PCRScriptTM AMP SK(+) cloning kit was from Stratagene, and the YeastmakerTM yeast transformation system was obtained from CLONTECH. DNA sequencing kits were obtained from Applied Biosystems. The DNA size ladder used for agarose gel electrophoresis was purchased from Life Technologies, Inc.. The baculovirus transfer vector pVL1392 was obtained from Invitrogen. Triton X-100 and bovine serum albumin were purchased from Sigma. Lipids were purchased from Avanti Polar Lipids, Sigma, and Biomol. Radiochemicals were purchased from NEN Life Science Products. Scintillation counting supplies were from National Diagnostics. Silica Gel 60 thin layer chromatography plates were from EM Science. Escherichia coli DG kinase was obtained from Lipidex Inc.
Methods
Strains, Plasmids, and Growth ConditionsThe strains
and plasmids used in this work are listed in Tables
I and II,
respectively. Methods for yeast growth, sporulation, and tetrad
analysis were performed as described previously (31, 32). Yeast
cultures were grown in YEPD medium (1% yeast extract, 2% peptone, 2%
glucose) or in complete synthetic medium minus inositol (33) containing 2% glucose at 30 °C. The appropriate amino acid of complete
synthetic medium was omitted for selection purposes. E. coli
strain DH5
was grown in LB medium (1% tryptone, 0.5% yeast
extract, 1% NaCl (pH 7.4)) at 37 °C. Ampicillin (100 µg/ml) was
added to cultures of DH5
carrying plasmids. Media were supplemented
with either 2% (yeast) or 1.5% (E. coli) agar for growth
on plates. Yeast cell numbers in liquid media were determined by
microscopic examination with a hemacytometer or spectrophotometrically
at an absorbance of 600 nm. The inositol excretion phenotype (34) of
yeast strains was examined on complete synthetic medium (minus
inositol) by using growth of the inositol auxotrophic indicator strain
MC13 (ino1) (33) as described by McGee et al.
(35).
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DNA Manipulations, Amplification of DNA by PCR, and DNA
Sequencing--
Plasmid and genomic DNA preparation, restriction
enzyme digestion, and DNA ligations were performed by standard methods
(32). Transformation of yeast (36, 37) and E. coli (32) were
performed as described previously. Conditions for the amplification of
DNA by PCR were optimized as described previously (38). The annealing temperature for the PCRs was 55 °C, and extension times were
typically between 2.0 and 2.5 min at 72 °C. PCRs were routinely run
for a total of 30 cycles. DNA sequencing reactions were performed with
the Prism DyeDeoxy Terminator Cycle sequencing kit and analyzed with an
automated DNA sequencer. Plasmid maintenance and amplifications were
performed in E. coli strain DH5.
Isolation of the LPP1 Gene-- A computer search of the Saccharomyces Genome Data base indicated that the LPP12 gene (locus, YDR503C) (GenBankTM accession no. U33057) flanked the 3'-end of the PSP1 gene. We obtained plasmid pSK5+ (from Dr. Timothy Formosa) that contained genomic copies of the PSP1 and LPP1 genes. A 1.8-kb insert, containing the LPP1 gene, 755 bp of the promoter region, and 200 bp of the untranslated region, was released from pSK5+ by digestion with HpaI. This DNA fragment was ligated into the SmaI site of pRS426, a multicopy E. coli/yeast shuttle vector containing the URA3 gene (39) to form plasmid pWB1-LPP1. This construct was transformed into W303-1B and the indicated mutants for the overexpression of the LPP1 gene product.
Construction of an lpp1 Mutant--
The plasmid pSK5+ was
digested with HpaI/SalI to remove the
LPP1 gene. This DNA fragment was ligated into the
SmaI/SalI sites of pCRScriptTM AMP
SK(+) to form pCR-LPP1. The pCR-LPP1 construct was digested with
StyI/HindIII to remove the entire LPP1
coding sequence. The sites on the digested plasmid were converted to
blunt ends using the Klenow fragment of DNA polymerase I. A 2.5-kb
HIS3/Kanr disruption cassette, derived from
plasmid pJA50 (40) by SmaI digestion, was inserted into the
blunt-ended plasmid to form plasmid pCR-lpp1
. A linear 2.8-kb
LPP1 deletion cassette was released from pCR-lpp1
by
digestion with XhoI/NotI. This DNA fragment was
transformed into W303-1B to delete the chromosomal copy of the
LPP1 gene by the one-step gene replacement technique (41). Transformants were selected for their ability to grow on complete synthetic medium without histidine. The deletion of the chromosomal copy of the LPP1 gene was confirmed by PCR (primers,
5'-GAATGTCAATGAGTTTCGCAGAAGACG-3' and 5'-GTATTTTGGCTTCGGTTAATATCTGG-3')
using 30 cycles at 55 °C annealing temperature with a 3.5-min
extension time at 72 °C. The template for the PCR used to confirm
the LPP1 deletion was genomic DNA isolated from transformed
colonies. The PCR template for the native LPP1 gene was
genomic DNA isolated from W303-1B. One of the lpp1
mutants that we isolated was designated strain WBY1.
Construction of the lpp1 dpp1
Double Mutant--
The
lpp1
mutant was crossed with a dpp1
mutant
to form a diploid that was heterozygous for the LPP1 and
DPP1 alleles. Putative lpp1
dpp1
double mutants were selected for their ability to grow on complete
synthetic media lacking both histidine and tryptophan. The deletion of
the chromosomal copies of the LPP1 gene and the DPP1 gene (16) was confirmed by PCR as described above.
Strain TBY1 was one of the haploid lpp1
dpp1
double mutants that were isolated.
Recombinant Viral Expression of the S. cerevisiae LPP1 Gene in
Insect Cells--
Plasmid pWB1-LPP1 was digested with
BamHI/SalI to release the entire coding sequence
of the LPP1 gene. This DNA fragment was ligated into the
BamHI/SalI sites of the YEp352 vector resulting in the formation of plasmid pDT1-LPP1. This construct was then digested
with Bsu36I/SalI to reduce the remaining promoter
sequence to 26 bp upstream of the protein coding sequence. These sites on the plasmid were converted to blunt ends using the Klenow fragment of DNA polymerase I. The blunt ends were ligated together to form plasmid pDT2-LPP1. The pDT2-LPP1 plasmid was digested with
BamHI/PstI to release the LPP1 open
reading frame. This DNA fragment was then ligated into the
BamHI/PstI site of the baculovirus vector PVL1392
to form plasmid pWW1-LPP1. The pWW1-LPP1 plasmid was subsequently co-transfected with BaculoGoldTM Autographa
californica DNA (PharMingen) into a monolayer of Sf-9 cells using
the CaCl2 method. The Sf-9 cells were routinely grown in
TMNFH medium (42) containing 10% heat-inactivated fetal bovine serum.
General procedures for the growth, maintenance, and infection of Sf-9
cells followed the methods described by O'Reilly et al. (42). Routine infection of Sf-9 cells for LPP1 expression
used 1-2 × 107 cells grown in 75-cm2
tissue culture flasks. The cells were infected at a viral multiplicity of 10 and grown in TMNFH medium with 10% heat-inactivated fetal bovine
serum for 48 h. The infected cells were collected by gentle trituration with medium, harvested by centrifugation, and washed twice
with phosphate-buffered saline. The final cell pellet was snap-frozen
over dry ice and stored at 80 °C.
Preparation of Cell Extracts and Subcellular Fractions-- All steps were performed at 5 °C. Yeast cells were disrupted with glass beads with a Mini-Bead-Beater (Biospec Products) in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM Na2EDTA, 0.3 M sucrose, and 10 mM 2-mercaptoethanol (43). Glass beads and cell debris were removed by centrifugation at 1,500 × g for 5 min. The supernatant was used as the cell extract. Insect cells were disrupted by sonic oscillation in 50 mM Tris-HCl buffer (pH 7.5) containing 0.3 M sucrose, 1 mM Na2EDTA, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethanesulfonyl fluoride, 1 mM benzamide, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml pepstatin (16). The disrupted cell suspension was centrifuged at 1,500 × g for 5 min to remove unbroken cells and cell debris. The supernatant was used as the cell extract. The cell extract was centrifuged at 100,000 × g for 1.5 h to obtain the cytosolic (supernatant) and total membrane fractions.
Preparation of Substrates--
[32P]PA and
[32P]LPA were synthesized enzymatically from DG and
monoacylglycerol, respectively, using E. coli DG kinase (44) as described previously (6). DGPP standard and
[-32P]DGPP were synthesized enzymatically using
purified Catharanthus roseus PA kinase as described by Wu
et al. (15).
Enzyme Assays and Protein
Determination--
Mg2+-Independent PA phosphatase
activity was measured by following the release of water-soluble
32Pi from chloroform-soluble
[32P]PA (10,000 cpm/nmol) (45). The reaction mixture
contained 50 mM Tris maleate buffer (pH 6.5), 0.1 mM PA, 1 mM Triton X-100, 2 mM
Na2EDTA, 10 mM 2-mercaptoethanol, and enzyme in
a total volume of 0.1 ml. Mg2+-Dependent PA phosphatase
activity was measured with 50 mM Tris maleate buffer (pH
7.0), 10 mM 2-mercaptoethanol, 2 mM
MgCl2, 1 mM Triton X-100, 0.1 mM
[32P]PA, and enzyme protein (45). LPA phosphatase
activity was measured by following the release of water-soluble
32Pi from chloroform-soluble
[32P]LPA (20,000 cpm/nmol) (45). The reaction mixture
contained 50 mM Tris maleate buffer (pH 6.5), 0.1 mM LPA, 1 mM Triton X-100, 2 mM
Na2EDTA, 10 mM 2-mercaptoethanol, and enzyme
protein in a total volume of 0.1 ml. DGPP phosphatase activity was
measured by following the release of water-soluble
32Pi from chloroform-soluble
[-32P]DGPP (5,000-10,000 cpm/nmol) as described by Wu
et al. (15). The reaction mixture contained 50 mM citrate buffer (pH 5.0), 0.1 mM DGPP, 2 mM Triton X-100, 10 mM 2-mercaptoethanol, and
enzyme protein in a total volume of 0.1 ml. All enzyme assays were
conducted at 30 °C in triplicate. The average S.D. of the assays
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 nmol of product/min.
Specific activity was defined as units/mg of protein. Protein
concentration was determined by the method of Bradford (46) using
bovine serum albumin as the standard.
Labeling and Analysis of Phospholipids-- Steady-state labeling of phospholipids with 32Pi was performed as described previously (47-49). Lipids were extracted from labeled cells by the method of Bligh and Dyer (50) as described by Morlock et al. (8). Phospholipids were analyzed by two-dimensional thin layer chromatography on high performance silica gel thin layer chromatography plates using chloroform/methanol/glacial acetic acid (65:25:10, v/v) as the solvent for dimension one and chloroform/methanol, 88% formic acid (65:25:10, v/v) as the solvent for dimension two (51). The 32P-labeled phospholipids were analyzed by autoradiography and by PhosphorImager analysis. The position of the labeled lipids on chromatography plates was compared with standard lipids after exposure to iodine vapor. The amount of each labeled lipid was determined by liquid scintillation counting of the corresponding spots on the chromatograms.
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RESULTS |
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Isolation of the S. cerevisiae LPP1 Gene and the Deduced Primary Structure of Its Encoded Protein-- The LPP1 (lipid phosphate phosphatase) gene was identified in the Saccharomyces Genome Data Base on the basis that its deduced protein product showed homology to the DPP1-encoded DGPP phosphatase (16) and to the mouse Mg2+-independent PA phosphatase (24). The homologous regions of these proteins have been shown to constitute a novel phosphatase sequence motif (52). Based on this information we hypothesized that the gene encoded a Mg2+-independent PA phosphatase. The LPP1 gene is located on the right arm of chromosome IV (53). The LPP1 gene and its flanking sequences were isolated from plasmid pSK5+, a multicopy plasmid that contains LPP1 on a 7.8-kb insert of genomic DNA. The LPP1 gene was sequenced twice by automated DNA sequence analysis and was shown to match the sequence in the data base.
The LPP1 DNA sequence does not have any sequence motifs that would suggest the existence of introns in the gene. The predicted protein product is 274 amino acids in length, has a minimum subunit molecular mass of 31.6 kDa, and is predicted to be an integral membrane protein (Fig. 1). This protein is predicted to have six transmembrane spanning regions distributed over the entire polypeptide sequence (Fig. 1B). The phosphatase sequence motif is comprised of three domains (52), and these domains (Fig. 1A) are predicted to be localized to the hydrophilic surface of the membrane (Fig. 1B). The PSORT computer program3 predicts possible endoplasmic reticulum, plasma membrane, and Golgi body localization for the deduced amino acid sequence of the LPP1 gene. The PROSITE Motif program4 predicts that the LPP1 gene product has six protein kinase C and four casein kinase II phosphorylation target sites.
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Mg2+-Independent PA Phosphatase, LPA Phosphatase, and DGPP Phosphatase Activities in S. cerevisiae Cells Overexpressing the LPP1 Gene-- The LPP1 gene was used to construct a multicopy plasmid for the overexpression of the LPP1 gene product in S. cerevisiae. Cells bearing the multicopy plasmid were grown to the exponential phase of growth, and cell extracts were prepared and assayed for Mg2+-independent PA phosphatase. The plasmid containing the LPP1 gene directed a 1.9-fold overexpression of Mg2+-independent PA phosphatase activity when compared with cells not bearing the plasmid (Fig. 2A). We also examined the ability of the LPP1 gene product to utilize LPA and DGPP as substrates. These substrates were used for this analysis since they are also substrates for the DGPP phosphatase from S. cerevisiae (15, 54) and the Mg2+-independent PA phosphatase from rat liver (26, 27). The LPA phosphatase and DGPP phosphatase activities in cells bearing the LPP1 gene on the multicopy plasmid were 1.45- and 1.4-fold higher, respectively, when compared with the control cells (Fig. 2A).
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Mg2+-Independent PA Phosphatase, LPA Phosphatase, and DGPP Phosphatase Activities in Sf-9 Insect Cells Overexpressing the LPP1 Gene-- To test further the hypothesis that the LPP1 gene was the structural gene encoding a Mg2+-independent PA phosphatase, we used heterologous expression of the gene in Sf-9 insect cells. The LPP1 gene was placed within the genome of baculovirus under control of the polyhedrin promoter and expressed by viral infection of Sf-9 cells. Infection of the cells with the baculovirus containing the LPP1 gene resulted in the massive overexpression of PA phosphatase, LPA phosphatase, and DGPP phosphatase activities when compared with uninfected cells (Fig. 2A). These data provided strong evidence that the LPP1 gene encoded a Mg2+-independent PA phosphatase enzyme. These studies also indicated that the highest activity was obtained when PA was used as the substrate (Fig. 2A).
The computer analysis predicted that the LPP1 gene product is an integral membrane protein. We examined this hypothesis using insect cells overexpressing the LPP1 gene. The cytosolic and total membrane fractions were isolated from the cell extract and used for the assay of Mg2+-independent PA phosphatase activity. The specific activity of the enzyme in the total membrane fraction was 24-fold higher than the activity in the cytosolic fraction (Fig. 2B). These data supported the conclusion that the LPP1 gene product was indeed a membrane-associated enzyme. We examined the effects of ceramide 1-phosphate and sphingosine 1-phosphate on the Mg2+-independent PA phosphatase activity encoded by the LPP1 gene. These compounds are substrates and inhibitors of the rat liver Mg2+-independent PA phosphatase (26). The concentrations of ceramide 1-phosphate and sphingosine 1-phosphate were varied in the assay up to 16 mol % using uniform Triton X-100/lipid mixed micelles (3). At a final concentration of 16 mol %, ceramide 1-phosphate inhibited Mg2+-independent PA phosphatase activity by only 26%. This indicated that ceramide 1-phosphate was not a good inhibitor and was likely a poor substrate for the enzyme. Sphingosine 1-phosphate had no effect on activity at concentrations up to 16 mol %.Deletion of the LPP1 Gene--
The LPP1 gene was
deleted to further support the hypothesis that its gene product encoded
a Mg2+-independent PA phosphatase enzyme. In addition, the
availability of an lpp1 mutant would allow us to examine
whether the LPP1 gene was essential for cell growth and to
examine possible phenotypes that would shed light on the physiological
role of its gene product. A genomic construct containing the
LPP1 gene was manipulated to delete all of the coding
sequences and small amounts of the 5'- and 3'-noncoding sequences. The
LPP1 deletion construct was introduced into the genome of
haploid cells by homologous recombination as described under
"Experimental Procedures." Haploid lpp1
mutant cells
were viable and exhibited growth properties similar to wild-type control cells when grown vegetatively in complete synthetic medium and
in YEPD medium at 30 °C. In addition, mating and sporulation were
unaffected by the deletion of the LPP1 gene. Microscopic examination of lpp1
mutant cells showed no apparent gross
morphological differences when compared with wild-type cells. Overall,
these results indicated that the LPP1 gene was not essential
for cell growth under typical laboratory growth conditions.
Deletion of Both LPP1 and DPP1 Genes--
The DPP1 gene
encodes a DGPP phosphatase enzyme (16) that utilizes a variety of lipid
phosphate substrates including DGPP, PA, and LPA (15, 54). We
constructed an lpp1 dpp1
double mutant to
examine the effects of the deletion of both LPP1 and DPP1 genes on cell viability and to examine the phosphatase
activities contributed by each gene product. The double mutant was
constructed by crossing the lpp1
mutant with a
dpp1
mutant as described under "Experimental
Procedures." Diploid cells were induced to sporulate and analyzed for
the segregation of the HIS3 and TRP1 genes that
were used to disrupt LPP1 and DPP1 (16),
respectively. Analysis of 12 tetrads showed that the cross yielded two
parental ditype (i.e. all four spores of parental types), 8 tetratype (i.e. one spore of each parental genotype and one
spore of each recombinant type), and 2 nonparental ditype (all four
spores of the recombinant genotypes) tetrads for the HIS3
and TRP1 genes. This segregation pattern indicated that the
LPP1 and DPP1 genes segregated independently. Haploid lpp1
dpp1
double mutants were
viable and exhibited growth properties similar to wild-type control
cells and to lpp1
and dpp1
mutant cells
when grown vegetatively in complete synthetic medium and in YEPD medium
at 30 °C.
Mg2+-Independent PA Phosphatase, LPA Phosphatase, and
DGPP Phosphatase Activities in the S. cerevisiae lpp1 Mutant,
dpp1
Mutant, and lpp1
dpp1
Double Mutant--
The
lpp1
mutant was grown to exponential phase, and cell
extracts were prepared and assayed for phosphatase activities using PA,
LPA, and DGPP as substrates. The Mg2+-independent PA
phosphatase, LPA phosphatase, and DGPP phosphatase activities in the
lpp1
mutant were reduced by 35, 22, and 20%, respectively, when compared with the activities found in the wild-type parent (Fig. 3). Transformation of the
lpp1
mutant with the multicopy plasmid containing the
LPP1 gene resulted in a small but reproducible overexpression of Mg2+-independent PA phosphatase
(1.56-fold), LPA phosphatase (1.22-fold), and DGPP phosphatase
(1.27-fold) activities when compared with the activity exhibited by the
mutant (Fig. 3).
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Mg2+-Dependent PA Phosphatase Activity in the S. cerevisiae lpp1 Mutant, dpp1
Mutant, and lpp1
dpp1
Double
Mutant--
Two membrane-associated forms of the
Mg2+-dependent type of PA phosphatase have been
purified and characterized from S. cerevisiae (5-7).
However, the genes encoding these enzymes have not been isolated. We
examined the expression of Mg2+-dependent PA
phosphatase activity in the lpp1
mutant,
dpp1
mutant, and lpp1
dpp1
double mutant. The level of expression of the
Mg2+-dependent PA phosphatase activity
paralleled the expression of the Mg2+-independent PA
phosphatase activity in wild-type cells and in the lpp1
and dpp1
mutants (Fig. 4).
However, the Mg2+-dependent PA phosphatase
activity was 4.35-fold higher than the Mg2+-independent
activity in the lpp1
dpp1
double mutant
(Fig. 4). These results confirmed that separate genes encoded the
Mg2+-dependent and Mg2+-independent
types of PA phosphatase. The availability of the lpp1
dpp1
double mutant should facilitate the isolation of
genes encoding the Mg2+-dependent enzymes.
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Phospholipid Composition of the lpp1 Mutant, dpp1
Mutant, and
lpp1
dpp1
Double Mutant--
The effects of the mutations in the
LPP1 and DPP1 genes on phospholipid composition
were examined. Wild-type cells and the lpp1
mutant,
dpp1
mutant, and lpp1
dpp1
double mutant were labeled to steady-state with
32Pi. Phospholipids were extracted from the
cells and then analyzed by two-dimensional thin layer chromatography as
described under "Experimental Procedures." The major changes
observed in the phospholipid composition of the lpp1
mutant were a 38% decrease in the amount of phosphatidylinositol and a
47% increase in the amount of DGPP when compared with wild-type cells
(Fig. 5). The deletion of the DPP1 gene resulted in a 49% decrease in
phosphatidylinositol, a 38% increase in PA, and a 67% increase in
DGPP when compared with the control cells (Fig. 5). The deletion of
both LPP1 and DPP1 genes together resulted in a
61% decrease in phosphatidylinositol, a 45% increase in PA, and a
69% increase in DGPP when compared with wild-type cells (Fig. 5). The
lpp1
dpp1
mutant also showed small changes
in the relative amounts of phosphatidylcholine (10% decrease),
phosphatidylethanolamine (18% increase), and phosphatidylserine (28%
decrease).
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DISCUSSION |
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The Mg2+-independent type of PA phosphatase is
postulated to play a role in lipid signaling pathways in mammalian
cells (18, 26, 56). Owing to its tractable molecular genetic system, we
are using S. cerevisiae as a model eukaryote to study this enzyme and determine its role in phospholipid metabolism.
Mg2+-Independent PA phosphatase activity has previously
been shown to be an activity of the DPP1-encoded (16) DGPP
phosphatase enzyme (15). Biochemical analysis of a dpp1
mutant has revealed that the DPP1 gene is not responsible
for all of the Mg2+-independent PA phosphatase activity in
S. cerevisiae (16). We hypothesized that the LPP1
gene encoded a Mg2+-independent PA phosphatase enzyme. This
was based on the fact that the predicted product of the gene contains a
novel phosphatase sequence motif (52) found in the yeast
DPP1-encoded DGPP phosphatase (16) and in the mammalian
Mg2+-independent PA phosphatase (24).
The LPP1 gene was isolated and expressed in wild-type cells
on a multicopy plasmid. The amount of overexpression of
Mg2+-independent PA phosphatase activity was relatively low
when compared with other phospholipid metabolic enzymes that have been
expressed in S. cerevisiae (16, 57-62). The low level of
activity may have been due to regulation of LPP1 gene
expression and/or regulation of its encoded activity. In addition, the
presence of the DPP1-encoded DGPP phosphatase enzyme in
wild-type cells masked the expression of the LPP1 gene
product. The expression of the LPP1 gene in the dpp1 mutant and in the lpp1
dpp1
double mutant resulted in an overexpression of
Mg2+-independent PA phosphatase activity of 3.36- and
12.9-fold, respectively. Moreover, the heterologous expression of the
S. cerevisiae LPP1 gene in Sf-9 insect cells resulted in a
715-fold overexpression of Mg2+-independent PA phosphatase
activity relative to control insect cells and a 65-fold overexpression
of activity relative to wild-type yeast. This activity was associated
with the membrane fraction of the cell. Taken together, these data
provided a conclusive level of evidence for the identification of the
LPP1 gene as the structural gene encoding a
membrane-associated Mg2+-independent PA phosphatase. The
expressed LPP1 gene product in S. cerevisiae and
in insect cells also exhibited LPA phosphatase and DGPP phosphatase
activities. The order of substrate preference based on the assay
conditions described here was PA > LPA > DGPP. Detailed
substrate specificity studies await the purification of the
LPP1-encoded Mg2+-independent PA phosphatase
enzyme.
The LPP1-encoded Mg2+-independent PA phosphatase was similar to the DPP1-encoded DGPP phosphatase (15, 16, 54) and the mammalian Mg2+-independent PA phosphatase (24-27) insofar as these enzymes are Mg2+-independent, utilize a variety of lipid phosphate molecules as substrates, and contain a novel phosphatase sequence motif (52). However, other than the phosphatase sequence motif, these enzymes show relatively little (~23% identity) overall amino acid sequence homology. These enzymes also differ with respect to their substrate specificity. For example, the LPP1-encoded Mg2+-independent PA phosphatase had a preference for PA over DGPP by about 4-fold, whereas the DPP1-encoded DGPP phosphatase has a 10-fold higher specificity constant for DGPP when compared with PA (15). The rat liver Mg2+-independent PA phosphatase utilizes PA and DGPP with about equal specificity (27). These data may suggest that each of these enzymes play different roles in phospholipid metabolism.
The great advantage of using S. cerevisiae to study the
Mg2+-independent PA phosphatase is the relative ease with
which null allele mutants can be constructed (41). The construction of the lpp1 mutant revealed that the LPP1 gene
was not essential for cell growth in S. cerevisiae. The
lpp1
mutant lacked an identifiable phenotype. The
vegetative growth and cell morphology of the mutant were
indistinguishable from its wild-type parent. The construction of the
lpp1
mutant, the dpp1
mutant (16), and the
lpp1
dpp1
double mutant facilitated
biochemical studies to examine the contribution of the LPP1
and DPP1 gene products to the lipid phosphate phosphatase activities in S. cerevisiae. The analysis of the mutants
showed that the DPP1 gene product was responsible for most
of the Mg2+-independent PA phosphatase and LPA phosphatase
activities, and all of the detectable DGPP phosphatase activity in the
cell. Together, the LPP1 and DPP1 gene products
accounted for 92% of the Mg2+-independent PA phosphatase,
90% of the LPA phosphatase activities, and all of the DGPP phosphatase
activity in the cell. Thus, additional gene(s) exist in S. cerevisiae to account for the remaining
Mg2+-independent PA phosphatase and LPA phosphatase
activities. As described previously (16), the level of DGPP was
elevated in the dpp1
mutant, and as expected, DGPP was
elevated in the lpp1
dpp1
double mutant.
However, the 20% reduction in DGPP phosphatase activity and the
increase in DGPP content in the lpp1
mutant were
surprising. All of the detectable DGPP phosphatase activity was absent
in the dpp1
mutant. It is unclear whether the expression of the phosphatase activities in the lpp1
mutant was a
reflection of the loss of the LPP1 gene alone and/or a
reflection of the regulation of the DPP1 gene in the
lpp1
mutant background. Additional studies will be
required to address this question.
The phospholipid composition analysis of the mutants revealed that the
LPP1 and DPP1 gene products played a role in the
regulation of phospholipid metabolism. All three mutants showed
decreases in the amounts of phosphatidylinositol. The most dramatic
decrease was observed in the lpp1 dpp1
double mutant. The dpp1
mutant and lpp1
dpp1
double mutant also showed significant increases in
PA levels. The mechanism responsible for the changes in
phosphatidylinositol and PA in the mutants is unknown. It is known that
DGPP, which was elevated in the mutants, potently inhibits
Mg2+-independent PA phosphatase activity (15). This
inhibition of activity may attribute to the increase in PA levels that
were observed in the dpp1
mutant and lpp1
dpp1
double mutant. In turn, PA is a potent activator of
phosphatidylserine synthase activity (63). The activation of
phosphatidylserine synthase activity could have drawn upon the cellular
pools of CDP-diacylglycerol, which is also a substrate for the enzyme
phosphatidylinositol synthase (2-4). Reduction in CDP-diacylglycerol
results in a decrease in phosphatidylinositol synthase activity and
phosphatidylinositol content in S. cerevisiae (64, 65). In
addition, the elevation of phosphatidylethanolamine in the double
mutant was consistent with a stimulation of phosphatidylserine
synthesis at the expense of phosphatidylinositol. These explanations
for the changes in phospholipid composition may be an
oversimplification of the regulation occurring in the mutants. The
regulation of phospholipid metabolism in S. cerevisiae is
complex and involves both genetic and biochemical mechanisms (2-4,
55).
Phosphatidylinositol is an essential membrane phospholipid in S. cerevisiae (4) that serves as the precursor for sphingolipids and
the D-3 and D-4 phosphoinositides (4, 66).
These inositol-containing lipids and their hydrolysis products are
prominent lipid signaling molecules in S. cerevisiae and in
mammalian cells (66-70). PA regulates the activity of several
lipid-dependent enzymes in S. cerevisiae and
mammalian cells (63, 71-74) and exhibits mitogenic effects in
mammalian cells (28, 74-77). The fact that the levels of
phosphatidylinositol and PA changed in the dpp1 and
lpp1
dpp1
mutants may suggest that the
LPP1 and DPP1 gene products play a role in lipid
signaling pathways. Clearly, additional studies are needed to gain
insight into the role(s) the LPP1 and DPP1 gene
products play in phospholipid metabolism. The availability of the genes
and mutants described in this work will facilitate future studies on
these important phosphatase enzymes and how they impact on cell
physiology.
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ACKNOWLEDGEMENTS |
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We acknowledge Dr. Hideo Kanoh for providing
us with the observation that the mouse Mg2+-independent PA
phosphatase showed homology to the predicted product of the
LPP1 gene prior to the publication of his paper (24). Dr.
Geri Marie Zeimetz is acknowledged for contributions to the construction of plasmid pCR-lpp1 and the observation that the LPP1 gene flanked the PSP1 gene. We thank Dr.
Timothy Formosa for providing us with the plasmid pSK5+. We also
acknowledge Deirdre A. Dillon, Xiaoming Chen, and Darin B. Ostrander
for their technical contributions to this work.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health USPHS Grants GM-28140 (to G. M. C.) and GM-32453 (to D. R. V.) and the Charles and Johanna Busch Memorial Fund (to G. M. C.). This is New Jersey Agricultural Experiment Station Publication D-10531-1-98.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence and reprint requests should be addressed. Tel.: 732-932-9611 (ext. 217); Fax: 732-932-6776; E-mail: carman{at}aesop.rutgers.edu.
1 The abbreviations used are: PA, phosphatidate; LPA, lysophosphatidate; DGPP, diacylglycerol pyrophosphate; DG, diacylglycerol; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair.
2 The LPP1 gene was previously referred to as the DPP2 gene (27).
3 Available on-line at the following address: http://psort.nibb.ac.jp/form.html.
4 Available on-line at the following address: http://www.genome.ad.jp/sit/motif.html.
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REFERENCES |
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