From the Department of Food Science, Cook College,
New Jersey Agricultural Experiment Station, Rutgers University, New
Brunswick, New Jersey 08903, § 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, and the ¶ Department of Food Science and
Nutrition, University of Rhode Island,
West Kingston, Rhode Island 02892
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ABSTRACT |
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Diacylglycerol pyrophosphate (DGPP) is involved
in a putative novel lipid signaling pathway. DGPP phosphatase (DGPP
phosphohydrolase) is a membrane-associated 34-kDa enzyme from
Saccharomyces cerevisiae which catalyzes the
dephosphorylation of DGPP to yield phosphatidate (PA)
and then catalyzes the dephosphorylation of PA to yield diacylglycerol. Amino acid sequence information derived from DGPP phosphatase was used
to identify and isolate the DPP1
(diacylglycerol pyrophosphate phosphatase) gene encoding the enzyme. Multicopy plasmids
containing the DPP1 gene directed a 10-fold overexpression
of DGPP phosphatase activity in S. cerevisiae. The
heterologous expression of the S. cerevisiae DPP1 gene in
Sf-9 insect cells resulted in a 500-fold overexpression of DGPP
phosphatase activity over that expressed in wild-type S. cerevisiae. DGPP phosphatase possesses a
Mg2+-independent PA phosphatase activity, and its
expression correlated with the overexpression of DGPP phosphatase
activity in S. cerevisiae and in insect cells. DGPP
phosphatase was predicted to be an integral membrane protein with six
transmembrane-spanning domains. The enzyme contains a novel phosphatase
sequence motif found in a superfamily of phosphatases. A
dpp1 mutant was constructed by deletion of the
chromosomal copy of the DPP1 gene. The dpp1
mutant was viable and did not exhibit any obvious growth defects. The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold)
DGPP. Analysis of the mutant showed that the DPP1 gene was
not responsible for all of the Mg2+-independent PA
phosphatase activity in S. cerevisiae.
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INTRODUCTION |
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Diacylglycerol pyrophosphate (DGPP)1 is a novel phospholipid that contains a pyrophosphate group attached to diacylglycerol (DG) (Fig. 1) (1). DGPP has been found in a variety of plants (2, 3) and in the yeast Saccharomyces cerevisiae (4). This phospholipid is synthesized from phosphatidate (PA) and ATP via the reaction catalyzed by the membrane-associated enzyme PA kinase (1) and is dephosphorylated to PA via the reaction catalyzed by the membrane-associated enzyme DGPP phosphatase (Fig. 1) (4). The amounts of DGPP in wild-type S. cerevisiae and in plants are barely detectable (3, 4). For example, DGPP accounts for only 0.18 mol % of the major phospholipids in S. cerevisiae (4). The low abundance of DGPP is reminiscent of lipid signaling molecules such as the inositol-containing phospholipids (5-9). Recent studies indicate that the metabolism of DGPP is involved in a novel lipid signaling pathway. DGPP accumulates in plant tissues upon G protein activation through the stimulation of PA kinase activity (3), and metabolic labeling studies with Catharanthus roseus cells have shown that DGPP is metabolized rapidly to PA and then to DG (10). It has been suggested that DGPP may function as a signaling molecule (3, 4). Alternatively, the formation of DGPP may serve to attenuate the signaling functions of PA (11, 12).
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DGPP phosphatase activity has been identified in S. cerevisiae, C. roseus, Escherichia coli, rat
liver, pig liver, pig brain, and bovine brain (4, 10). The discovery of
DGPP phosphatase in such a wide range of organisms suggests that it
plays an important role in cell function. DGPP phosphatase has been
purified to homogeneity from S. cerevisiae and has been
characterized with respect to its enzymological and kinetic properties
(4). The enzyme has a subunit molecular mass of 34 kDa (4). When DGPP
is supplied as a substrate for the pure enzyme, it removes the phosphate of DGPP to generate PA, and then removes the phosphate of PA
to generate DG (Fig. 1) (4). Indeed, DGPP phosphatase can utilize PA as
a substrate in the absence of DGPP, although the enzyme has a 10-fold
higher specificity constant for DGPP (4). PA does not alter DGPP
phosphatase activity (4). However, DGPP does competitively inhibit the
PA phosphatase activity of the DGPP phosphatase enzyme (4). The DGPP
phosphatase and PA phosphatase activities of the DGPP phosphatase
enzyme are Mg2+-independent and
N-ethylmaleimide-insensitive (4). In addition, DGPP
phosphatase activity is inhibited potently by Mn2+ ions
(4). The PA phosphatase activity of the DGPP phosphatase enzyme is
distinct from the conventional PA phosphatase enzymes (13-15) that are
proposed to be used for the synthesis of phospholipids and
triacylglycerols in S. cerevisiae (4). The conventional PA
phosphatases (45-, 75-, and 104-kDa forms) have a Mg2+ ion
requirement and are sensitive to inhibition by
N-ethylmaleimide (13, 14, 16). The 45- and 104-kDa forms of
the Mg2+-dependent PA phosphatases do not
utilize DGPP as a substrate (4). In fact, the 104-kDa PA phosphatase
activity is stimulated by DGPP (4, 16).
DGPP phosphatase is an interesting enzyme insofar as the product of one
reaction becomes the substrate for the subsequent reaction (Fig. 1).
The regulation of this enzyme could control specific cellular pools of
DGPP, PA, and DG and thus influence lipid signaling as well as overall
lipid metabolism. The isolation of the gene encoding DGPP phosphatase
is required for defined studies to examine the physiological roles of
DGPP and DGPP phosphatase in eukaryotic cells. In this paper we report
the isolation and initial characterization of the DPP1
(diacylglycerol pyrophosphate phosphatase) gene encoding DGPP phosphatase in S. cerevisiae. This work represents the first report of the isolation
of a eukaryotic gene encoding DGPP phosphatase. In addition, the
chromosomal copy of the DPP1 gene was deleted from S. cerevisiae to produce a dpp1 mutant. The initial
characterization of the dpp1
mutant showed that the
DPP1 gene was not essential for cell growth and that DPP1 was responsible for all of the detectable DGPP
phosphatase activity in the cell.
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EXPERIMENTAL PROCEDURES |
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Materials
All chemicals were reagent grade. Growth medium supplies were
purchased from Difco Laboratories. Protein assay reagent, molecular mass standards for SDS-polyacrylamide gel electrophoresis, and electrophoresis reagents were purchased from Bio-Rad. Polyvinylidene difluoride paper was purchased from Millipore. 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 and Sigma. Radiochemicals were
purchased from NEN Life Science Products. Scintillation counting
supplies and acrylamide for electrophoresis were from National
Diagnostics. Silica Gel 60 thin-layer chromatography plates were from
EM Science. E. coli DG kinase was obtained from Lipidex
Inc.
Methods
Strains, Plasmids, and Growth Conditions--
The strains and
plasmids used in this work are listed in Tables
I and II,
respectively. Methods for yeast growth, sporulation, and tetrad
analysis were performed as described previously (17, 18). Yeast
cultures were grown in YEPD medium (1% yeast extract, 2% peptone, 2%
glucose) or in complete synthetic medium minus inositol (19) 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 hemocytometer or spectrophotometrically
at an absorbance of 600 nm. The inositol excretion phenotype (20) of
yeast strains was examined on complete synthetic medium (minus
inositol) by using growth of the inositol auxotrophic indicator strain
MC13 (ino1) (19) as described by McGee et al.
(21).
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Amino Acid Composition and Amino Acid Sequence Analyses of DGPP Phosphatase-- Pure DGPP phosphatase (160 pmol) was subjected to SDS-polyacrylamide gel electrophoresis (22) and transferred to polyvinylidene difluoride paper (23). A portion (11 pmol) of the sample was subjected to amino acid composition analysis (24). The remainder of the sample on polyvinylidene difluoride paper was digested with sequencing grade trypsin for 20 h at 37 °C as described by Aebersold et al. (25) using the buffer system of Fernandez et al. (26). The digested sample was washed twice with 0.06% trifluoroacetic acid. The digest was then subjected to narrowbore reverse phase high performance liquid chromatography (HPLC) using a Zorbax C-18 column (1 × 150 mm, inner diameter) as described by Lane et al. (24). Strategies for the selection of peptide fragments and their sequencing by automated Edman degradation were performed as described by Lane et al. (24). The amino acid composition and amino acid sequencing analyses were performed at the Harvard Microchemistry Facility (Cambridge, MA).
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
(18). Transformation of yeast (27, 28) and E. coli (18) were
performed as described previously. Conditions for the amplification of
DNA by PCR were optimized as described by Innis and Gelfand (29). The
annealing temperature for the PCRs was 50 °C, and extension times
were typically between 2.0 and 2.5 min at 72 °C. PCRs were run
routinely 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.
Amplification of the plasmid pDT1-DPP1 was performed in E. coli strain Epicurian ColiRXL-1.
Isolation of the DPP1 Gene--
We identified an open reading
frame DNA sequence in the Saccharomyces Genome Data Base
(locus YDR284C) whose predicted amino acid sequence matched exactly the
amino acid sequences derived from the DGPP phosphatase protein. This
gene was named DPP1. A 1.9-kb DNA fragment containing 600 bp
of the putative DPP1 promoter, its entire protein coding
sequence, and 500 bp of the 3-flanking sequence was obtained by PCR
(primers: 5
-GTTACATTGTATCAGTCACAGGTACGG-3
and
5
-GTCGACATTTATACATAGTATGTGTTAAGG-3
) using strain W303-1A genomic DNA
as a template. The PCR product was ligated into the SrfI
site of the pCRScriptTM AMP SK(+) cloning vector resulting
in the formation of pDT1-DPP1. This plasmid was digested with
SalI, which released a 2.0-kb fragment containing the open
reading frame and approximately 600 bp of the promoter region and 500 bp of the 3
-untranslated region. This fragment was ligated into the
SalI site of YEp351, a multicopy E. coli/yeast
shuttle vector containing the LEU2 gene (30), to form
plasmid pDT2-DPP1. This construct was then transformed into W303-1A
(W303-1A/pDT2-DPP1) for the overexpression of the DPP1 gene
product.
Construction of a dpp1 Mutant--
The plasmid pDT1-DPP1 was
digested with SnaBI/BstZ17I to remove the entire
DPP1 coding sequence. A 2.8-kb
TRP1/Kanr disruption cassette, derived from
plasmid pJA52 (33) by SmaI digestion, was inserted into the
blunt-ended SnaBI/BstZ17I sites of plasmid
pDT1-DPP1 to form the plasmid pDT3-dpp1
. A linear 3.5-kb
DPP1 deletion cassette was released from pDT3-dpp1
by digestion with SalI. This DNA fragment was transformed into
W303-1A to delete the chromosomal copy of the DPP1 gene by
the one-step gene replacement technique (34). Transformants were
selected for their ability to grow on complete synthetic medium without tryptophan. Deletion of the chromosomal copy of the DPP1
gene was confirmed by PCR (35) using the primers listed above with the
extension time increased to 3.5 min. The template for the PCRs used to
confirm the DPP1 deletion was genomic DNA isolated from
transformed colonies that grew on medium without tryptophan. One of the
dpp1
mutants that we isolated was designated strain DTY1.
DTY1 was transformed with pBZ1-DPP1 (DTY1/pBZ1-DPP1) to compliment the
dpp1
mutant.
Recombinant Viral Expression of the S. cerevisiae DPP1 Gene in
Insect Cells--
Plasmid pBZ1-DPP1 was digested with
MfeI/SalI to release the entire coding sequence
of the DPP1 gene. This DNA fragment was ligated into the
EcoRI/SalI sites of the pCRScriptTM
AMP SK(+) vector resulting in the formation of plasmid pDT4-DPP1. This
plasmid was then digested with KpnI/NotI, which
released the DPP1 open reading frame. The DPP1
gene was then ligated into the KpnI/NotI site of
the baculovirus vector PVL1392 to form plasmid pWW1-DPP1. The pWW1-DPP1
plasmid was subsequently cotransfected 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 (36) 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. (36). Routine infection of Sf-9 cells
for DGPP phosphatase 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. Cells that were not infected with virus, cells infected
with virus containing the vector without the DPP1 gene, and
cells infected with recombinant virus containing the S. cerevisiae choline kinase gene (37) served as controls. The
expression of the S. cerevisiae choline kinase gene in
insect cells will be described elsewhere.
Preparation of Enzymes-- DGPP phosphatase was purified to homogeneity from S. cerevisiae as described by Wu et al. (4). The protein used for amino acid sequencing was derived from Mono Q II (4). PA kinase was purified from suspension-cultured C. roseus cells as described by Wissing and Behrbohm (2). Yeast cell extracts (38) and total membranes (13) were prepared as described previously. Cell extracts derived from Sf-9 insect cells were prepared at 5 °C. Cells were washed in 50 mM Tris-HCl, pH 7.5, 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 and suspended (0.3 g, wet weight, of cells/ml) in the same buffer. Cells were disrupted by sonic oscillation for seven 30-s bursts, with a 1.5-min pause between bursts. The disrupted cell suspension was then centrifuged at 1,500 × g for 5 min to remove unbroken cells and cell debris.
Preparation of Substrates--
DGPP standard and
[-32P]DGPP were synthesized enzymatically using
purified C. roseus PA kinase as described by Wu et
al. (4). [32P]PA was synthesized enzymatically from
DG using E. coli DG kinase (39) as described previously
(13).
Enzyme Assays--
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. (4). 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. DGPP
phosphatase activity has been measured at pH 6.5 using Tris-maleate
buffer (4). Reexamination of the pH optimum for the reaction showed
that maximum activity was obtained at pH
5.0.2
Mg2+-independent PA phosphatase activity was measured by
following the release of water-soluble 32Pi
from chloroform-soluble [32P]PA (10,000 cpm/nmol) (40).
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. All enzyme
assays were conducted at 30 °C in triplicate. The average standard
deviation 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 (41) using bovine serum albumin as the standard.
Mass Analysis of DGPP-- Phospholipids were extracted from S. cerevisiae cells using the solvent system consisting of 95% ethanol/water/diethyl ether/pyridine/ammonium hydroxide (15:15:5:1:0.018) as described by Hanson and Lester (42). Samples were dried in vacuo, dissolved in chloroform/methanol/water (15:15:5), and subjected to analytical normal phase HPLC as described by Wu et al. (4). The identity of DGPP was determined by comparing its elution profile with that of authentic DGPP (4). The cellular concentration of DGPP was calculated relative to the concentration of the major phospholipids in the extract (4).
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RESULTS |
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Isolation of the S. cerevisiae DPP1 Gene and the Deduced Primary
Structure of Its Encoded Protein--
The amino acid sequence analysis
of the pure DGPP phosphatase protein yielded an
NH2-terminal amino acid sequence of MNRVSFIKTPFNIGAKWRLE and two internal amino acid sequences of QPVEGLPLDTLFTAK and
FPPIDDPLPFKPLMD. These amino acid sequences aligned perfectly with the
deduced amino acid sequence of an identified open reading frame DNA
sequence in the Saccharomyces Genome Data Base (locus
YDR284C) (Fig. 2A). In
addition, the amino acid composition of the deduced protein matched the
amino acid composition of pure DGPP phosphatase (data not shown). We
named this gene DPP1 for diacylglycerol
pyrophosphate phosphatase. The DPP1
gene is located on the right arm of chromosome IV (43). The
DPP1 gene coding sequence along with its 5- and 3
-flanking
sequences was isolated by PCR amplification using genomic DNA from
strain W303-1A as the template. The DPP1 gene and its
flanking sequences were also isolated from plasmid p1219, a CEN-based
plasmid that contains the ZIP1 gene (31) on a 20-kb insert
of genomic DNA. The PCR-derived and genomic-derived genes were
sequenced twice by automated DNA sequence analysis. This analysis
showed that both versions of the gene were identical and matched the
sequence in the data base.
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DGPP Phosphatase and PA Phosphatase Activities in S. cerevisiae Cells and in Insect Cells Overexpressing the DPP1 Gene Product-- The PCR-derived and the genomic-derived versions of the DPP1 gene were used to construct multicopy plasmids for the overexpression of the DPP1 gene product in S. cerevisiae. Cells bearing these multicopy plasmids were grown to the exponential phase of growth, and cell extracts were prepared and assayed for DGPP phosphatase activity. The plasmids containing both versions of the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity compared with cells not bearing a plasmid (Fig. 3A). Cells bearing multicopy plasmids without the DPP1 gene also exhibited wild-type levels of DGPP phosphatase activity (Fig. 3A).
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Deletion of the DPP1 Gene and Initial Characterization of the
dpp1 Mutant--
The DPP1 gene was deleted to examine
whether the gene was essential for cell growth and to examine
phenotypes that would shed light on the physiological role of DGPP
phosphatase. The gene was deleted in vitro and introduced
into the genome of haploid cells by homologous recombination as
described under "Experimental Procedures." The strategy for the
deletion of the chromosomal copy of the DPP1 gene by
replacement with the TRP1/Kanr disruption
cassette is shown in Fig. 4A.
PCR amplification reactions using genomic DNA of mutant cells as a
template confirmed that the TRP1/Kanr disruption
cassette had integrated at the DPP1 locus of the chromosome (Fig. 4B). Haploid dpp1
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. Microscopic examination of dpp1
mutant cells showed no apparent gross morphological differences
compared with wild-type cells. Overall, these results indicated that
the DPP1 gene was not essential for cell growth under
typical laboratory growth conditions.
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DISCUSSION |
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DGPP phosphatase is a novel enzyme recently purified and
characterized from S. cerevisiae (4). The enzyme catalyzes
the dephosphorylation of the phosphate of DGPP to form PA and then catalyzes the dephosphorylation of the PA product to form DG (4). The
substrates and products of the DGPP phosphatase reaction, namely DGPP,
PA, and DG, have been shown to be involved in lipid signaling pathways
(3, 11, 12, 47, 48). Thus DGPP phosphatase could play a major role in
lipid signaling by regulating specific pools of these lipids. To gain
insight into the function and regulation of DGPP phosphatase in
eukaryotic cells we isolated the DPP1 gene. The deduced
amino acid sequence of the DPP1 gene matched perfectly the
amino acid sequences derived from pure DGPP phosphatase. Multicopy plasmids containing the DPP1 gene directed the
overexpression of DGPP phosphatase and Mg2+-independent PA
phosphatase activities in S. cerevisiae. Moreover, the
heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a massive overexpression of DGPP phosphatase and Mg2+-independent PA phosphatase activities.
The relative difference in the specific activities of DGPP phosphatase
and Mg2+-independent PA phosphatase from yeast cells and
from insect cells overexpressing the DPP1 gene was
consistent with the relative difference in the
Vmax values determined for the pure DGPP
phosphatase enzyme using DGPP and PA as substrates (4). Finally, the
deletion of the DPP1 gene in S. cerevisiae
resulted in the loss of detectable DGPP phosphatase activity as well as
a 4-fold accumulation in the cellular mass of DGPP. Collectively, these
data provided a conclusive level of evidence for the identification of
the DPP1 gene as the structural gene encoding DGPP
phosphatase in S. cerevisiae.
The loss of DGPP phosphatase activity in the dpp1 mutant
does not rule out the possibility of another gene in S. cerevisiae which encodes a DGPP phosphatase activity. A second
gene may exist whose product was not expressed under the growth
conditions used in our experiments. Alternatively, a DGPP phosphatase
may exist with assay requirements very different from those used in our experiments to measure DGPP phosphatase activity. For example, the
deletion of the S. cerevisiae PSD1 gene results in the loss of detectable phosphatidylserine decarboxylase activity (35, 49). Yet
S. cerevisiae has a second gene (PSD2) that
encodes a phosphatidylserine decarboxylase which is expressed at very low levels and has assay requirements different from the
phosphatidylserine decarboxylase encoded by the PSD1 gene
(50, 51). The dpp1
mutant exhibited a 50% reduction in
Mg2+-independent PA phosphatase activity compared with the
activity in wild-type cells. This indicated that the DPP1
gene product was not responsible for all of the
Mg2+-independent PA phosphatase activity in the cell. We
have isolated another gene from S. cerevisiae whose product
exhibits Mg2+-independent PA phosphatase activity. Its
isolation and characterization will be the subject of a future paper.
The pgpB gene encodes DGPP phosphatase activity in E. coli (52). The E. coli DGPP phosphatase has been partially purified and characterized (52). The biochemical properties of the E. coli enzyme are similar to the pure DGPP phosphatase from S. cerevisiae (4) with respect to its substrate specificity for DGPP and for PA (52). In addition, the DGPP phosphatase and PA phosphatase activities exhibited by the pgpB gene product are Mg2+-independent and N-ethylmaleimide-insensitive (52). The deduced protein products of the E. coli pgpB gene and the S. cerevisiae DPP1 gene show regions of high homology which constitute the novel phosphatase sequence motif (44). Other than the phosphatase motif, these gene products show very little overall amino acid sequence homology (17% identity). The DGPP phosphatases from S. cerevisiae and E. coli are members of a superfamily of phosphatases that share amino acid sequence homology in the phosphatase sequence motif (44). This superfamily also includes the Mg2+-independent and N-ethylmaleimide-insensitive form of PA phosphatase from mouse (53) and rat liver.3 The rat liver Mg2+-independent PA phosphatase can utilize DGPP as a substrate (54). However, this enzyme differs from the DGPP phosphatases from S. cerevisiae (4) and from E. coli (52) with respect to its substrate specificity for DGPP and for PA (54). Moreover, the mouse (53) and rat liver3 Mg2+-independent PA phosphatases share very little overall amino acid sequence homology with the DGPP phosphatases of S. cerevisiae (24 and 25% identity, respectively) and E. coli (19 and 20% identity, respectively).
Zhang et al. (55) have reported recently that Wunen, the product of the wunen gene in Drosophila, shows localized regions (that constitute the novel phosphatase sequence motif (44)) of high homology with the deduced protein products of the mouse Mg2+-independent PA phosphatase cDNA (53) and an unidentified open reading frame DNA sequence in S. cerevisiae. We found here that the deduced yeast protein identified by Zhang et al. (55) is encoded by the DPP1 gene. Wunen repels migrating germ cells during embryonic development (55). Based on the predicted amino acid sequence homologies of the wunen gene and of the mouse Mg2+-independent PA phosphatase cDNA (53), Zhang et al. (55) have speculated that Wunen mediates its function through lipid signaling pathways involving Mg2+-independent PA phosphatase activity. Wunen has not been examined for Mg2+-independent PA phosphatase activity nor for DGPP phosphatase activity.
Construction of the dpp1 mutant showed that the
DPP1 gene was not essential for cell growth in S. cerevisiae. The vegetative growth and cell morphology of the
dpp1
mutant were indistinguishable from those of its
wild-type parent. The mutant could mate with a wild-type strain, and a
DPP1/dpp1
heterozygote could sporulate. Although the dpp1
mutant lacked a dramatic phenotype,
DGPP and DGPP phosphatase are ubiquitous in nature and have been shown in other systems possibly to play a role in cellular regulation (3,
10). The availability of the DPP1 gene and the
dpp1
mutant will permit a combination of genetic,
molecular, and biochemical approaches to gain more a in-depth
understanding of the roles DGPP phosphatase and DGPP play in lipid
signaling and cell physiology.
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ACKNOWLEDGEMENTS |
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We acknowledge Dr. Geri Marie Zeimetz for identifying the DPP1 gene in the yeast data base, for performing a computer analysis to predict the structural features of the DPP1 gene product, and for contributions to the construction of plasmid pBZ1-DPP1. We also acknowledge Dr. Zeimetz for a critical review of this work. We thank Dr. William Lane and colleagues at the Harvard Microchemistry Facility for the amino acid composition and amino acid sequence analysis of the DGPP phosphatase protein. We thank Dr. Shirleen Roeder for providing plasmid p1219. We thank Drs. Bettina Riedel and Dr. Josef Wissing for providing purified PA kinase. We also acknowledge Ken Howard for providing a copy of his manuscript describing the wunen gene before its publication.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service, National Institutes of Health Grants GM-28140 (to G. M. C.), GM-32453 (to D. R. V.), and GM-49214 (to A. S. F.) and the Charles and Johanna Busch Memorial Fund (to G. M. C.). This is New Jersey Agricultural Experiment Station Publication D-10531-2-97 and Rhode Island Agricultural Experiment Station Contribution 3520.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 732-932-9611 (ext. 217); Fax: 732-932-6776; E-mail: carman{at}aesop.rutgers.edu.
1 The abbreviations used are: DGPP, diacylglycerol pyrophosphate; DG, diacylglycerol; PA, phosphatidate; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pairs.
2 X. Chen and G. M. Carman, unpublished data.
3 D. W. Waggoner and D. N. Brindley, personal communication.
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REFERENCES |
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