From the Department of Food Science, Cook College,
New Jersey Agricultural Experiment Station, Rutgers University, New
Brunswick, New Jersey 08901 and § Institut für
Biochemie, Technische Universität Graz, Petersgasse 12/2,
A-8010 Graz, Austria
Received for publication, December 19, 2000
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ABSTRACT |
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The DPP1 gene, encoding
diacylglycerol pyrophosphate (DGPP) phosphatase from
Saccharomyces cerevisiae, has recently been identified as a
zinc-regulated gene, and it contains a putative zinc-responsive element
(UASZRE) in its promoter. In this work we examined the hypothesis that expression of DGPP phosphatase was regulated by zinc
availability. The deprivation of zinc from the growth medium resulted
in a time- and dose-dependent induction of
The DPP1-encoded diacylglycerol pyrophosphate
(DGPP)1 phosphatase (1) is a
membrane-associated enzyme from the yeast Saccharomyces cerevisiae that catalyzes the removal of the DPP1-encoded DGPP phosphatase also exhibits a PA phosphatase
activity (2), but DGPP is the preferred substrate, with a specificity
constant 10-fold higher than that of PA (2). The DGPP phosphatase
protein (1) contains a three-domain phosphatase sequence motif (12-14)
that is conserved in a superfamily of lipid phosphatase enzymes
(15-19). Conserved Arg125, His169, and
His223 residues within domains 1, 2, and 3, respectively,
play important roles in the phosphatase reactions catalyzed by the
enzyme (20). The DGPP phosphatase enzyme also utilizes
lysophosphatidate (15), sphingolipid phosphates (21), and
isoprenoid phosphates (22) as substrates in vitro. However,
only DGPP and PA have been shown to be substrates in vivo
(1).
We have begun to examine the regulation of DGPP phosphatase expression
in S. cerevisiae to gain an understanding of DGPP function. The enzyme is induced by inositol in both exponential and stationary phase cells (23). DGPP phosphatase expression is greater in stationary
phase cells, and the inositol- and growth phase-dependent regulation of the enzyme is additive (23). These growth conditions have
profound effects on the expression of many phospholipid biosynthetic enzymes and on the regulation of phosphatidylinositol metabolism (5,
24-26). Interestingly, DGPP phosphatase regulation by inositol occurs
in a manner that is opposite that of many phospholipid biosynthetic
enzymes (23). Studies performed with a dpp1 In this work, we showed that DGPP phosphatase expression was also
regulated by zinc availability. Zinc deprivation induced DGPP
phosphatase in wild-type cells. This regulation was mediated by the
Zap1p transcriptional activator through a zinc-responsive element
(UASZRE) in the promoter of the DPP1 gene. The
pattern of DGPP phosphatase regulation in mutants defective in plasma membrane and vacuolar membrane zinc transporters indicated that enzyme
expression was sensitive to the cytoplasmic levels of zinc. DGPP
phosphatase activity was inhibited by zinc through a mechanism that
involved formation of DGPP-zinc complexes. We also showed that DGPP
phosphatase was localized to the vacuolar membrane.
Materials--
All chemicals were reagent grade. Growth medium
supplies were purchased from Difco Laboratories. Yeast nitrogen base
lacking zinc sulfate was purchased from BIO 101 by custom order. Triton X-100, bovine serum albumin, benzamidine, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, pepstatin, O-nitrophenyl
Strains and Growth Conditions--
The strains used in this work
are listed in Table I. Methods for yeast
growth were performed as described previously (27, 28). Yeast cells
were grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose)
or in synthetic complete (SC) medium containing 2% glucose at
30 °C. For selection of cells bearing plasmids, appropriate amino
acids were omitted from SC medium. Zinc-deplete medium was SC medium
prepared with yeast nitrogen base lacking zinc sulfate. Glassware was
washed with Liqui-Nox and rinsed several times with distilled water
followed by a final rinse with deionized distilled water.
Escherichia coli strain DH5 DNA Manipulations, Amplification of DNA by PCR, Site-directed
Mutagenesis, and DNA Sequencing--
Plasmid DNA preparation,
restriction enzyme digestion, and DNA ligations were performed by
standard methods (28). Transformation of yeast (29) and E. coli (28) were performed as described previously. Conditions for
the amplification of DNA by PCR were optimized as described previously
(30). Site-directed mutagenesis was performed by PCR using overlap
extension (31) or using the QuikChange site-directed mutagenesis kit.
DNA sequencing reactions were performed by the dideoxy method using
Taq polymerase (28).
Plasmid Constructions--
The plasmids used in this study are
listed in Tables II. Plasmid pJO2, which
contains the DPP1 promoter fused to the lacZ gene
of E. coli, was constructed as described previously (23). Plasmid pGH209 was constructed by replacing the EcoRI
fragment of pJO2 with the DPP1 promoter containing mutations
in the putative UASZRE (ACCTGAAAGGT Preparation of Cell Extracts, DGPP Phosphatase, and Protein
Determination--
Cells were disrupted with glass beads (32) 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 µg/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 (1) was purified to
near homogeneity as described by Wu et al. (2). Protein
concentration was determined by the method of Bradford (33) using
bovine serum albumin as the standard.
Enzyme Assays--
DGPP phosphatase activity was measured by
following the release of water-soluble 32Pi
from chloroform-soluble [ Immunoblot Analysis--
SDS-polyacrylamide gel electrophoresis
(35) using either 10 or 12% slab gels and immunoblotting (36) using
polyvinylidene difluoride membranes were performed as described
previously. The molecular mass standards used were phosphorylase B
(97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa),
carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). Rabbit polyclonal anti-DGPP phosphatase antibodies (37) and
mouse monoclonal anti-HA antibodies were used at a dilution of 1:1000.
Anti-rabbit and mouse IgG-alkaline phosphatase conjugate were used as
secondary antibodies at a dilution of 1:5000. DGPP phosphatase protein
was detected on immunoblots using the enhanced chemifluorescence
Western blotting detection kit as described by the manufacturer. The
DGPP phosphatase protein on immunoblots was acquired by
fluorimaging analysis. The relative density of the protein was
analyzed using ImageQuant software. Immunoblot signals were in the
linear range of detectability.
Analysis of DGPP-Zinc Complexes--
The ability of DGPP to form
complexes with zinc ions was analyzed with the metal indicator reagent
4-(2-pyridylazo)resorcinol by a modification of a method described by
Hunt et al. (38). Reaction mixtures contained 40 mM HEPES buffer (pH 7.0), 50 µM 4-(2-pyridylazo)resorcinol, 100 µM ZnCl2, and
various concentrations of dioctanoyl DGPP. In this assay, the
reaction of 4-(2-pyridylazo)resorcinol with zinc ions produced a red
color that was measured spectrophotometrically at 500 nm. Compounds
that form complexes with zinc, such as Na2EDTA, compete
with 4-(2-pyridylazo)resorcinol for zinc and cause a decrease in absorbance.
Isolation and Characterization of Subcellular
Fractions--
Organelles were isolated from wild-type cells grown in
YEPD growth medium to early stationary phase at 30 °C under aerobic conditions. Mitochondria, microsomes 1 (30,000 × g
fraction), 2 (40,000 × g fraction), 3 (100,000 × g fraction), cytosol, vacuoles, and lipid particles were
isolated from spheroplasts, which were prepared by standard methods
(39). The mitochondrial and microsomal fractions were isolated from
cell extracts by differential centrifugation (40). The cytosol was the
supernatant of the 100,000 × g fraction (40). Vacuoles
and lipid particles were isolated by several steps of flotation and
density gradient centrifugation as described by Uchida et
al. (41) with the modifications of Leber et al. (42).
Plasma membranes were prepared from cells disrupted with glass beads
using a combination of differential centrifugation and density gradient
centrifugation (43). Relative enrichment and cross-contamination of
organelle fractions were assessed by immunoblot analysis (36) as
described by Zinser and Daum (44). Antibodies were directed against
porin (mitochondria), Sec61p (microsomes, endoplasmic reticulum),
carboxypeptidase Y (vacuoles), Erg6p (lipid particles), plasma membrane
ATPase (plasma membrane), and glyceraldehyde 3-phosphate dehydrogenase (cytosol).
Immunofluorescence Microscopy--
Immunofluorescent staining of
cells was performed as described by Pringle et al. (45) with
minor modifications. Wild-type strain W303-1A, carrying either plasmid
pGH201 or plasmid pGH202, was grown to the late exponential phase of
growth in SC medium and then fixed for 1 h with 3.7%
formaldehyde. Fixed cells were treated with zymolyase (1 unit/ml) for
15 min and then attached to a polylysine-coated glass slide. Cells were
treated with 0.1% Triton X-100 for 10 min followed by incubation for
20 min with phosphate-buffered saline (pH 7.5) containing 5% bovine
serum albumin. The cells on the glass slide were incubated for 1 h
with anti-HA antibodies (15 µg/ml), washed with phosphate-buffered saline, and then incubated for 1 h with fluorescein-conjugated anti-IgG (H+L) antibodies (7.5 µg/ml). Images were observed and recorded using an Olympus BH2-RFCA fluorescence microscope equipped with a Photometrics Sensys KAF-1400 CCD camera.
Effect of Zinc Deprivation on the Expression of
To further examine the effect of zinc on the expression of the
DPP1 gene, we measured
The specific activity of Effect of the Putative UASZRE on the Expression of
Regulation of DPP1 by Zinc Deprivation in a Mutant Defective in the
ZAP1 Regulatory Gene--
Zap1p is a transcriptional activator that is
maximally expressed in zinc-deplete cells and repressed in zinc-replete
cells (49). Zap1p binds to the UASZRE of the
ZRT1, ZRT2, and ZRT3 genes for
increased expression in zinc-deplete media (47, 49, 50). We examined
whether the regulation of DPP1 by zinc was dependent on
Zap1p function. A zap1 Effect of Zinc Deprivation on the Levels of DGPP Phosphatase
Activity and Protein--
We examined the regulation of expression of
the DPP1-encoded DGPP phosphatase by zinc. Wild-type cells
were first precultured in zinc-deplete media followed by incubation in
growth media containing zinc. The depletion of zinc from the growth
media led to a dose-dependent induction of DGPP phosphatase
activity (Fig. 5A). DGPP
phosphatase specific activity in cells grown in the absence of zinc was
10-fold greater than the activity from cells grown with 5 µM zinc (Fig. 5A).
We also examined the levels of the DGPP phosphatase protein in cells
grown in the absence and presence of zinc. For these experiments, we
used an epitope-tagged DPP1 allele in the single-copy plasmid pGH201. In this plasmid, the sequence for HA was inserted into
the N terminus of the DPP1-coding sequence. Plasmid pGH201 was expressed in a dpp1 Regulation of DGPP Phosphatase Activity and Protein by Zinc in
Mutants Defective in Genes Encoding Zinc Transporters--
The
regulation of DGPP phosphatase was examined in mutants defective in the
ZRT1, ZRT2, ZRT3, COT1, and
ZRC1 genes. Zrt1p (51) and Zrt2p (52) are high affinity and
low affinity plasma membrane zinc transporters, respectively. Zrt3p
(50) is a vacuolar membrane zinc efflux transporter, whereas Cot1p and
Zrc1p are vacuolar membrane zinc influx transporters (50, 53). For
cells grown in the presence of zinc, expression of DGPP phosphatase activity was 4.3- and 2-fold greater in a zrt1 Effect of Zinc on DGPP Phosphatase Activity and Formation of
DGPP-Zinc Complexes--
We examined the effect of zinc ions on DGPP
phosphatase activity. The addition of zinc to the assay system resulted
in a dose-dependent inhibition of DGPP phosphatase activity
(Fig. 7). The zinc-mediated inhibition of
activity followed cooperative kinetics (Fig. 7, inset).
Analysis of the data according to the Hill equation yielded an
IC50 value for zinc of 150 µM and a Hill
number of 2.5. Owing to the fact that DGPP contains a pyrophosphate
group, we questioned whether the mechanism of inhibition involved
formation of DGPP-zinc complexes. Formation of DGPP-zinc complexes was
examined using the metal indicator reagent 4-(2-pyridylazo)resorcinol.
For these experiments, we used dioctanoyl DGPP because of its
solubility in aqueous solutions. Dioctanoyl DGPP is enzymatically
active when employed as a substrate for pure DGPP phosphatase (10). The
addition of DGPP to a 50 µM 4-(2-pyridylazo)resorcinol,
100 µM ZnCl2 mixture resulted in a
dose-dependent decrease in absorbance at 500 nm, indicating
the formation of DGPP-zinc complexes (data not shown). The
concentration of DGPP that resulted in half maximum complex formation
was 90 µM. This concentration was within the range of the
IC50 value for zinc. Formation of Na2EDTA-zinc
complexes was used as a control. Under the same conditions, the
concentration of Na2EDTA that resulted in half-maximum
Na2EDTA-zinc complex formation was 30 µM.
Subcellular Localization of DGPP Phosphatase--
DGPP phosphatase
has been purified from crude microsomal membranes (2). However, the
subcellular location of DGPP phosphatase has not been addressed. To
better understand the functional role(s) of DGPP phosphatase, we
examined the localization of the enzyme. Subcellular fractions of
S. cerevisiae were isolated and characterized as described
under "Experimental Procedures." The fractions were then used for
immunoblot analysis with anti-DGPP phosphatase antibodies. The 34-kDa
DGPP phosphatase protein (Dpp1p) was highly enriched in isolated
vacuoles (Fig. 8). The subcellular
location of DGPP phosphatase was examined further using wild-type cells
that expressed the HA-tagged version of the enzyme directed by the
single-copy and multicopy plasmids pGH201 and pGH202, respectively.
Cells were grown to exponential phase, fixed, and probed for the HA epitope by indirect immunofluorescence microscopy. The fluorescence signal in cells with the single-copy plasmid was difficult to detect
when compared with the signal in cells with the multicopy plasmid.
HA-DGPP phosphatase fluorescence in cells with the multicopy plasmid
outlined the periphery of the vacuole (Fig.
9B), which could be observed
by phase contrast microscopy (Fig. 9A). In separate experiments, identification of the vacuole was confirmed by red fluorescence resulting from the styryl dye FM 4-64, which specifically stains the vacuolar membrane (54). These data provided evidence that
the DGPP phosphatase enzyme was localized to the vacuolar membrane.
The DPP1 gene was first identified by Toke et
al. (1) as the gene encoding DGPP phosphatase. This identification
was based on amino acid sequence information derived from the purified
enzyme (1). Recent studies identify DPP1 as a gene that is
regulated by zinc deprivation (46, 48). The work presented here
advances the understanding of the regulation of DGPP phosphatase by
zinc and the localization of the enzyme. Using a
PDPP1-lacZ reporter gene, we showed that the
expression of DPP1 was induced in a time- and
dose-dependent manner when wild-type cells were deprived of zinc. The DPP1 promoter contains a putative
UASZRE (47), which is present in several genes that are
regulated by zinc deprivation (48). The regulation of DPP1
expression by zinc was dependent on this UASZRE. Mutations
in the element, as well as the deletion of the element from the
PDPP1-lacZ reporter gene, precluded the regulation by zinc deprivation. The DPP1 gene is also
induced when cells are supplemented with inositol and when they enter the stationary phase of growth. This regulation is not dependent on the
UASZRE.2
Induction of UASZRE-containing genes such as
ZRT1, ZRT2, and ZRT3, in response to
zinc deprivation (47) is mediated by the Zap1p transcriptional
activator (49). Indeed, Zap1p mediated DPP1 regulation by
zinc deprivation. As previously reported (46, 48), the regulation of
DPP1 by zinc deprivation was lost in a zap1 We examined the regulation of DGPP phosphatase expression in response
to zinc directly by measuring the levels of DGPP phosphatase activity
and protein. The depletion of zinc from the growth medium of wild-type
cells resulted in a 10-fold increase in DGPP phosphatase activity,
which corresponded to a 10-fold increase in the level of the DGPP
phosphatase protein. The magnitude of induction of the DGPP phosphatase
enzyme was considerably less than the 64-fold induction of
Zinc homeostasis in S. cerevisiae is largely controlled by
Zrt1p and Zrt2p, which are plasma membrane proteins that transport extracellular zinc into the cytoplasm (51, 52). Expression of Zrt1p and
Zrt2p is induced when the extracellular concentration of zinc is low,
and their expression is repressed when the extracellular concentration
of zinc is high (51, 52). Moreover, when the zinc concentration is low,
the Zrt1p transporter is a stable plasma membrane protein (55).
However, when the zinc concentration is high, Zrt1p is ubiquitinated
(56) and removed from the plasma membrane by endocytosis and vacuolar
degradation (55). Cytoplasmic levels of zinc are controlled further by
the vacuolar membrane efflux (Zrt3p) and influx (Cot1p and Zrc1p) zinc
transporters (50). Expression of DGPP phosphatase activity in the
zrt1 DPP1-encoded DGPP phosphatase activity is independent of any
divalent cation requirement (2). However, DGPP phosphatase activity is
inhibited by manganese, calcium, and magnesium ions (2). In this work,
we showed that the enzyme was inhibited by zinc ions. The inhibitor
constant for zinc ions (150 µM) was within its cellular
concentration (150-1500 µM, based on a cell volume of
5 × 10 Based on studies using well characterized subcellular fractions and
indirect immunofluorescence microscopy, the DGPP phosphatase enzyme was
localized to the vacuolar membrane. DGPP phosphatase is predicted to be
an integral membrane protein with six transmembrane-spanning regions
distributed over its entire protein sequence (1). The three domains
that comprise the active site of DGPP phosphatase (20) are positioned
on the same side of the membrane (1). The internal pH of the vacuole
and the pH optimum for DGPP phosphatase activity are acidic, suggesting
that the active site of the enzyme may be located on the internal
surface of the vacuolar membrane. Additional studies will be required
to address this hypothesis and establish the topology of the enzyme
within the membrane.
Zinc is an essential mineral (58). It is a cofactor for more than 300 enzymes, including superoxide dismutase, carbonic anhydrase, and
alcohol dehydrogenase, as well as several proteases (58). In addition,
several proteins, including transcription factors, require zinc as a
structural cofactor for folding of specific domains (59).
Notwithstanding its essential nature, zinc can be toxic if it is
accumulated in excess amounts (58). The cytoplasmic levels of zinc are
controlled by a variety of mechanisms, including cellular influx (60),
efflux (61, 62), and chelation by metallothioneins (63). As discussed
above, the plasma membrane zinc transporters play a major role in this regulation. Why is a vacuolar membrane-associated DGPP phosphatase activity regulated in a coordinate manner with zinc transporters whose
main function is to regulate zinc homeostasis? The DPP1 gene
is not essential (1). Moreover, dpp1-galactosidase activity driven by a
PDPP1-lacZ reporter gene. This regulation was
dependent on the UASZRE in the DPP1 promoter
and was mediated by the Zap1p transcriptional activator. Induction of
the DGPP phosphatase protein and activity by zinc deprivation was
demonstrated by immunoblot analysis and measurement of the
dephosphorylation of DGPP. The regulation pattern of DGPP phosphatase
in mutants defective in plasma membrane (Zrt1p and Zrt2p) and vacuolar
membrane (Zrt3p) zinc transporters indicated that enzyme expression was sensitive to the cytoplasmic levels of zinc. DGPP phosphatase activity
was inhibited by zinc by a mechanism that involved formation of
DGPP-zinc complexes. Studies with well characterized subcellular fractions and by indirect immunofluorescence microscopy revealed that
the DGPP phosphatase enzyme was localized to the vacuolar membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phosphate from DGPP to form PA and then removes the phosphate from PA to form DG (2).
DGPP is a minor phospholipid in S. cerevisiae (2) that
contains a pyrophosphate group attached to DG (3). DGPP is derived from
PA via the reaction catalyzed by PA kinase (2, 4). DGPP is postulated
to function in a novel lipid-signaling pathway (5, 6). Metabolic
labeling studies with plants, where DGPP was first discovered (3), show
that DGPP accumulates upon G protein activation (7) and upon
hyperosmotic stress (8, 9). Exogenous DGPP augments secretion of
prostaglandins in mouse macrophages (10). This occurs by a mechanism
that involves the activation of cytosolic phospholipase
A2 via the mitogen-activated protein kinase pathway, an
important event in the immunoinflammatory response of leukocytes (10).
The accumulation of DGPP in plants is short-lived (9); it is rapidly
converted to PA and then to DG (11), consistent with the reactions
catalyzed by yeast DGPP phosphatase (2).
mutant reveal
that DGPP phosphatase plays a role in the regulation of phospholipid
metabolism by inositol as well as regulating the cellular levels of
DGPP, PA, and phosphatidylinositol (16, 23). Moreover, inhibition of
DGPP phosphatase activity by CDP-diacylglycerol and stimulation of
phosphatidylserine synthase activity by DGPP may contribute to this
regulation on a biochemical level (23).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside, 4-(2-pyridylazo)resorcinol, and
poly-D-lysine were purchased from Sigma. Restriction
endonucleases, modifying enzymes, and Vent DNA polymerase were obtained
from New England Biolabs. Oligonucleotides for PCR and DNA sequencing
were prepared by Genosys Biotechnologies, Inc. Protein assay reagent,
electrophoresis reagents, and immunochemical reagents were purchased
from Bio-Rad. DNA and protein molecular mass markers were from Life
Technologies and Bio-Rad, respectively. Polyvinylidene difluoride
membranes, protein A-Sepharose, and the enhanced
chemifluorescence Western blotting detection kit were purchased
from Amersham Pharmacia Biotech. Mouse monoclonal anti-HA antibodies
(12CA5) and ImmunoPure fluorescein-conjugated goat anti-mouse IgG (H+L)
antibodies were purchased from Roche Molecular Biochemicals and Pierce,
respectively. FM 4-64 was purchased from Molecular Probes, Inc.
Radiochemicals were purchased from PerkinElmer Life Sciences.
Scintillation counting supplies were from National Diagnostics. Lipids
were purchased from Avanti Polar Lipids. Liqui-Nox detergent was from
Alconox, Inc. Zymolyase 20T was from Seikagaku America Inc.
[
-32P]DGPP was synthesized enzymatically using
purified Catharanthus roseus PA kinase as described by Wu
et al. (2).
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 bacterial cultures 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 spectrophotometrically at an absorbance of
600 nm.
Strains used in this work
AGCTGAAGTCG), which was generated by PCR using
overlap extension. A 0.39-kb 5'-fragment of the DPP1
promoter containing the mutation was amplified by PCR (primers:
5'-GTGAAGAAGCAGGAATTCATAAAGGGACAACACGG-3' and
5'-TAACCAGCTGAAGTCGACTATCTGAAAAAGTGTTG-3'). A 0.48-kb 3'- fragment of
the DPP1 promoter containing the mutation was amplified by
PCR (primers: 5'-TAGTCGACTTCAGCTGGTTAGAGTGCGATCCCTTT-3' and 5'-GTTTTAATAAACGAAACTGAATTCATTTTGGTCG-3'). The two PCR products were
mixed, denatured, annealed, and extended. The overlap-extended DNA
fragment was then amplified by PCR (primers:
5'-GTGAAGAAGCAGGAATTCATAAAGGGACAACACGG-3' and
5'-GTTTTAATAAACGAAACTGAATTCATTTTGGTCG-3'). The 0.85-kb PCR products of
the DPP1 promoter were digested with EcoRI and
substituted for the EcoRI fragment of pJO2. Plasmid pGH210
was constructed by replacing the EcoRI fragment of pJO2 with
the 0.44-kb DPP1 promoter lacking the UASZRE and
its upstream sequence, which was generated by PCR (primers:
5'-CCGGAATTCGGTTAGAGTGCGATCCCTTTA-3' and
5'-GTTTTAATAAACGAAACTGAATTCATTTTGGTCG-3'). Plasmids pGH201 and pGH202
contain the DPP1 gene with sequences for a HA epitope tag
inserted after the start codon. A 0.69-kb fragment containing the
DPP1 promoter, the start codon, and an HA epitope sequence was amplified with the primers 5'-CTCTAGAGTCGACGGTATCGATAAGC-3' and
5'-AGCGTAGTCTGGGACGTCGTATGGGTACATTTTGGTCGTTTGCTATGATTTAATTCT-3' using
pDT2-DPP1 (1) as template. The 1.4-kb fragment containing an HA epitope
sequence, the DPP1 coding sequence, and its 5'-untranslated region was amplified with primers
5'-TACCCATACGACGTCCCAGACTACGCTAACAGAGTTTCGTTTATTAAAACGCCTTTC-3' and
5-GCTTGCATGCCTGCAGGTCGACATT-3'. The two PCR products were mixed,
denatured, annealed, and extended. The overlap-extended HA-DPP1 DNA fragment was then amplified using
primers 5'-CTCTAGAGTCGACGGTATCGATAAGC-3' and
5'-GCTTGCATGCCTGCAGGTCGACATT-3'. The 2-kb PCR products of the
HA-DPP1 DNA were digested with SalI
and inserted into the same restriction site in plasmids pRS415 and
YEp351, generating pGH201 and pGH202, respectively. All plasmid
constructions were confirmed by DNA sequencing.
Plasmids used in this work
-32P]DGPP (10,000-15,000
cpm/nmol) as described by Wu et al. (2). The reaction
mixture contained 50 mM citrate buffer (pH 5.0), 10 mM 2-mercaptoethanol, 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 O-nitrophenyl
-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 (34). 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. DGPP phosphatase and
-galactosidase assays were conducted at 30 and 25 °C,
respectively. The average S.D. of the enzyme assays (performed in
triplicate) was ± 5%. The enzyme reactions were linear with time
and protein concentration. A unit of DGPP phosphatase activity and
-galactosidase activity was defined as the amount of enzyme that
catalyzed the formation of 1 nmol and 1 µmol, respectively, of
product/min. Specific activity was defined as units/mg of protein.
Statistical analyses were performed with SigmaPlot 5.0 software.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Galactosidase Activity in Cells Bearing the PDPP1-lacZ
Reporter Gene--
Yuan (46) recently identified the DPP1
gene in a genetic screen designed to identify genes that are regulated
by zinc deprivation. In addition, inspection of the DPP1
gene revealed that it contains a sequence (ACCTGAAAGGT) in its promoter
(
442 to
452) that is closely related to a consensus
UASZRE (47). The UASZRE is a cis-acting element that affects the transcriptional
activation of several genes (48), including ZRT1,
ZRT2, and ZRT3, in response to zinc deprivation
(47). These observations were the basis for the hypothesis that DGPP
phosphatase was regulated by zinc availability. We examined the
expression of DPP1 in zinc-limited growth media. This
analysis was facilitated by the use of plasmid pJO2 that bears a
PDPP1-lacZ reporter gene where the
DPP1 promoter is fused in-frame with the coding sequence of
the E. coli lacZ gene (23). The expression of
-galactosidase activity in cells with plasmid pJO2 is dependent
on transcription driven by the DPP1 promoter (23). Wild-type
cells bearing plasmid pJO2 were grown to the exponential phase of
growth in SC (zinc replete) medium. The cells were washed, resuspended
in SC medium without zinc, and incubated for 12 h. Control cells
were resuspended in SC medium. The removal of zinc from the growth
medium resulted in a time-dependent increase in
-galactosidase activity (Fig. 1). At
the time of maximum expression, the
-galactosidase activity from
cells grown in the absence of zinc was 3-fold greater than the activity
from the control cells grown in the presence of zinc (Fig. 1). Over the
time course of this experiment, there was no difference in the growth
rate of the two cultures (data not shown).
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Fig. 1.
Time-dependent induction of
-galactosidase activity in cells bearing the
PDPP1-lacZ reporter gene in response to
zinc deprivation. Wild-type (strain W303-1A) cells bearing the
PDPP1-lacZ reporter plasmid pJO2 were grown to
the exponential phase of growth (A600 nm ~ 0.5) in SC medium. Cells were washed three times with deionized
distilled water. Half of the culture was resuspended in an equal volume
of zinc-deplete growth medium, and the other half was resuspended in SC
medium. Both cultures were incubated for 12 h at 30 °C. At the
indicated time intervals, cell extracts were prepared and used for the
assay of
-galactosidase activity. Each data point represents the
average of triplicate enzyme determinations from a minimum of two
independent experiments ± S.D.
-galactosidase activity from
wild-type cells bearing plasmid pJO2 that were grown with various
concentrations of zinc. In these experiments and those described below,
cells were first grown for 24 h in zinc-deplete media to reduce
the intracellular levels of zinc. This regimen was used to accentuate the regulation of the DPP1 gene by zinc deprivation. Zinc
was then added to the final concentrations indicated. Reduction for zinc in the growth medium resulted in a dose-dependent
increase in
-galactosidase activity. The activity found in cells
grown in the absence of zinc was 64-fold greater that the activity in cells grown in the presence of 5 µM zinc (Fig.
2).
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[in a new window]
Fig. 2.
Dose-dependent induction of
-galactosidase activity in cells bearing the
PDPP1-lacZ reporter gene in response to
zinc deprivation. Wild-type (strain W303-1A) cells bearing the
PDPP1-lacZ reporter plasmid pJO2 were grown for
24 h in zinc-deplete media. Cultures were then diluted to 1 × 106 cells/ml in zinc-deplete media and grown to the
exponential phase of growth (A600 nm ~ 0.5)
in the absence and presence of the indicated concentrations of zinc
sulfate. Cell extracts were prepared and used for the assay of
-galactosidase activity. Each data point represents the average of
triplicate enzyme determinations from a minimum of two independent
experiments ± S.D.
-galactosidase in cells grown in the
presence of 1.5 µM zinc, the approximate concentration of
zinc in SC medium, was 0.09 units/mg. There was a 1.8-fold reduction in
-galactosidase activity when the concentration of zinc was increased
to 5 µM (Fig. 2). Concentrations of zinc up to 1 mM did not cause a further reduction in
-galactosidase
activity (data not shown).
-Galactosidase Activity in Cells Bearing the PDPP1-lacZ Reporter
Gene in Response to Zinc Deprivation--
The effect of mutations in
the putative UASZRE in the DPP1 promoter on
the regulation by zinc was examined. The putative UASZRE in
the PDPP1-lacZ reporter plasmid pJO2 was changed
to a nonconsensus sequence by transition and transversion mutations
(plasmid pGH209) and was deleted from the DPP1 promoter
(plasmid pGH210). Cells bearing these reporter plasmids were
precultured in zinc-deplete media and then incubated in media without
and with 1.5 µM zinc. These mutations did not affect the
expression of
-galactosidase activity when cells were grown in the
presence of zinc (Fig. 3). In contrast to
the control, cells bearing the mutations in the UASZRE and
lacking the UASZRE did not exhibit the great induction of
-galactosidase activity when grown in the absence of zinc (Fig.
3).
View larger version (13K):
[in a new window]
Fig. 3.
Effect of UASZRE mutations on the
regulation of DPP1 by zinc deprivation. Wild-type
(strain W303-1A) cells bearing the PDPP1-lacZ
reporter plasmid pJO2, pGH209, or pGH210 were grown for 24 h in
zinc-deplete media. Cultures were then diluted to 1 × 106 cells/ml in zinc-deplete media and grown to the
exponential phase of growth (A600 nm ~ 0.5)
in the absence and presence of 1.5 µM zinc sulfate. Cell
extracts were prepared and used for the assay of -galactosidase
activity. Each data point represents the average of triplicate enzyme
determinations from a minimum of two independent experiments ± S.D. Mutations in the UASZRE in plasmid pGH209 are
underlined.
mutant bearing plasmid pJO2 was
grown in the absence and presence of 1.5 µM zinc followed by the measurement of
-galactosidase activity. In contrast to wild-type cells, the absence of zinc in the growth medium did not
result in the induction of
-galactosidase activity (Fig. 4). As a control, we examined the
expression of
-galactosidase activity in wild-type and
zap1
mutant cells bearing the reporter plasmid pDg2. This
plasmid contains one UASZRE from the ZRT1 gene (47). As described previously (47),
-galactosidase activity was
induced in wild-type cells but was not induced in zap1
mutant cells when they were grown in the absence of zinc (Fig. 4).
View larger version (14K):
[in a new window]
Fig. 4.
Effect of the zap1
mutation on the regulation of DPP1 by zinc
deprivation. Wild-type (strain W303-1A) and zap1
mutant (strain ZHY6) cells bearing plasmid pJO2
(PDPP1-lacZ) or plasmid pDg2
(PCYC1-lacZ containing one UASZRE of
ZRT1) were grown for 24 h in zinc-deplete media.
Cultures were then diluted to 1 × 106 cells/ml in
zinc-deplete media and grown to the exponential phase of growth
(A600 nm ~ 0.5) in the absence and presence
of 1.5 µM zinc sulfate. Cell extracts were prepared and
used for the assay of
-galactosidase activity. Each data point
represents the average of triplicate enzyme determinations from a
minimum of two independent experiments ± S.D.
View larger version (26K):
[in a new window]
Fig. 5.
Dose-dependent induction of the
levels of DGPP phosphatase activity and protein in response to zinc
deprivation. Wild-type (strain W303-1A) cells (panel A)
and dpp1 mutant (strain DTY1) cells bearing plasmid
pGH201 with the HA-tagged DPP1 gene (panel B)
were grown for 24 h in zinc-deplete media. Cultures were then
diluted to 1 × 106 cells/ml in zinc-deplete media and
grown to the exponential phase of growth
(A600 nm ~ 0.5) in the absence and presence
of the indicated concentrations of zinc sulfate. Cell extracts were
prepared and used for the assay of DGPP phosphatase activity
(panel A) and immunoblot analysis using a 1:1000 dilution of
anti-HA antibodies (panel B). Each activity data point
represents the average of triplicate enzyme determinations from a
minimum of two independent experiments ± S.D. A portion of the
immunoblot is shown, and the position of the HA-tagged DGPP phosphatase
protein (Dpp1pHA) is indicated.
mutant to avoid interference from
the DGPP phosphatase activity encoded by the genomic wild-type copy of
the DPP1 gene (1). This plasmid directed normal levels of DGPP phosphatase activity (data not shown). The epitope-tagged DGPP
phosphatase protein was specifically recognized by anti-HA antibodies
at the expected molecular mass of about 34 kDa. Immunoblot analysis
showed that the levels of the DGPP phosphatase protein increased
10-fold in a dose-dependent manner when zinc was depleted from the growth medium (Fig. 5B).
zrt2
double mutant and in a zrt3
mutant,
respectively, when compared with the wild-type control (Fig.
6A). Expression of DGPP
phosphatase activity in a cot1
zrc1
double
mutant grown in the presence of zinc was not significantly different
from that in wild-type cells (Fig. 6A). Deprivation of zinc
from the growth medium of the mutants resulted in a further increase in
DGPP phosphatase activity that was comparable with the level of
activity found in wild-type cells grown without zinc (Fig.
6A). Expression of the DGPP phosphatase protein was also
examined in the zinc transporter mutants. For these experiments, the
enzyme protein was analyzed by immunoblotting using anti-DGPP
phosphatase antibodies. The pattern of regulation for DGPP phosphatase
protein expression (Fig. 6B) in the mutants was consistent
with the activity found in these cells (Fig. 6A).
View larger version (16K):
[in a new window]
Fig. 6.
Effect of zrt1
zrt2
,
zrt3
, and cot1
zrc1
mutations on the regulation of DGPP
phosphatase activity and protein by zinc deprivation. Wild-type
(WT; strain DY1457) cells, zrt1
zrt2
(strain ZHY3), zrt3
(strain CM101),
and cot1
zrc1
(strain CM104) mutant cells
were grown for 24 h in SC growth media containing 5 µM zinc sulfate. Cultures were then diluted to 1 × 106 cells/ml in zinc-deplete media and grown to the
exponential phase of growth (A600 nm ~ 0.5)
in the absence and presence of 5 µM zinc sulfate. Cell
extracts were prepared and used for the assay of DGPP phosphatase
activity (panel A) and subjected to immunoblot analysis
using a 1:1000 dilution of anti-DGPP phosphatase antibodies
(panel B). Each activity data point represents the average
of triplicate enzyme determinations from a minimum of two independent
experiments ± S.D. The density of the DGPP phosphatase protein
(Dpp1p) bands on the immunoblot was quantified by scanning
densitometry. The amount of the DGPP phosphatase protein in wild-type
cells grown in the presence of zinc was set at 1. Data that was found
with wild-type strain CM100 was the same as that found with wild-type
strain DY1457. The immunoblot data shown is representative of two
independent experiments.
View larger version (19K):
[in a new window]
Fig. 7.
Effect of zinc on DGPP phosphatase
activity. DGPP phosphatase activity was measured using pure enzyme
with the indicated concentrations of ZnCl2. The
inset is a replot of the zinc-mediated inhibition of DGPP
phosphatase activity.
View larger version (21K):
[in a new window]
Fig. 8.
Subcellular localization of DGPP
phosphatase. Subcellular fractions were isolated from wild-type
cells. Each fraction (8.5 µg) was subjected to SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose paper, and probed with a
1:1000 dilution of anti-DGPP phosphatase antibodies. A portion of the
immunoblot is shown, and the positions of the 34-kDa DGPP phosphatase
protein (Dpp1p) and the 31-kDa molecular mass standard are indicated.
The data shown is representative of two independent experiments.
View larger version (37K):
[in a new window]
Fig. 9.
Immunofluorescent localization of DGPP
phosphatase. Wild-type cells bearing the HA-tagged DGPP
phosphatase were grown to late exponential phase of growth, harvested,
fixed, probed for the HA epitope, and viewed by phase contrast
(panel A) and fluorescence (panel B) microscopy.
The bright structure within the cell shown in panel A
corresponds to the vacuole.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant background.
-galactosidase activity driven by the
PDPP1-lacZ reporter gene. However, the
regulation of the DGPP phosphatase enzyme was more consistent with that
of DPP1 mRNA abundance (46, 48).
zrt2
mutant grown in the presence of
zinc was significantly greater (4.3-fold) than that found in wild-type
cells. The zrt3
mutation in the vacuolar membrane zinc
efflux transporter also caused an increase (2-fold) in expression of
DGPP phosphatase activity, but to a smaller magnitude. These data were
consistent with the hypothesis that expression of DGPP phosphatase was
sensitive to the cytoplasmic levels of zinc.
13 liter/cell), determined for
cells grown with varying concentrations of zinc (47, 55). Thus,
regulation of DGPP phosphatase activity by zinc may be physiologically
relevant. Pyrophosphate compounds have the ability to chelate divalent
cations (57). Our studies indicated that DGPP had the ability to form
complexes with zinc. This observation was consistent with a mechanism
of inhibition of DGPP phosphatase that involved the formation of
DGPP-zinc complexes.
mutants do not
exhibit any dramatic phenotypes under a variety of growth conditions
(1), including fluctuations in zinc supplementation (data not shown) (46). Thus, the role of DGPP phosphatase in response to zinc deprivation would have to be complimentary to other mechanisms that
respond to this stress. DGPP phosphatase plays a role in regulating the
levels of DGPP, PA, and phosphatidylinositol in S. cerevisiae (16, 23). Although the function of DGPP is unclear, it
is tempting to speculate that it may function to chelate a specific
pool of zinc ions at the surface of the vacuolar membrane. This
function would be eliminated by the dephosphorylation of free DGPP by
the phosphatase, especially under zinc-limiting conditions. An
alternative role for the enzyme may be to control the levels of PA and
phosphatidylinositol in vacuolar membranes. These two phospholipids
play critical roles in the structure and function of membranes in
S. cerevisiae and in higher eukaryotic cells (5, 24, 25).
Accordingly, future studies will address the role DGPP phosphatase
plays in the regulation of vacuolar phospholipid metabolism in response
to zinc deprivation.
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ACKNOWLEDGEMENTS |
---|
We acknowledge Daniel S. Yuan for providing us with the observation that he identified the DPP1 gene based on its regulation by zinc deprivation before the publication of his paper (46). We thank David Eide for providing us with the observation that the DPP1 gene contained a putative UASZRE, for providing strains ZHY6, DY1457, ZHY3, CM101, CM104, and CM100, for plasmid pDg2, and for many helpful discussions.
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FOOTNOTES |
---|
* This work was supported in part by United States Public Health Service Grant GM-28140 from the National Institutes of Health (to G. M. C.) and by the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich project 13669 (to G. D.).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: Dept. of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901. Tel.: 732-932-9611 (ext. 217); Fax: 732-932-6776; E-mail: carman@aesop.rutgers.edu
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M011421200
2 J. Oshiro and G. M. Carman, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: DGPP, diacylglycerol pyrophosphate; PA, phosphatidate; DG, diacylglycerol; UASZRE, zinc-responsive element, PCR, polymerase chain reaction; HA, hemagglutinin; kb, kilobases; U, units.
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