THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 279, No. 7, Issue of February 13, pp. 5338 –5345, 2004
Printed in U.S.A.
Vacuole Membrane Topography of the DPP1-encoded
Diacylglycerol Pyrophosphate Phosphatase Catalytic Site
from Saccharomyces cerevisiae*
Received for publication, October 27, 2003, and in revised form, November 18, 2003
Published, JBC Papers in Press, November 20, 2003, DOI 10.1074/jbc.M311779200
Gil-Soo Han, Celeste N. Johnston, and George M. Carman‡
From the Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901
The Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase is a vacuole membrane-associated enzyme that catalyzes the removal of
the ␤-phosphate from diacylglycerol pyrophosphate to
form phosphatidate, and it then removes the phosphate
from phosphatidate to form diacylglycerol. The enzyme
has six putative transmembrane domains and a hydrophilic region that contains a phosphatase motif required for its catalytic activity. In this work, we examined the topography of diacylglycerol-pyrophosphate
phosphatase catalytic site within the transverse plane
of the vacuole membrane. Results of protease protection
analysis using endoproteinase Lys-C and labeling of cysteine residues using sulfhydryl reagents were consistent
with a model where the catalytic site of diacylglycerolpyrophosphate phosphatase was oriented to the cytosolic face of the vacuole membrane. In addition, diacylglycerol-pyrophosphate phosphatase activity was found
with intact vacuoles. The phospholipids diacylglycerol
pyrophosphate (0.6 mol %) and phosphatidate (1.4 mol
%) were found in the vacuole membrane, and their levels
decreased to an undetectable level and by 79%, respectively, when cells were depleted for zinc. The reduced
levels of diacylglycerol pyrophosphate and phosphatidate correlated with the induced expression of diacylglycerol-pyrophosphate phosphatase. This work suggested that diacylglycerol pyrophosphate phosphatase
functions to regulate the levels of diacylglycerol pyrophosphate and phosphatidate on the cytosolic face of
the vacuole membrane.
DGPP1 phosphatase, encoded by the DPP1 gene (1–3), is a
34-kDa vacuole membrane-associated enzyme (4) that was first
discovered in the yeast Saccharomyces cerevisiae (5). It catalyzes the removal of the ␤-phosphate from DGPP to form PA,
and it then removes the phosphate from PA to form diacylglycerol (Fig. 1) (5). DGPP is the preferred substrate, and PA
will not serve as a substrate in the presence of DGPP (5). DGPP
phosphatase also utilizes lyso-PA (6), sphingoid base phos-
* This work was supported in part by U. S. Public Health Service,
National Institutes of Health Grant GM-28140. The costs of publication
of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: Dept. of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901.
Tel.: 732-932-9611 (ext. 217); E-mail: [email protected].
1
The abbreviations used are: DGPP, diacylglycerol pyrophosphate;
PA, phosphatidate; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; HA, hemagglutinin; AMS, 4-acetamido-4⬘-maleimidylstilbene-2,2⬘-disulfonic
acid; MPB, N␣-(3-maleimidylpropionyl) biocytin.
phates (7), and isoprenoid phosphates (2) as substrates in vitro.
However, only DGPP and PA have been shown to be substrates
in vivo (8). In fact, the products of the LCB3/LBP1/YSR2 (9 –11)
and the CWH8 (12) genes are responsible for the dephosphorylation of sphingoid base phosphates and isoprenoid phosphates in vivo, respectively.
The DGPP molecule was originally identified in plants as the
product of the PA kinase enzyme (13). Research with plants
indicates that DGPP may function as a signaling molecule
under stress conditions. It accumulates upon G-protein activation (14), hyperosmotic stress (15), dehydration (15), Rhizobium-secreted nodulation factors (16), and general elicitors such
as xylanase (17). DGPP accumulation is transient and coincides with a rise in PA levels (15, 17). Because of the reactions
catalyzed by DGPP phosphatase, the enzyme may play a role
during stress conditions to regulate specific cellular pools of
DGPP and PA (18). Indeed, the Arabidopsis thaliana homolog
(AtLPP1) of the yeast DPP1 gene is induced by genotoxic stress
(␥ and UV-B radiation), G-protein activation, and oxidative
stress (19). Consistent with these observations, the S. cerevisiae DPP1-encoded DGPP phosphatase is induced by the stress
conditions of zinc depletion (4) and stationary phase (20) and by
inositol supplementation (20). Moreover, the expression of the
DPP1 homolog in the pathogenic yeast Candida albicans is
induced in clinical isolates that are resistant to azole antifungal agents (21).
The catalytic activity of DGPP phosphatase is conferred by a
three-domain lipid phosphatase motif (22) found in the lipid
phosphatase superfamily (23, 24). The conserved Arg125,
His169, and His223 residues within domains I, II, and III, respectively, are required for both the DGPP phosphatase and PA
phosphatase activities of the enzyme (25). The lipid phosphatase activities of DGPP phosphatase are Mg2⫹-independent
and N-ethylmaleimide-insensitive (5). The PA phosphatase activity of the DGPP phosphatase is distinct (5) from the Mg2⫹dependent and N-ethylmaleimide-sensitive PA phosphatase
enzyme (26, 27) that is presumably responsible for the synthesis of phospholipids and triacylglycerols in S. cerevisiae (28). In
addition to yeast, DGPP phosphatase activity is found in a wide
range of organisms (e.g. plants, bacteria, and mammalian cells
(19, 29)), suggesting an important role for the enzyme in cell
physiology (28).
Deletion of the DPP1 gene results in altered phospholipid
composition (8). dpp1⌬ mutant cells exhibit increased amounts
of DGPP and PA and a decrease in the amount of PI (8).
Moreover, DPP1 is one of the most highly regulated genes that
respond to zinc depletion in the S. cerevisiae genome (3, 30).
However, the physiological role of the DGPP phosphatase enzyme is not fully understood because dpp1⌬ mutant cells do not
exhibit dramatic phenotypic changes under standard laboratory conditions (1–3). The subcellular location of DGPP phos-
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This paper is available on line at http://www.jbc.org
Topography of Yeast DGPP Phosphatase Catalytic Site
FIG. 1. Reactions catalyzed by DGPP phosphatase. The figure
shows the reactions catalyzed by the DPP1-encoded DGPP phosphatase
(Dpp1p) enzyme. DAG, diacylglycerol.
phatase to the vacuole membrane suggests that the functional
role of the enzyme utilizing phospholipid substrates should be
governed by the topography of its catalytic site. In this report,
we provided evidence based on protease protection analysis,
chemical labeling, and enzyme activity measurements that the
DGPP phosphatase catalytic site was oriented to the cytosolic
face of the vacuole membrane. In addition, we demonstrated
that the enzyme substrates DGPP and PA were present in the
vacuole membrane and that their levels were significantly reduced when cells were depleted for zinc.
EXPERIMENTAL PROCEDURES
Materials—All of the chemicals were reagent grade. Growth medium
supplies were purchased from Difco Laboratories. Protease inhibitors
(aprotinin, benzamidine, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride), Ficoll 400, lyticase, 4-(2-pyridylazo)resorcinol, bovine serum albumin, urea, Triton X-100, and endoproteinase Lys-C were purchased from Sigma. Protein assay reagent, electrophoresis reagents,
and molecular mass markers were purchased from Bio-Rad. Mouse
monoclonal anti-HA antibodies (12CA5) and agarose-conjugated anti-HA antibodies were purchased from Roche Applied Science. FM4 – 64,
4-acetamido-4⬘-maleimidylstilbene-2,2⬘-disulfonic acid (AMS), N␣-(3maleimidylpropionyl)biocytin (MPB), monoclonal antibodies against
yeast carboxypeptidase Y, and vacuolar H⫹-ATPase were purchased
from Molecular Probes. Alkaline phosphatase-linked goat anti-rabbit
(or mouse) IgG antibodies were purchased from Pierce. Radiochemicals
were from PerkinElmer Life Sciences. Scintillation counting supplies
were from National Diagnostics. Liqui-Nox was from Alconox, Inc.
Polyvinylidene difluoride membranes and the enhanced chemifluorescence Western blotting detection kit were purchased from Amersham
Biosciences. Silica Gel 60 high performance TLC plates were from EM
Science, and phospholipids were obtained from Avanti Polar Lipids.
Strains and Growth Conditions—S. cerevisiae strain W303–1A
(MATa ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1) (31) is
the parent of the dpp1⌬ mutant strain DTY1 (MATa ade2–1 can1–100
his3–11,15 leu2–3,112 trp1–1 ura3–1 dpp1⌬::TRP1/Kanr) (1). The HAtagged version of the DPP1 gene was expressed in DTY1 from plasmid
pGH202 (4), which is a derivative of YEp351 (32). Cultures were grown
at 30 °C in YPDA medium (1% yeast extract, 2% peptone, 2% glucose,
and 25 mg/liter adenine) or in synthetic complete medium (33) containing 2% glucose. The appropriate amino acid of synthetic complete medium was omitted for selection purposes. Yeast cell numbers in liquid
media were determined spectrophotometrically at an absorbance of 600
nm. Cultures were grown to the exponential phase (1–2 ⫻ 107 cells/ml)
5339
and harvested by centrifugation at 1,500 ⫻ g for 5 min. For zincdepleted cultures, cells were first grown for 24 h in synthetic complete
medium supplemented with 1.4 ␮M zinc sulfate. Cultures were harvested, washed in de-ionized distilled water, diluted to 1 ⫻ 106 cells/ml
in media containing 0 or 1.4 ␮M zinc sulfate, and grown for 24 h.
Cultures were then diluted to 1 ⫻ 106 cells/ml and grown in media
containing 0 or 1.4 ␮M zinc sulfate. This process was used to deplete
internal stores of zinc. For zinc-free glassware, items were washed with
Liqui-Nox and rinsed several times with distilled water followed by a
final rinse with de-ionized distilled water.
Preparation of Cell Extracts and Vacuoles—Cell extracts were prepared as described previously (34, 35). Cells were suspended in 50 mM
Tris maleate buffer (pH 7.0) containing 1 mM EDTA, 0.3 M sucrose, 10
mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, 5 ␮g/ml aprotinin, 5 ␮g/ml leupeptin, and 5 ␮g/ml pepstatin. Cells were disrupted by homogenization with chilled glass beads
(0.5-mm diameter) using a Biospec Products bead beater. Samples were
homogenized for 10 1-min bursts followed by a 2-min cooling between
bursts at 4 °C. The cell extract (supernatant) was obtained by centrifugation of the homogenate at 1,500 ⫻ g for 10 min. Vacuoles were
isolated from spheroplasts by several steps of flotation and density
gradient centrifugation as described by Uchida et al. (36). The protein
concentration of the cell extract and vacuoles was determined by the
method of Bradford (37) using bovine serum albumin as the standard.
Staining of Vacuoles with FM4 – 64 —Vacuolar membranes were
stained with the specific styryl dye FM4 – 64 (38) at a final concentration of 50 ␮M. Fluorescent images were observed and recorded using an
Olympus BH2-RFCA fluorescence microscope equipped with a Photometrics Sensys KAF-1400 CCD camera.
SDS-PAGE and Immunoblot Analysis—Proteins were separated by
SDS-PAGE (39) using 10% gels or 10 –20% gradient gels. Proteins were
transferred to polyvinylidene difluoride membranes for immunoblotting
as described by Haid and Suissa (40). Membranes were probed with an
appropriate dilution of the indicated primary antibody. Goat anti-rabbit
or anti-mouse IgG alkaline phosphatase conjugate was used as a secondary antibody against primary antibodies produced in rabbits or
mice, respectively, at a dilution of 1:5000. Antibody interactions were
detected using the enhanced chemifluorescence Western blotting detection kit as described by the manufacturer. Signals on the membrane
were acquired by fluoroimaging analysis. The relative density of the
protein signals was analyzed using ImageQuant software. Immunoblot
signals were in the linear range of detectability.
Endoproteinase Lys-C Treatment of Vacuoles—Vacuoles (5 ␮g of protein) were mixed with 1 ␮g of endoproteinase Lys-C and incubated for
2 h at 37 °C in a total volume of 15 ␮l. The reaction mixture contained
50 mM Tris-HCl (pH 8.5), 0.3 M sucrose, and 3 M urea. Urea was
included in the reaction mixture, because it facilitated exposure of
lysine residues to endoproteinase Lys-C and provided some protection
from the endogenous vacuolar protease(s) that digested the DGPP phosphatase in the detergent-solubilized vacuole. Vacuoles were ruptured
by including 0.4% Triton X-100 in the reaction mixture. After incubation, 5 mM phenylmethylsulfonyl fluoride was added to the mixture to
inhibit the protease. The reaction products were then subjected to
SDS-PAGE with a 10 –20% gradient gel followed by transfer to a polyvinylidene difluoride membrane. The membrane was probed with anti-HA antibodies or with antibodies raised against the C-terminal
epitope (residues 263–279) of DGPP phosphatase (20).
Chemical Labeling of Cysteine Residues—The cysteine residues of
vacuole membrane proteins were labeled with sulfhydryl reagents as
described by Leng et al. (41) with some modifications. Vacuoles (15 ␮g
of protein) were suspended in 200 ␮l of Tris-HCl buffer (pH 7.4) containing 0.3 M sucrose and incubated for 10 min at 25 °C in the absence
or presence of 100 ␮M AMS, a membrane-impermeable reagent used to
label cysteine residues. The reaction mixture was then diluted to 1 ml
with vacuole suspension buffer and incubated for 20 min at 25 °C with
250 ␮M MPB, a membrane-permeable reagent used to label cysteine
residues with biotin. The labeling reaction was terminated by the
addition of 20 mM 2-mercaptoethanol. The sulfhydryl reagent-treated
vacuoles were collected by centrifugation at 37,000 ⫻ g for 30 min at
4 °C. The vacuoles were lysed in 500 ␮l of radioimmunoprecipitation
buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5%
sodium deoxycholate, and 0.1% SDS) (42) and mixed with 100 ␮l of 10%
slurry of agarose-conjugated anti-HA antibodies for 2 h at 4 °C. The
anti-HA affinity matrix was collected by centrifugation at 5,000 ⫻ g for
30 s, washed twice with radioimmunoprecipitation buffer, and resuspended in 20 ␮l of SDS-PAGE treatment buffer. After boiling and a brief
centrifugation, the supernatant was subjected to SDS-PAGE with a
10 –20% gradient gel followed by transfer to polyvinylidene difluoride
5340
Topography of Yeast DGPP Phosphatase Catalytic Site
membrane. The membrane was probed with alkaline phosphataseconjugated NeutrAvidin to detect biotinylated proteins. The membrane
was then stripped and re-probed with anti-HA antibodies to detect
DGPP phosphatase.
Preparation of Labeled DGPP Substrates—32P-Labeled DGPP was
enzymatically synthesized from PA and ATP with purified Catharanthus roseus PA kinase as described by Wu et al. (5). [␤-32P]DGPP was
synthesized from PA and [␥-32P]ATP, whereas [␣-32P]DGPP was synthesized from [32P]PA and ATP. The [32P]PA used in the latter reaction
was synthesized from diacylglycerol and [␥-32P]ATP using Escherichia
coli diacylglycerol kinase (43). The 32P-labeled DGPP substrates were
purified by thin-layer chromatography on potassium oxalate-treated
plates using the solvent system chloroform/acetone/methanol/glacial
acetic acid/water (50:15:13:12:4) (5).
DGPP Phosphatase Assays—DGPP phosphatase activity was routinely measured by following the release of water-soluble 32Pi from chloroform-soluble [␤-32P]DGPP (10,000 cpm/nmol) (5). Unless otherwise indicated, the standard reaction mixture contained 50 mM citrate buffer (pH
5.0), 0.1 mM [␤-32P]DGPP, 2 mM Triton X-100, and enzyme protein in a
total volume of 0.1 ml. The function of Triton X-100 in the assay system is
to solubilize the DGPP substrate (5). All of the enzyme assays were
conducted in triplicate at 30 °C. The mean ⫾ S.D. of the assays was ⫾ 5%.
DGPP phosphatase reactions were linear with time and protein concentration. A unit of enzymatic activity was defined as the amount of enzyme
that catalyzed the formation of 1 ␮mol of product/min.
DGPP phosphatase activity was also measured with DGPP incorporated into phospholipid vesicles that were prepared by sonication (44).
The phospholipid vesicles contained 0.18 mM PC, 0.125 mM PE, 0.125
mM PI, 0.062 mM PS, and 0.1 mM [␣-32P]DGPP. The molar ratio of
PC/PE/PI/PS in the vesicles was 3:2:2:1, the approximate composition of
the major phospholipids in wild type S. cerevisiae (45). The molar ratio
of the PC/PE/PI/PS mixture to DGPP was 5:1. In this assay, DGPP
phosphatase activity was measured by following the formation of
[32P]PA from [␣-32P]DGPP (10,000 cpm/nmol) (5). Following the reaction, phospholipid vesicles (supernatant) were separated from vacuoles
by centrifugation at 37,000 ⫻ g for 30 min. Phospholipids were extracted (46) and analyzed by thin-layer chromatography using chloroform/acetone/methanol/glacial acetic acid/water (50:15:13:12:4) as the
solvent system (5). The identity of the labeled phospholipids on the
chromatography plates was confirmed by comparison with standard
radioactive DGPP and PA. Radiolabeled DGPP and PA were visualized
by phosphorimaging analysis, and their relative quantities were analyzed using ImageQuant software.
Analysis of Vacuole Membrane Phospholipids—Phospholipids were
extracted from vacuoles by the method of Bligh and Dyer (46). The
aqueous phase and mat of solid material between the two phases of this
extraction were removed with a Pasteur pipette and subjected to a
second extraction with chloroform. The chloroform phases were combined, dried under nitrogen, and resuspended in 500 ␮l of chloroform/
methanol (9:1, v/v). A 25-␮l aliquot was used to determine the total
phosphate content of the sample. The remainder was dried to a minimal
volume under nitrogen. The sample was spotted onto an oxalate-EDTA
(1.2% potassium oxalate, 2 mM EDTA dissolved in methanol/water (2:3,
v/v)) treated high performance TLC Silica Gel 60 plate, which had been
heated at 110 °C overnight and cooled to room temperature immediately prior to the application of the sample. The first dimension was
developed using chloroform/methanol/ammonium hydroxide/water (30:
16.7:1.3:2, v/v). The plate was allowed to completely dry in the hood for
at least 1 h. It was then dried in vacuo for an additional 2 h. The second
dimension was developed using chloroform/methanol/acetic acid/water
(32:4:5:1, v/v). The plate was allowed to dry completely overnight in the
hood. Phospholipids were visualized by acid charring (47). Plates were
sprayed with sulfuric acid/methanol (1:1, v/v) and heated at 120 °C for
20 min. This treatment caused the phospholipids to become purple-gray
in color while the rest of the plate remained white. Vacuole phospholipids were identified by comparison to standard phospholipids. Silica
gel containing phospholipids was scraped from the plate and collected
directly into glass ignition tubes. Distilled, de-ionized water (300 ␮l)
and 70% perchloric acid (150 ␮l) were added directly to the silica gel.
The samples were heated at 150 °C overnight to digest the phospholipids. After cooling to room temperature, the digested sample was filtered
with an Acrodisc 4-mm syringe filter and subjected to phosphate determination as described by Ames and Dubin (48). Potassium phosphate
was used as the standard.
Analyses of Data—Statistical analyses were performed with SigmaPlot software. Statistical significance was determined by performing
the Student’s t test. p values ⬍ 0.05 were taken as a significant difference.
FIG. 2. Models for the topography of DGPP phosphatase in the
vacuole membrane. The figure shows predicted topography models of
DPP1-encoded DGPP phosphatase in the vacuole membrane with the
catalytic site facing the lumen (upper) or facing the cytosol (lower). The
residues in the regions that comprise domains I, II, and III of the
phosphatase motif are indicated as black circles. The hydrophilic loops
are designated L1–L3.
RESULTS
Models for the Topography of the DPP1-encoded DGPP Phosphatase Catalytic Site—Hydropathy analysis and analogy to
the mammalian lipid phosphatase Dri 42 (49) predicts that
DPP1-encoded DGPP phosphatase has six transmembrane regions with hydrophilic N- and C-terminal sequences localized
to the same side of the membrane (Fig. 2) (1). With the exception of the transmembrane regions, most of the amino acid
residues between the first and last transmembrane regions
comprised hydrophilic loops designated L1–L3. These loops are
located on the opposite side of the membrane to the hydrophilic
N- and C-terminal amino acid residues (Fig. 2) (1). The three
domains of the lipid phosphatase motif (1, 22, 25) are oriented
to the same side of the membrane (1). In the model shown in
Fig. 2, domains I and II are present in loop L2, whereas domain
III is present in loop L3. The catalytic site of the DGPP phosphatase enzyme may be oriented to the cytosolic or the luminal
surface of the vacuole membrane (Fig. 2).
Enrichment of the HA-tagged DPP1-encoded DGPP phosphatase in Purified Vacuoles—To determine the topography of the
DGPP phosphatase enzyme in the vacuole membrane, we used
vacuoles purified from dpp1⌬ mutant cells expressing a DPP1HA
allele on a multicopy plasmid. The N-terminal HA-tagged DGPP
phosphatase was functional (4) and was overexpressed to facilitate its analysis by immunological methods and chemical labeling. The enzyme is specifically recognized by anti-HA antibodies
at the expected molecular mass of ⬃34 kDa (4). Our preparations
were highly enriched for vacuoles as assessed by immunoblot
analysis using specific antibodies to the vacuole membrane H⫹ATPase and the vacuole luminal carboxypeptidase Y (Fig. 3A).
The enrichment of carboxypeptidase Y confirmed that the procedure (36) used to isolate the vacuoles resulted in a preparation
with a right-side-out orientation.
Immunoblot analysis using anti-HA antibodies showed that
the DGPP phosphatase protein was enriched 12-fold in the
vacuole membrane when compared with that observed in the
Topography of Yeast DGPP Phosphatase Catalytic Site
5341
FIG. 4. Intact vacuoles stained with the florescent dye FM4 –
64. Vacuoles were isolated from cells as described under “Experimental
Procedures.” The purified vacuoles were mixed with the fluorescent dye
FM4 – 64 at a final concentration of 50 ␮M and visualized by fluorescence microscopy.
FIG. 3. Enrichment of the HA-tagged DPP1-encoded DGPP
phosphatase in purified vacuoles. dpp1⌬ mutant cells with and
without the DPP1HA allele on a multicopy plasmid were grown to
exponential phase. Cells were harvested, and vacuoles were isolated as
described under “Experimental Procedures.” Panel A, 5 ␮g of cell extract (E) and vacuoles (V) were subjected to SDS-PAGE using a 10%
slab gel followed by transfer to polyvinylidene difluoride membrane.
The membrane was probed with anti-HA antibodies to detect the HAtagged DGPP phosphatase (Dpp1pHA) and antibodies against the 100kDa subunit of the vacuole H⫹-ATPase (Vph1p) and the 61-kDa carboxypeptidase Y (Prc1p). The data are representative of three
independent vacuole preparations. Panel B, DGPP phosphatase activity
was measured with the indicated fractions under standard assay conditions using [␤-32P]DGPP as substrate. Each data point represents the
average of triplicate determinations ⫾ S.D.
cell extract (Fig. 3A). Faster migrating forms of the DGPP
phosphatase protein in the vacuoles were recognized by the
anti-HA antibodies (Fig. 3A). This indicated that the enzyme
was subject to proteolytic cleavage at its C-terminal end during
the purification of vacuoles. The presence of protease inhibitors
during the vacuole preparation did not prevent this proteolysis.
The enrichment of DGPP phosphatase activity in the purified vacuoles was examined. Cell extracts and vacuoles from
the dpp1⌬ mutant expressing the HA-tagged enzyme were
assayed for DGPP phosphatase activity under standard assay
conditions. The specific activity of DGPP phosphatase in vacuoles (2.6 units/mg) was 16.8-fold greater than the activity
(0.154 units/mg) in the cell extract (Fig. 3B). The LPP1 gene of
S. cerevisiae encodes a Golgi membrane-associated (50) lipid
phosphatase enzyme that also exhibits DGPP phosphatase activity (8). To examine the possible contribution of the LPP1encoded enzyme to the DGPP phosphatase activity in vacuoles,
we measured activity in vacuoles purified from dpp1⌬ mutant
cells that did not bear the DPP1HA allele. The vacuoles derived
from the dpp1⌬ mutant did not possess any measurable DGPP
phosphatase activity (Fig. 3B).
DGPP Phosphatase Activity of Intact and Ruptured Vacuoles—Activity measurements of a membrane-associated phospholipid synthetic enzyme before and after organelle disruption with detergent have been used to determine the
topography of the enzyme catalytic site (51, 52). The lack of
significant enzyme activity with an intact organelle preparation and a significant amount of latent activity following organelle rupture implies that the catalytic site faces the lumen
of that organelle (51, 52). Likewise, the expression of a significant amount of activity with the intact organelle coupled to a
lack of latent activity following rupture implies that the catalytic site faces the external face of the organelle (51, 52). To
address whether the DGPP phosphatase catalytic site was
oriented to the cytosolic or the luminal surface of the vacuole
membrane, enzyme activity was measured using intact and
ruptured vacuoles. Throughout this work, vacuole integrity
was confirmed by microscopic examination of vacuoles stained
with the fluorescent dye FM4 – 64. Intact vacuoles exhibited a
fluorescent signal that was concentrated at the periphery of the
vacuoles (Fig. 4). Upon rupture, the fluorescent signal of the
dye dissipated. A significant amount of DGPP phosphatase
activity (1.3 units/mg) was exhibited with intact vacuoles (Fig.
5). For this experiment, the concentrations of DGPP and Triton
X-100 were reduced from the standard assay conditions to 0.04
and 0.4 mM (0.024%), respectively. Under these conditions, the
concentration of detergent was high enough to solubilize the
phospholipid substrate delivered to the assay system but was
not high enough to rupture the vacuole. These results indicated
that the enzyme catalytic site was oriented to the cytosolic face
of the vacuole membrane. Vacuole membranes were then ruptured with Triton X-100 and assayed for DGPP phosphatase
activity. For this experiment, the concentrations of DGPP and
Triton X-100 were 0.04 and 8 mM (0.4%), respectively. The activity (7 units/mg) found in detergent-solubilized vacuoles was
5-fold greater than that observed with the intact vacuoles (Fig. 5).
To further examine whether the catalytic site of DGPP phosphatase was exposed to the cytosolic face of the vacuole, enzyme activity was measured with intact vacuoles in an assay
system that did not utilize Triton X-100 to solubilize the DGPP
substrate. Instead, ␣-32P-labeled DGPP was delivered to the
assay in phospholipid vesicles. Following the reaction, the
phospholipid vesicles were separated from intact vacuoles by
centrifugation. Phospholipids were extracted with chloroform
and analyzed by thin-layer chromatography. The DGPP phosphatase in the intact vacuoles catalyzed a time-dependent conversion of DGPP to PA (Fig. 6). Vacuoles derived from the
dpp1⌬ mutant expressing the vector control did not exhibit
DGPP phosphatase activity (Fig. 6).
Peptide Analysis of DGPP Phosphatase After the Treatment
of Vacuoles with Endoproteinase Lys-C—There are six lysine
5342
Topography of Yeast DGPP Phosphatase Catalytic Site
FIG. 5. DGPP phosphatase activity exhibited by intact and
ruptured vacuoles. DGPP phosphatase activity was measured with
50 ng of intact and ruptured vacuoles using 0.04 mM [␤-32P]DGPP in
Triton X-100 mixed micelles. The final concentrations of Triton X-100 in
the assay systems for the intact and ruptured vacuoles were 0.4 mM
(0.024%) and 8 mM (0.4%), respectively. Each data point represents the
average of triplicate determinations ⫾ S.D. The integrity of the intact
vacuoles was monitored by staining with florescent dye FM4 – 64.
FIG. 6. DGPP phosphatase activity exhibited by intact vacuoles using the DGPP substrate incorporated into phospholipid
vesicles. DGPP phosphatase activity was measured for the indicated
time intervals with 2 ␮g of intact vacuoles isolated from dpp1⌬ mutant
cells with (solid circles) and without (open circles) the DPP1HA allele on
a multicopy plasmid using 0.1 mM [␣-32P]DGPP in PC/PE/PI/PS vesicles. Following the reaction, the phospholipid vesicles were separated
from intact vacuoles by centrifugation at 37,000 ⫻ g for 30 min. The
formation of 32P-labeled PA from [␣-32P]DGPP in the phospholipid
vesicles (supernatant) was determined after extraction and analysis by
thin-layer chromatography. The data shown are the result of a representative experiment.
residues found in the DGPP phosphatase enzyme. The lysine
residues at the N-terminal (Lys8 and Lys16) and C-terminal
(Lys257) ends are located on one side of the membrane, whereas
the lysine residues in loop L2 (Lys118, Lys147, and Lys153) are
on the other side of the membrane (Fig. 7A). We analyzed
DGPP phosphatase peptides that were produced from intact
vacuoles after treatment with endoproteinase Lys-C, a protease that cleaves the carboxyl side of peptide bonds of lysine
residues. In the first set of experiments, peptides were analyzed by Western blotting using anti-HA antibodies to detect
fragments containing the N-terminal HA epitope. If loop L2
faces the cytosolic side of the vacuole, we would expect that
peptide fragments of 19, 18.4, and 15 kDa would be generated.
On the other hand, if the N- and C-terminal lysine residues face
the cytosolic side of the vacuole, peptide fragments of 31, 2.9,
and 2.1 kDa would be generated. Immunoblot analysis showed
that 18- and 15-kDa peptides were produced from intact vacuoles by digestion with endoproteinase Lys-C (Fig. 7B, left). This
result suggested that loop L2 was on the cytosolic face of the
intact vacuole. Endoproteinase Lys-C digestion of detergentsolubilized vacuoles resulted in a great reduction of the HAcontaining peptides (Fig. 7B, left). As indicated above, cleavage
of DGPP phosphatase at Lys8 or Lys16 would produce 2- or
FIG. 7. Peptide analysis of DGPP phosphatase after treatment
of vacuoles with endoproteinase Lys-C. Panel A, the figure shows
the predicted topography of DGPP phosphatase in the membrane with
its lysine residues indicated as black circles. The positions of the HA
epitope and the C-terminal epitope residues of which antibodies were
directed against are shown in gray. Panel B, vacuoles (5 ␮g) containing
the HA-tagged DGPP phosphatase were incubated with 1 ␮g of endoproteinase Lys-C for 2 h at 37 °C in the absence (intact) or presence
(ruptured) of 0.4% Triton X-100. The samples were then subjected to
SDS-PAGE using a 10 –20% slab gel followed by transfer to a polyvinylidene difluoride membrane. The HA-containing peptides derived
from DGPP phosphatase were detected by probing the membrane with
anti-HA antibodies (left). The peptides containing the C-terminal portion of DGPP phosphatase were detected by probing the membrane with
antibodies (Anti-Dpp1p-C) directed against the C-terminal epitope of
the enzyme (right).
3-kDa peptides containing an HA epitope. However, these fragments were not be detected in our immunoblot analysis, probably because of poor retention of the peptides on the membrane
during the electrophoretic transfer. Attempts to solve this problem by using membranes with smaller pore sizes were unsuccessful. The striking reduction of the HA-containing peptides
after endoproteinase Lys-C treatment of detergent-solubilized
vacuoles suggested that the N-terminal lysine residues were
localized in the vacuole lumen.
The peptides derived from endoproteinase Lys-C treatment
of intact vacuoles were analyzed with antibodies directed
against the C-terminal epitope of DGPP phosphatase. Immunoblot analysis revealed that C-terminal peptides of 19 and 16
kDa were produced by the protease treatment (Fig. 7B, right).
This was consistent with the cleavage of Lys118 and Lys147
(and/or Lys153) in loop L2 and that these residues faced the
cytosolic side of the intact vacuoles. If the N- and C-terminal
lysine residues faced the cytosolic side of the vacuole, peptide
Topography of Yeast DGPP Phosphatase Catalytic Site
5343
FIG. 8. Cysteine labeling of DGPP
phosphatase. Panel A, the figure shows
the predicted topography of DGPP phosphatase in the membrane with its cysteine residues indicated as black circles.
The position of the HA epitope is indicated in gray. Panel B, vacuoles (15 ␮g)
containing the HA-tagged DGPP phosphatase were incubated for 10 min at
25 °C in reaction buffer (50 mM Tris-HCl
(pH 7.4), 0.25 M sucrose) with and without
100 ␮M AMS. The reaction mixture was
diluted 5-fold with the same buffer and
incubated with 250 ␮M MPB for 20 min at
25 °C. The reaction was terminated by the
addition of 20 mM 2-mercaptoethanol.
Vacuoles were collected by centrifugation
and lysed, and DGPP phosphatase was
immunoprecipitated with anti-HA antibodies. The enzyme was then subjected to
SDS-PAGE followed by transfer to polyvinylidene difluoride membrane. The membrane was probed with alkaline phosphatase-conjugated NeutrAvidin to detect
biotinylated enzyme (left) and then reprobed with anti-HA antibodies to detect
the HA-tagged DGPP phosphatase
(right). The presence of the multiple
DGPP phosphatase bands was due to endogenous proteolytic cleavage.
fragments of 32, 31, and 4 kDa would have been generated.
Interestingly, the peptides produced from detergent-solubilized
vacuoles were the same size as those produced from intact vacuoles (Fig. 7B, right). This result indicated that the lysine residues
(Lys200 and Lys257) in the C-terminal region of DGPP phosphatase were less susceptible to endoproteinase Lys-C digestion.
A 20-kDa peptide was detected in the detergent-solubilized
vacuoles with the anti-HA antibodies that were not treated
with endoproteinase Lys-C (Fig. 7B, left). The HA-containing
peptide was presumably generated from an endogenous vacuole protease whose identity was unknown. Peptide(s) containing the C-terminal epitope were not detected under the same
condition (Fig. 7B, right). This may be attributed to the loss of the
C-terminal epitope by proteolytic cleavage or to the generation of
small peptides that were not detected by immunoblot analysis.
Cysteine Labeling of DGPP Phosphatase—DGPP phosphatase contains three cysteine residues in the central region of
the protein. Cys132 and Cys150 are located in hydrophilic loop
L2 and Cys185 is located in the fourth transmembrane region
(Fig. 8A). The asymmetric distribution of these cysteine residues permitted the examination of the topography of the DGPP
phosphatase using the sulfhydryl reagents MPB and AMS (41).
The biotinylated reagent MPB is permeable to membranes,
whereas the AMS reagent is impermeable to membranes (41).
Intact vacuoles containing the HA-tagged DGPP phosphatase
enzyme were labeled with MPB with and without pretreatment
with AMS. Pretreatment of the vacuoles with AMS was performed to block MPB labeling if the cysteine residues were
located on the cytosolic face of the vacuoles. Following the
treatments with these sulfhydryl reagents, the DGPP phosphatase was immunoprecipitated from the ruptured vacuoles with
anti-HA antibodies and subjected to immunoblot analysis using
alkaline phosphatase-conjugated NeutrAvidin. This reagent was
used to identify cysteine residues that were labeled with MPB.
The MPB labeling of intact vacuoles without AMS pretreatment resulted in the biotinylation of DGPP phosphatase (Fig.
8B, left). However, pretreatment of the intact vacuoles with
AMS greatly reduced the extent of MPB labeling (Fig. 8B, left).
The ratio of the biotinylated signals of the samples treated with
and without AMS was ⬃3:1, consistent with the stoichiometry
of cysteine residues that can be labeled in the presence of AMS.
The presence of the DGPP phosphatase protein on the blots
treated with the alkaline phosphatase-conjugated NeutrAvidin
reagent was confirmed by immunoblot analysis using anti-HA
antibodies (Fig. 8B, right). This difference in MPB labeling
indicated that Cys132 and Cys150 were located on the external
surface of the intact vacuole membrane. The MPB labeling that
occurred when vacuoles were pretreated with AMS would be
due to the reaction of Cys185 with MPB. If loop L2 were exposed
to the luminal side of the vacuole membrane, the extent of MPB
labeling would be the same whether or not the vacuoles were
pretreated with the AMS reagent.
Effect of Zinc on Vacuole Membrane Phospholipid Composition—DGPP has been identified in total cellular extracts from
S. cerevisiae (5, 8, 20, 35). However, it has not been identified in
vacuole membranes where DGPP phosphatase is localized. To
5344
Topography of Yeast DGPP Phosphatase Catalytic Site
FIG. 9. Effect of zinc on vacuole membrane phospholipid composition. Vacuoles were isolated from wild type cells grown in the
presence and absence of zinc. Phospholipids were extracted from vacuoles, separated by two-dimensional thin-layer chromatography, and
analyzed for phosphate content as described under “Experimental Procedures.” Each data point represents the average of two independent
vacuole preparations ⫾ S.D.
address this issue, phospholipids were extracted from vacuoles
of wild type cells and analyzed by two-dimensional thin-layer
chromatography as described under “Experimental Procedures.” DGPP was present in the vacuolar membrane and
accounted for 0.6 mol % of the total vacuolar membrane phospholipids (Fig. 9, inset). As described previously (53), the major
vacuolar membrane phospholipids included PC, PE, PI, PS,
and PA (Fig. 9).
Previous studies have shown that zinc availability regulates
the expression of the DPP1-encoded DGPP phosphatase (4).
Under zinc-depleted conditions, the transcription factor Zap1p
binds the DPP1 promoter and induces the expression of DGPP
phosphatase (4). Accordingly, we questioned whether zinc
availability affected the level of DGPP in vacuole membranes.
Vacuoles were isolated from cells grown in the presence and
absence of zinc and analyzed for phospholipid composition. Zinc
depletion resulted in a decrease in DGPP content from 0.6 mol
% to an undetectable level (Fig. 9, inset). This finding was
consistent with the increase in expression of DGPP phosphatase activity in response to zinc depletion (4). PA, which is also
a substrate for the DGPP phosphatase enzyme (5), was also
affected by zinc depletion. The amount of PA in vacuoles of
zinc-supplemented cells (1.4 mol %) was reduced (79%) to 0.3
mol % in vacuoles isolated from zinc-depleted cells (Fig. 9,
inset). The depletion of zinc from the growth medium also
affected the levels of major vacuolar membrane phospholipids
(Fig. 9). When compared with the vacuoles of cells grown in the
presence of zinc, the level of PE decreased by 36%, whereas the
levels of PI and PS increased by 44 and 29%, respectively.
DISCUSSION
The DPP1-encoded (1) DGPP phosphatase of S. cerevisiae
has been purified to homogeneity and characterized with respect to its enzymological and kinetic properties (2, 4 –7, 20,
25). The enzyme catalyzes the dephosphorylation of DGPP to
yield PA and then catalyzes the dephosphorylation of PA to
yield diacylglycerol (5). The metabolism of DGPP in plants
plays a role in lipid signaling under conditions of stress (14, 15,
15–17), and the substrate and products of the DGPP phosphatase have lipid messenger roles in mammalian cells (23, 54, 55).
The expression of the yeast DPP1 gene is induced by the stress
conditions of stationary phase and zinc depletion and by inositol supplementation (4, 20). The DGPP phosphatase is localized
to the vacuole membrane in the cell (4). Its subcellular location
suggests that the DGPP phosphatase might play a role in
vacuole function under stress conditions by regulating vacuole
membrane pools of DGPP and PA. Our initial approach in
addressing this hypothesis was to examine the topography of
the DGPP phosphatase catalytic site.
A significant amount of DGPP phosphatase activity was
observed in intact vacuoles. This suggested that the catalytic
site of the enzyme faced the cytosol. However, the apparent
latent enzyme activity observed in detergent-solubilized vacuoles raised the question of whether the vacuole contained a
population of enzyme with its catalytic site facing the lumen of
the vacuole. Alternatively, the latent activity may result from
detergent-mediated stimulation of the enzyme that contains its
catalytic site exposed to the cytosol. We favored the latter
explanation because of the fact that pure DGPP phosphatase
(5) is one of several phospholipid-dependent enzymes that is
stimulated by Triton X-100 and exhibits surface dilution kinetics (56). Moreover, the solubilization of the enzyme (5) from the
vacuole membrane may have activated the enzyme by increasing enzyme-substrate accessibility (56, 57).
Protease protection analysis and chemical labeling have
been used to study the topography of membrane-associated
proteins (41, 58 – 60). We utilized these methods to further
examine the topography of the DGPP phosphatase catalytic
site. Digestion with endoproteinase Lys-C and labeling of cysteine residues with sulfhydryl reagents substantiated the conclusion that the catalytic site of DGPP phosphatase was located
on the cytosolic face of the vacuole membrane. The sizes of the
N- and C-terminal peptides derived from intact and ruptured
vacuoles after treatment with endoproteinase Lys-C were consistent with the topographical model of the DGPP phosphatase
catalytic site facing the cytosol. Moreover, the overall labeling
of the cysteine residues of the enzyme in the intact vacuoles
was strongly diminished by the membrane-impermeable sulfhydryl reagent (AMS) that blocks the MPB labeling of cysteine
residues on the cytosolic face of the vacuole. The topography of
the N and C termini was not defined directly because of the small
size of HA-containing cleavage products and because of reduced
efficiency of cleavage at the C-terminal end of the protein.
The topography of the yeast DGPP phosphatase catalytic site
differs from that of the closely related lipid phosphate phosphatase enzymes of mammalian cells (24, 61). These lipid phosphatase family members are primarily localized to the plasma
membrane, and their catalytic sites are oriented to the external
leaflet of the membrane (24, 61). The mammalian lipid phosphatases also differ by their N-linked glycosylation on the
hydrophilic loop between transmembrane regions three and
four on the external leaflet of the membrane (24, 61). Studies
with various cell types indicate that this orientation is important for the degradation of bioactive lipid phosphate molecules
(e.g. lyso-PA) in both the extracellular space and outer leaflet of
the plasma membrane (23, 24, 61– 64). The topography of the
yeast DGPP phosphatase catalytic site suggests that the enzyme functions to regulate the levels of DGPP and PA on the
leaflet of the vacuole membrane exposed to the cytosol. Our
enzyme activity data using DGPP incorporated into phospholipid vesicles also indicated that the enzyme could metabolize
DGPP in membrane structures external to the vacuole.
The functional role of the DGPP phosphatase enzyme in the
vacuole is supported by the presence of its substrates in the
vacuole membrane. DGPP and PA accounted for 0.6 and 1.4
mol %, respectively, of the total vacuole membrane phospholipids. DGPP was not identified as a vacuole membrane phospholipid in the earlier work of Zinser et al. (53). The fact that DGPP
is a recently discovered phospholipid in S. cerevisiae (5) suggests that it may have been grouped within a relatively large
percentage of unidentified vacuole membrane phospholipids
labeled as “others” in this earlier work (53). Interestingly, the
Topography of Yeast DGPP Phosphatase Catalytic Site
amount of DGPP diminished to an undetectable level when
cells were depleted for zinc, a stress condition that greatly
induces (10-fold) DGPP phosphatase expression (4). Zinc depletion also caused a significant reduction (79%) in the amount of
PA in the vacuole membrane. This was also consistent with the
induction of DGPP phosphatase under this stress condition.
Along with changes in DGPP and PA, zinc depletion resulted in
changes in the amounts of the major vacuole membrane phospholipids PE, PI, and PS. Upon zinc depletion, there was a 36%
decrease in PE and 44 and 29% increases in PI and PS, respectively. These changes, however, were not dependent on the
regulation of DPP1 gene expression. Similar changes in PE, PI,
and PS composition are also exhibited in the vacuoles of dpp1⌬
mutant cells depleted for zinc.2 Instead, changes in these phospholipids can be attributed to zinc-mediated regulation of other
enzymes of phospholipid metabolism.3
The yeast vacuole is an acidic organelle (analogous to the
mammalian lysosome) that contains a cadre of hydrolytic enzymes required for turnover of macromolecules as well as cellular organelles (i.e. autophagy) (65– 67). It is also a reservoir
for several small molecules including amino acids, purines,
ions, and polyphosphates (65, 66). Vacuole function is important during times of stress when cells respond to nutrient
limitation by recycling cellular materials (65– 69). The pathways that organelles, proteins, and small molecules follow to
the vacuole are well characterized (65– 69). Phospholipids and
in particular polyphosphoinositides have been shown to play a
major role in vesicle trafficking (68, 70, 71). In the stress
condition of zinc depletion that induces DPP1 expression, genes
involved in autophagy are also induced (30). Whether the removal of DGPP in the vacuole membrane is related to the
process of autophagy or other vesicle transport processes is
unknown. The studies reported here provide a foundation for
addressing these questions.
Acknowledgments—We acknowledge William Dowhan for the suggestion to take advantage of the cysteine residues in DGPP phosphatase for the chemical labeling experiments. We also thank Avula
Sreenivas for many helpful discussions.
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