Tài liệu Báo cáo Y học: Characterization and regulation of yeast Ca2+-dependent phosphatidylethanolamine-phospholipase D activity docx

Characterization and regulation of yeast Ca
2+
-dependent
phosphatidylethanolamine-phospholipase D activity
Xiaoqing Tang, Michal Waksman, Yona Ely and Mordechai Liscovitch
Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel
An unconventional phospholipase D (PLD) activity was
identified recently in Saccharomyces cerevisiae which is
Ca
2+
-dependent, preferentially hydrolyses phosphatidyl-
ethanolamine (PtdEtn) and phosphatidylserine and does not
catalyse a transphosphatidylation with primary short-chain
alcohols. We have characterized the cytosolic and mem-
brane-bound forms of the yeast PtdEtn-PLD and examined
the regulation of its activity under certain growth, nutritional
and stress conditions. Both forms of PtdEtn-PLD activity
were similarly activated by Ca
2+
ions in a biphasic manner.
Likewise, other divalent cations affected both cytosolic and
membrane-bound forms to the same extent. The yeast
PtdEtn-PLD activity was found to interact with immobilized
PtdEtn in a Ca
2+
-dependent manner. The partially purified
cytosolic form and the salt-extracted membrane-bound form
of yeast PtdEtn-PLD exhibited a similar elution pattern on
size-exclusion chromatography, coeluting as low apparent
molecular weight peaks. PtdEtn-PLD activity was stimu-
lated, along with Spo14p/Pld1p activity, upon dilution of

stationary phase cultures in glucose, acetate and galactose
media, but PtdEtn-PLD activation was less pronounced.
Interestingly, PtdEtn-PLD activity was found to be elevated
by  40% in sec14
ts
mutants at the restrictive temperature,
whereas in other sec mutants it remained unaffected. The
activity of PtdEtn-PLD was reduced by 30–40% upon
addition to the medium of inositol (75 l
M
) in either wild-type
yeast or spo14D mutants and this effect was seen regardless
of the presence of choline, suggesting that transcription of
the PtdEtn-PLD gene is down-regulated by inositol. Finally,
exposure of yeast cells to H
2
O
2
resulted in a transient
increase in PtdEtn-PLD activity followed by a profound,
nearly 90% decrease in activity. In conclusion, our results
indicate that yeast PtdEtn-PLD activity is highly regulated:
the enzyme is acutely activated upon entry into the cell cycle
and following inactivation of sec14
ts
, and is inhibited under
oxidative stress conditions. The implications of these find-
ings are discussed.
Keywords: oxidative stress; phosphatidylethanolamine;
phospholipase D; phospholipid metabolism; yeast.

The ability of cells to respond to changes in their environ-
ment depends on multiple adaptive mechanisms. Many such
mechanisms require the formation, inside the cells, of
specific molecules that act as messengers, informing various
cell systems of the need to change their activity or modify
their function. Phospholipase D (PLD) is an enzyme that
generates such a messenger, phosphatidic acid (PtdA), in
response to environmental signals and thus plays an
important role in regulating cell function [1–3]. A number
of eukaryotic PLD genes have been molecularly cloned in
recent years. These PLD genes all belong to an extended
gene family, termed the HKD family, that also includes
certain bacterial PLDs, as well as non-PLD phosphati-
dyltransferases [2,4–6]. Although the activation of PLD
enzymes has been implicated in signal transduction and
membrane traffic events, their precise cellular localization
and function are still poorly defined [7,8]. Furthermore,
forms of PLD that do not belong to the HKD family may
also exist. A yeast PLD gene, SPO14/PLD1, encodes a
Ca
2+
-independent PLD that hydrolyses phosphatidylcho-
line (PtdCho) and is stimulated by phosphatidylinositol 4,5-
bisphosphate (PtdInsP
2
) [9–11]. Spo14p function is essential
for sporulation [9]. Upon induction of sporulation the
enzyme is relocalized from the cytosol onto the spindle pole
bodies and then encircles the mature spores membranes [12].
Spo14p is also essential for SEC14-independent secretion,

i.e. in sec14
ts
-bypass mutants [13,14]. A second PLD activity
present in the yeast Saccharomyces cerevisiae was recently
identified [15,16]. The second yeast PLD enzyme, provi-
sionally designated ScPLD2, has distinct catalytic proper-
ties. Its activity is Ca
2+
-dependent; it preferentially
hydrolyses phosphatidylethanolamine (PtdEtn) and phos-
phatidylserine (PtdSer); and its activity is not stimulated by
PtdInsP
2
. In addition, unlike Spo14p/Pld1p and most other
eukaryotic PLDs (but similar to certain bacterial PLDs
[17]), the yeast Ca
2+
-dependent PLD is incapable of
catalysing the characteristic transphosphatidylation reac-
tion with primary short-chain alcoholic acceptors [15,16].
This PLD activity was assayed with PtdEtn as substrate and
is therefore abbreviated herein as PtdEtn-PLD. Important-
ly, SPO14/PLD1 is the sole PLD representative of the
HKD gene family that is present in the yeast genome [18].
The yeast Ca
2+
-dependent PtdEtn-PLD activity must
Correspondence to M. Liscovitch, Department of Biological
Regulation, Weizmann Institute of Science, PO Box 26,
Rehovot 76100, Israel.

Fax: + 972 8934 4116, Tel.: + 972 8934 2773,
E-mail:
Abbreviations: PLD, phospholipase D;
PtdA, phosphatidic acid; PtdCho, phosphatidylcholine; PtdEtn,
phosphatidylethanolamine; PtdSer, phosphatidylserine; PtdInsP
2
,
phosphatidylinositol 4,5-bisphosphate; C
6
-NBD, [6-N-(7-nitrobenzo-
2-O-1,3-diazol-4-yl)-amino]-caproyl; PtdIns, phosphatidylinositol;
YNB, yeast nitrogen base; SC, synthetic complete minimal medium.
(Received 26 November 2001, revised 15 May 2002,
accepted 25 June 2002)
Eur. J. Biochem. 269, 3821–3830 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03073.x
therefore be encoded by a distinct non-HKD family gene
which is likely to be a member of a novel PLD gene family,
but the gene that encodes it has not been identified yet. In
the present study we have further characterized the cytosolic
and membrane-bound forms of yeast PtdEtn-PLD and
examined the regulation of PtdEtn-PLD activity under
certain growth, nutritional and stress conditions.
MATERIALS AND METHODS
Chemicals
1-Acyl-2-[6-N-(7-nitrobenzo-2-O-1,3-diazol-4-yl)-amino]-
caproyl-glycero-3-phosphorylcholine (C
6
-NBD-PtdCho)
and 1-acyl-2-[6-N-(7-nitrobenzo-2-O-1,3-diazol-4-yl)-ami-
no]-caproyl-glycero-3-phosphorylethanolamine (C

6
-NBD-
PtdEtn) were from Avanti Polar Lipids (Alabaster, AL,
USA). TLC glass-backed plates precoated with silica gel
60A were from Whatman. Yeast Nitrogen Base (YNB)
lacking amino acids and ammonium sulfate were from
Difco. Dioleoyl-PtdEtn, PtdInsP
2
and all other reagents
were from Sigma.
Yeast strains
The wild-type yeast strain utilized for preparation of total
cell lysates and subcellular fractions was W303–1B (MATa
ade2-1 his3-11,15 leu2-3,112 ura3-1 trp1-1)[19].Thespo14D
strain used was the strain designated pld1-FS-1 (MATa
ade2-1 leu2-3,112 ura3-1 trp1-1 pld1::HIS3) [10]. The diploid
wild-type strain utilized in the carbon source experiments
was W303-1D (MATa/MATa ade2-1/ade2-1 his3-11,15/
his3-11,15 leu2-3,112, leu-2-3112 ura3-1/ura3-1 trp1-1/trp1–
1). sec mutants included: RSY979 (MATa ura3-52 sec7-5),
RSY961 (MATa ura3-52 leu2-3,112 sec12-1), RSY314
(MATa ura3-52 sec13-3), RSY1010 (MATa ura3-52 leu2-
3112 sec21-1) and RSY324 (MATa ura3-52 sec22-2)[20].
The sec14-1
ts
strain used here was CTY1-1A (MATa ura3-
52 hi 3-200 lys2-801 sec14-1
ts
) [21].
Media

Wild-type yeast cells were maintained on synthetic complete
minimal medium (SC). Spo14D cells were maintained on SC
drop-out medium lacking histidine. SC media were pre-
pared from YNB essentially according to Rose et al.[22].
Where indicated, SC medium was supplemented with 75 l
M
inositol (I
+
) and/or 1 m
M
choline (C
+
). Other amino acid-
rich media included: YPD [yeast extract and Bactopeptone
(YP) containing 2% dextrose]; YPA (YP containing 0.05%
glucose and 2% potassium acetate); and YPG (YP
containing 3.5% galactose).
Phospholipase D assays
Spo14p/Pld1p and PtdEtn-PLD activities can be assayed
separately from the same samples, with PtdCho as substrate
in the presence of EGTA and PtdInsP
2
(Spo14p/Pld1p) or
with PtdEtn in the presence of Ca
2+
(PtdEtn-PLD) [16].
Total cell lysates were prepared as described previously [10].
To solubilize C
6
-NBD-PtdEtn, 1.5 m

M
Triton X-100 was
added. The final concentration of Triton X-100 in assay
reactions containing C
6
-NBD-PtdEtn was 0.25 m
M
.The
hydrolysis of C
6
-NBD-PtdEtn was monitored by the
production of C
6
-NBD-PtdA, essentially as described by
Danin et al. [23]. The Spo14p/Pld1p reaction mixture
contained 0.3 mgÆmL
)1
yeast protein, 35 m
M
Na-Hepes
pH 7.4, 150 m
M
NaCl, 400 l
M
C
6
-NBD-PtdCho, 1 m
M
EDTA, 5 m
M

EGTA and 4 mol% PtdInsP
2
.(Note:the
surface concentration of PtdInsP
2
is expressed as a
percentage of the total lipid concentration.) The standard
PtdEtn-PLD reaction mixture contained 0.3 mgÆmL
)1
pro-
tein, 35 m
M
Na-Hepes pH 7.4, 150 m
M
NaCl, 40 l
M
C
6
-NBD-PtdEtn, 1 m
M
EDTA, 5 m
M
EGTA, 7 m
M
CaCl
2
andnoPtdInsP
2
. In experiments in which the free Ca
2+

concentration in the presence of EGTA and EDTA was
modified it was calculated utilizing the
CALCON
software
(Version 4.0, for MS-DOS). The reaction mixtures were
incubated at 30 °C for 30 min in a final volume of 120 lL.
Termination of the reaction, TLC separation and quanti-
fication of the fluorescent lipid products were conducted as
described [10,23]. Activity is expressed as the mean of
two duplicate samples measured in arbitrary fluorescence
units. Where indicated, specific activity is expressed as the
PtdA-derived fluorescence units per mg or lgprotein.
Subcellular fractionation and size-exclusion column
chromatography
Total cell lysates were prepared as described previously [10].
The lysate was centrifuged at 8000 g for 10 min to remove
cell wall debris. The supernatant was collected and ultra-
centrifuged at 100 000 g for 90 min. The supernatant
(cytosol) was collected and the resultant pellet (total
membranes) was washed as above and resuspended in salt
extraction buffer (2
M
NaCl, 35 m
M
Na-Hepes buffer
pH 7.4, 10 lgÆmL
)1
aprotinin and 10 lgÆmL
)1
leupeptin).

The membranes were salt-extracted for 1 h at 4 °C while
shaking and then were sedimented again by ultracentrifu-
gation at 100 000 g for 90 min The supernatant containing
the salt-extracted peripheral membrane proteins was col-
lected.
The partially purified cytosolic PtdEtn-PLD was pre-
pared as follows: the cytosolic fraction was applied to a
Q-Sepharose column (KR26/24, Pharmacia) equilibrated
with buffer A (50 m
M
NaCl, 35 m
M
Na-Hepes pH 7.4).
After washing with buffer A, enzyme was eluted in 5-mL
fractions with an NaCl gradient (0.1–1
M
) in buffer A.
Eluates containing activity were collected and loaded onto a
Reactive Green-19-agarose column (HR16/5, Pharmacia)
equilibrated with buffer A containing 0.3
M
NaCl. The
column was then eluted with a NaCl gradient (0.3–3
M
)in
buffer A. Active fractions were combined and concentrated
to 2 mL by using an Amicon PM5 filter. Aliquots of
the crude cytosol, salt extracted membranes and partially
purified cytosolic fraction (2 mL) were applied to a
Superdex-75 size-exclusion chromatography column

(HiLoad
TM
16/60, Pharmacia) equilibrated with buffer A.
Proteins were eluted with the same buffer at a flow rate of
0.3 mLÆmin
)1
at 4 °C. Fractions (2 mL) were collected and
assayed for PtdEtn-PLD activity. Molecular weight mark-
ers (albumin, 67 kDa; ovalbumin, 43 kDaA; chymotrypsi-
nogen A, 25 kDa; ribonuclease A, 14 kDa) were run
separately under identical conditions. Further purification
of the cytosolic PtdEtn-PLD resulted in rapid loss of
activity.
3822 X. Tang et al. (Eur. J. Biochem. 269) Ó FEBS 2002
PtdEtn-polyacrylamide affinity chromatography
A PtdEtn-polyacrylamide affinity column was prepared
essentially as described in [24] except that PtdEtn was used
instead of PtdSer. The PtdEtn-polyacrylamide particles
(2 mL) were loaded onto a small Poly Prep column
(0.8 · 4 cm, Bio-Rad) and equilibrated with loading buffer
containing 0.4
M
NaCl, 35 m
M
Na-Hepes pH 7.4, 5 m
M
dithiothreitol and 15 m
M
CaCl
2

.Asaltextractofyeast
membranes was diluted in the above buffer and loaded onto
the column. After incubating at 4 °Cfor30minwithgentle
shaking, the column was washed once with loading buffer,
followed by a two-step wash with the same buffer contain-
ing 5 m
M
CaCl
2
andthen0.1m
M
CaCl
2
.Elutionwas
carried out using a buffer containing 2 m
M
EGTA in place
of CaCl
2
. Samples of each fraction were assayed for PtdEtn-
PLD activity under standard conditions, with the final free
Ca
2+
concentration in the assay adjusted to 1 m
M
.
RESULTS
Previous work has demonstrated the existence in yeast of a
Ca
2+

-dependent PLD activity that hydrolyses PtdEtn and
PtdSer [15,16]. Both membrane-bound and cytosolic activ-
ities were observed, but the relationship between these two
forms remains unknown. Therefore, we have compared
some of the properties of membrane-bound and cytosolic
PtdEtn-PLD activities. Our studies demonstrate that their
dependence on free Ca
2+
concentration is quite similar,
both being stimulated in a biphasic manner, with an initial
activation phase at concentrations of 10
)6
to 10
)5
M
and a
second phase between 10
)3
and 10
)2
M
(Fig. 1). The
difference between PtdEtn-PLD activity at 10 l
M
and
10 m
M
free Ca
2+
was statistically significant (P < 0.001,

Student’s t-test). Next, we examined the effects of different
chloride salts of divalent cations on the membrane-bound
and cytosolic PtdEtn-PLD activities assayed in the absence
of added EDTA and EGTA, i.e. in the presence of  10
)5
M
of ambient free Ca
2+
. The divalent cations tested (at a
concentration of 1 m
M
) affected membrane-bound and
cytosolic PtdEtn-PLD activities in a similar manner. While
Ca
2+
ions further stimulated PtdEtn-PLD activity as
expected, Mg
2+
ions had no effect on the activity, whereas
the other divalent cations inhibited basal PtdEtn-PLD in the
following potency order: Co
2+
>Mn
2+
¼ Zn
2+
>Ba
2+
(Table 1). These data indicate that the pattern and extent
of stimulation of the membrane and soluble yeast PtdEtn-

PLD activity by Ca
2+
and their inhibition by other divalent
cations is highly comparable.
The mechanism of action of Ca
2+
ions in PtdEtn-PLD
activation may involve facilitation of substrate interaction,
stimulation of substrate hydrolysis, or both. To establish
whether the interaction of PtdEtn-PLD with its substrate
PtdEtn is stimulated by Ca
2+
ions, we examined its ability
to interact with PtdEtn, immobilized within polyacrylamide
beads, in a Ca
2+
-dependent manner, as previously demon-
strated for protein kinase C [24]. As shown in Fig. 2, loading
a yeast salt extract (see Materials and methods) on a column
containing immobilized PtdEtn in the presence of a high
Ca
2+
concentration (15 m
M
) resulted in retention of a
fraction of total yeast PtdEtn-PLD activity on the column,
which could then be released by adding EGTA. Thus, yeast
PtdEtn-PLD activity is able to interact with immobilized
PtdEtn in a Ca
2+

-dependent manner.
Soluble enzymes that utilize membrane phospholipids as
substrates or cofactors are often translocated to a mem-
brane compartment upon activation or during homogeni-
zation [25]. Their similar response to Ca
2+
and other
divalent cations, and the Ca
2+
-dependent interaction of
yeast PtdEtn-PLD with its PtdEtn substrate, raised the
possibility that the membrane PtdEtn-PLD activity repre-
sents a fraction of the cytosolic form that becomes bound to
membrane PtdEtn upon cell lysis. To determine if the
soluble PtdEtn-PLD activity may translocate to membranes
Fig. 1. Effect of increasing Ca
2+
concentration on membrane and
cytosolic PtdEtn-PLD activity. Cytosolic and membrane-bound
fractions were prepared as described in Materials and methods.
PtdEtn-PLD activity was measured with the indicated free Ca
2+
concentrations. The amount of cytosolic protein included in the assay
was 32 lg per reaction and the amount of membrane protein was
0.4 lg per reaction. Results (mean ± SD) are from four (cytosol) and
two (membrane-bound) replicates carried out in duplicate. The lack of
an error bar indicates an SD smaller than the size of the symbols.
Table 1. Effect of different divalent cations on cytosolic and membrane-
bound PtdEtn-PLD activity. Cytosolic and membrane-bound fractions
were prepared as described in Materials and methods. PtdEtn-PLD

activity measured without addition of EDTA, EGTA and any divalent
cations was considered as 100%. Different cation chloride salts were
added at a concentration of 1 m
M
. Results are from a representative
experiment carried out in duplicate and repeated twice.
Cation added
PtdEtn-PLD activity (% of control)
Cytosolic Membrane-bound
None 100 100
Ca
2+
153 561
Mg
2+
110 101
Co
2+
38 47
Ba
2+
86 75
Mn
2+
43 56
Zn
2+
54 53
Ó FEBS 2002 Characterization and regulation of yeast PtdEtn-PLD activity (Eur. J. Biochem. 269) 3823
in the presence of Ca

2+
we lysed the yeast cells in the
presence of Ca
2+
(10 m
M
)orEGTA(1m
M
) and examined
PtdEtn-PLD activity in the 100 000 g pellet (membranes)
and the 100 000 g supernatant (cytosol). Cell lysis in the
presence of Ca
2+
resulted in a marked decrease in PtdEtn-
PLD activity in the cytosol; however, there was no
corresponding increase in the activity found in the pellet
(Fig. 3). To rule out the possibility that the decrease in
cytosolic PtdEtn-PLD resulted from a Ca
2+
-dependent
membrane translocation of an essential cofactor, an EGTA
wash of the Ca
2+
-lysed membranes was reconstituted with
the Ca
2+
-lysed cytosol. However, the normal cytosolic
PtdEtn-PLD activity was not recovered even after reconsti-
tution (data not shown). The possibility that the translo-
cated enzyme might be masked by the presence of a

membrane-bound inhibitor is also excluded by this exper-
iment. These results indicate that the decrease in cytosolic
PtdEtn-PLD is not due to translocation to the membrane.
The decrease in cytosolic PtdEtn-PLD activity upon lysis in
the presence of Ca
2+
may occur because of stimulated
proteolytic degradation of the enzyme. This possibility was
not examined further.
To further elucidate the relationship between the mem-
brane-bound and cytosolic PtdEtn-PLD activities we
compared their chromatographic properties. Size-exclusion
column chromatography of a salt-extracted membrane
PtdEtn-PLD and the crude cytosolic PtdEtn-PLD activities
on Superdex-75 revealed that they exhibit a different
elution pattern. Whereas membrane-bound PLD eluted as
two major peaks, one of high apparent molecular mass
(peaking in fraction 6) and another of very low apparent
molecular mass (peaking in fraction 34) (Fig. 4A), the
crude cytosolic PtdEtn-PLD eluted as a single high
apparent molecular weight peak that paralleled the corre-
sponding peak of membrane PtdEtn-PLD (Fig. 4B).
However, after partial purification by Q-Sepharose and
Reactive Green-19-agarose, the partially purified cytosolic
PtdEtn-PLD eluted as a single low apparent molecular
weight peak that paralleled the corresponding peak of
membrane PtdEtn-PLD (Fig. 4C). In conclusion, it seems
that the two forms may share a common low apparent
molecular weight catalytic subunit, that mediates PtdEtn-
PLD response to Ca

2+
and other cations and may interact
with other component(s) in the high apparent molecular
weight peaks that determine their differential size and
subcellular localization. Only the future cloning of yeast
PtdEtn-PLD and its isozymes will confirm or refute this
conjecture.
To gain insight into the possible physiological role(s) of
yeast PtdEtn-PLD we examined the regulation of its activity
under different environmental and physiological conditions.
First, the effect of growth in media containing different
carbon sources (YPD, YPG and YPA, supplemented with
glucose, galactose and acetate, respectively) on Spo14p/
Pld1p activity and PtdEtn-PLD activity in vitro was
determined in parallel throughout culture growth. Dilution
of stationary phase diploid W303-1D wild-type cells in fresh
YPD media resulted in a 4.5-fold increase in Spo14p/Pld1p
activity within 30 min, which was followed by a second
peak of activation after 70 min The activity then declined
gradually to near basal levels after 2, 4 and 8 h (Fig. 5A).
PtdEtn-PLD activity similarly exhibited a transient 3.5-fold
activation which seemed to be biphasic, although the first
peak of activation was not as pronounced (Fig. 5A).
Spo14p/Pld1p activity was stimulated also upon exit from
Fig. 3. Effect of the presence of Ca
2+
during lysis on membrane and
cytosolic PtdEtn-PLD activity. Yeast cells were lysed in the presence of
EGTA (1 m
M

; left) or CaCl
2
(10 m
M
; right) and the membrane and
cytosol fractions were separated by centrifugation (100 000 g,60 min).
The fractions were then assayed for PtdEtn-PLD activity under stan-
dard conditions, with final free Ca
2+
concentration in the assay
adjusted to 1 m
M
. Results are from a representative experiment carried
out in duplicate and repeated twice.
Fig. 2. Ca
2+
-dependent retention of PtdEtn-PLD on a polyacrylamide-
immobilized PtdEtn affinity column. The PtdEtn-affinity column was
prepared as described in Materials and methods. A salt extract of yeast
membranes was then loaded onto the column (equilibrated with
15 m
M
CaCl
2
). A two-step wash with buffer containing 5 m
M
and
0.1 m
M
CaCl

2
was followed by elution with 2 m
M
EGTA. Fractions
were assayed for PtdEtn-PLD activity under standard conditions, with
final free Ca
2+
concentration in the assay adjusted to 1 m
M
.Results
are from a representative experiment carried out in duplicate and
repeated three times.
3824 X. Tang et al. (Eur. J. Biochem. 269) Ó FEBS 2002
stationary phase in YPG, but the second sixfold activation
peak was delayed somewhat and occurred after 120 min of
incubation (Fig. 5B). Here, the activation of PtdEtn-PLD
was smaller in magnitude (1.5-fold to twofold) but more
persistent (up to 4 h; Fig. 5B). In YPA, the pattern of
Spo14p/Pld1p activity was similar to that observed in YPD.
PtdEtn-PLD activity was stimulated rapidly nearly three-
fold and this was followed by a second, smaller activation
peak at 70 min of incubation (Fig. 5C). A biphasic activa-
tion of PtdEtn-PLD upon dilution (similar in terms of
magnitude and timing) was observed also in haploid wild-
type W303-1B cells (data not shown). These data clearly
indicate that both Spo14p/Pld1p and PtdEtn-PLD are
highly regulated enzymes that are turned on upon yeast
entry into the cell cycle.
Different lines of evidence support a biological role for
mammalian PLDs during vesicle formation, budding,

transport, docking and fusion to target membranes [2]. In
yeast, SPO14/PLD1 is required for SEC14-independent
vesicle transport (i.e. under sec14-bypass conditions) [13,14].
To explore the involvement of PtdEtn-PLD in secretion, we
screened 16 different secretion mutants, bearing mutations
at the early and late stages of the secretory pathway, for
changes in PtdEtn-PLD activity at room temperature and at
the restrictive temperature of 37 °C(atwhichthetemper-
ature-sensitive secretion phenotype is manifested). Fig. 6
shows PtdEtn-PLD activity in a selected subset of six
secretion mutants. sec14
ts
is the only one among 16 secretion
Fig. 5. Effect of carbon source on Spo14p and PtdEtn-PLD activity in
diploid cells during culture growth. PLD activity was determined at
different stages of growth in culture. A 48-h-old stationary phase
culture of W303-1D diploid cells was diluted in fresh YPD (A), YPG
(B) or YPA (C) media to 0.65 · 10
6
cellsÆmL
)1
and grown at 30 °C.
Samples were taken at the indicated times and Spo14p/Pld1p (d)and
PtdEtn-PLD activity (s) were assayed in duplicate. Results are
expressed as the percentage of the specific PLD activity at time 0 and
are taken from representative experiments that were repeated at least
twice.
Fig. 4. Size-exclusion chromatography of membrane (A), cytosolic (B)
and partially purified cytosolic (C) PtdEtn-PLD activities on Superdex-
75. Salt-extracted membrane, crude cytosolic, and cytosolic PtdEtn-

PLD partially purified on Q-Sepharose and Reactive Green-19 aga-
rose, were prepared and chromatographed on a Superdex-75 column
(see Materials and methods for details). Samples from each column
fraction were then assayed for PtdEtn-PLD activity in duplicate under
standard conditions. Molecular mass markers (arrows) were run sep-
arately under identical conditions. Results are from representative
experiments that were repeated at least twice.
Ó FEBS 2002 Characterization and regulation of yeast PtdEtn-PLD activity (Eur. J. Biochem. 269) 3825
mutants in which PtdEtn-PLD activity is elevated (by 37%)
at the restrictive temperature. All of the other secretion
mutants that we checked, and four wild-type cells that
served as additional controls, showed either a slight decrease
in PtdEtn-PLD activity at 37 °C or were unaffected by the
change in temperature, as compared with the room
temperature controls (data not shown). The activation in
sec14
ts
mutants suggests that Sec14p is involved, directly or
indirectly, in negative regulation of PtdEtn-PLD. Thus,
Sec14p may be a common negative regulator of both
Spo14p- and PtdEtn-PLD-mediated PtdA accumulation in
yeast. It should be noted that the effect on PtdEtn-PLD is
evident within 1 h of temperature elevation, indicating that
it may reflect a change in PtdEtn-PLD stability or in its
activation state rather than a change at the transcriptional
level.
Recent results indicate that SPO14/PLD1 may be
involved in regulating the expression of genes that are part
of the INO1 regulon [13]. Therefore, we examined the effect
of the presence of inositol and choline in the medium on

yeast PtdEtn-PLD activity. The results indicate that
Spo14p/Pld1p activity in wild-type cells (Fig. 7, left) and
PtdEtn-PLD activity in either wild-type yeast or spo14D
mutants (Fig. 7, right) are decreased by 30–40% upon
addition of inositol (75 l
M
) to the medium. This effect is
seen regardless of the presence of 1 m
M
choline in the
medium. The results suggest that under conditions of
repression of the INO1 regulon, both Spo14p/Pld1p and
PtdEtn-PLD activities are down-regulated, further impli-
cating both of these enzymes in regulating phospholipid
biosynthesis in yeast [13].
Mammalian PLD isoforms are activated upon exposure
to oxidative stress signals [26–29]. This prompted us to
examine the effect of H
2
O
2
on yeast Spo14p/Pld1p and
PtdEtn-PLD activities. A short exposure of yeast cells to
H
2
O
2
(30 min) caused a rapid but limited (25–30%)
stimulation of PtdEtn-PLD activity (Fig. 8A). Under these
conditions Spo14p/Pld1p activity was not significantly

stimulated. When yeast cells were exposed to H
2
O
2
for
2 h there was a profound decrease in PtdEtn-PLD activity
(up to 90%), that was evident at concentration of ‡ 1m
M
(Fig. 8B). Interestingly, although Spo14p/Pld1p activity is
also reduced by long exposure to H
2
O
2
it was affected less,
being reduced by  50% (Fig. 8B). The time course of the
changes in PtdEtn-PLD and Spo14p/Pld1p activity in
response to 2 m
M
H
2
O
2
demonstrates the biphasic nature
(i.e. a brief initial stimulation followed by a prolonged
inhibition) of the response of PtdEtn-PLD activity to this
oxidative stress (Fig. 8C).
DISCUSSION
Yeast PtdEtn-PLD is an unconventional PLD that differs
from prokaryotic and eukaryotic HKD family PLDs in its
inability to catalyse a transphosphatidylation reaction with

Fig. 6. PtdEtn-PLD activity in various sec mutants. Different strains
carrying mutations in various genes involved in secretion were grown
to stationary phase and then diluted in YPD and grown to mid log-
phase (6 h). The cultures were then divided into two portions and
further incubated either at room temperature or at 37 °C for 1 h. The
cells were then harvested and whole cell lysates were prepared and
assayed in duplicate for PtdEtn-PLD activity. Results are mean ± SD
of three independent experiments.
Fig. 7. Changes in Spo14p and PtdEtn-PLD
activity in response to inositol and/or choline in
the medium. Wild-type and spo14D mutant
strains were grown to mid log-phase in SC
medium in the absence or presence of choline
(1 m
M
) and inositol (75 l
M
) as indicated. The
cells were then harvested and whole cell lysates
were prepared and assayed in duplicate for
Spo14p/Pld1p and PtdEtn-PLD activity.
Results are from a representative experiment
that was repeated three times.
3826 X. Tang et al. (Eur. J. Biochem. 269) Ó FEBS 2002
short-chain primary alcohols. This difference is meaningful
because it implies that the yeast PtdEtn-PLD and HKD
family PLDs use different catalytic mechanisms. In HKD
family enzymes catalysis involves the formation of a
covalent phospho-enzyme intermediate that is formed on
the highly conserved active site histidine which is part of the

HKD family signature motif, HXKXXXXD [30]. It is
assumed that in HKD family PLDs the phosphatidyl-
histidine intermediate is attacked by an activated water
molecule to release PtdA, and that alcohols can compete
with water to form a phosphatidylalcohol product [31]. The
yeast genome includes only one HKD-family PLD gene,
namely, SPO14. Another HKD family gene found in the
yeast genome is PEL1/PGS1, encoding phosphatidylglyc-
erol phosphate synthase [32]. It is therefore highly likely that
yeast PtdEtn-PLD is encoded by a non-HKD gene and may
thus represent a novel PLD gene family. A prokaryotic
PLD activity similar to yeast PtdEtn-PLD that was
identified recently in Sterptoverticillium cinnamoneum and
was partially purified and characterized, may be another
member of this putative gene family [17]. With the exception
of alcohols (that act as competitive substrates) there are no
known active site-directed inhibitors of HKD-family PLDs.
Hence the existence of a distinct catalytic site in PtdEtn-
PLD cannot be tested directly at this time. Obviously,
identification of the gene that encodes yeast PtdEtn-PLD is
an important goal. So far, our earnest attempts to identify
this elusive gene, by using numerous genetic, genomic and
biochemical approaches, have proved unsuccessful
(X. Tang & M. Liscovitch, unpublished data). Therefore,
the present work was undertaken in order to obtain more
information about the yeast PtdEtn-PLD activity, its
properties and regulation.
In previous work we have shown that PtdEtn-PLD
activity can be found in both cytosolic and membrane-
bound forms [16]. The relationship between these two forms

was examined here in various ways. One of the characteristic
features of yeast PtdEtn-PLD is its almost absolute
dependence on Ca
2+
[15,16]. Our data indicate that the
response of the cytosolic and membrane-bound PtdEtn-
PLDs to increasing free Ca
2+
concentrations is almost
identical, both forms being activated in a biphasic manner.
Also, the two forms are similarly inhibited by the divalent
cations tested. This similarity suggests that the two forms
are catalytically related. Size exclusion column chromatog-
raphy of the membrane bound PtdEtn-PLD, solubilized by
treatment with high salt concentration, revealed that it
eluted as two major peaks. Intriguingly, the crude cytosolic
PtdEtn-PLD eluted as a single peak that corresponded to
the high apparent molecular weight peak of the membrane
form. However, following its partial purification, the cytosol
PtdEtn-PLD eluted as a single peak that corresponded to
the low apparent molecular weight peak of the membrane
form. These data are consistent with the hypothesis that the
cytosol and membrane forms of yeast PtdEtn-PLD share a
common catalytic subunit of low apparent molecular weight
that may interact with one or more subunits which could
determine their different cellular localization.
The stimulation of PtdEtn-PLD by Ca
2+
ions is biphasic.
This pattern raises the possibility that Ca

2+
may have a dual
mechanism of action in activating PtdEtn-PLD, e.g. Ca
2+
may participate in catalysis as well as facilitate enzyme–
substrate interaction. Our data, showing that PtdEtn-PLD
Fig. 8. Changes in Spo14p and PtdEtn-PLD activity in response to
oxidative stress. Wild-type cells were grown to mid log-phase in SC
medium. The cells were then aliquoted and incubated in the absence or
in the presence of H
2
O
2
at the indicated concentrations for 30 min (A)
and2h(B),orcellswereincubatedatthesameconcentrationofH
2
O
2
(2 m
M
) for different times (C). The cells were then harvested and whole
cell lysates were prepared and assayed in duplicate for Spo14p/Pld1p
and PtdEtn-PLD activity. Results are from a representative experi-
ment that was repeated three times.
Ó FEBS 2002 Characterization and regulation of yeast PtdEtn-PLD activity (Eur. J. Biochem. 269) 3827
may bind to immobilized PtdEtn in Ca
2+
-dependent
manner, strongly suggest that one mechanism of Ca
2+

action is to stimulate the interaction of PtdEtn-PLD with its
phospholipid substrate. It is not yet clear how Ca
2+
ions
promote enzyme–PtdEtn association. One possibility is that
yeast PtdEtn-PLD amino acid sequence contains a C2/
CaLB domain, homologous to the domains found in
mammalian proteins including protein kinase C, cytosolic
phospholipase A
2
and synaptotagmin [33]. We have there-
fore screened the yeast genome and identified three ORFs
encoding hypothetical unknown proteins with a distinct C2
domain. Specific disruptants of these ORFs, namely
YLR019w, YLL010c, and YOR086c, were obtained from
Research Genetics Inc. Lysates were prepared from these
disruption strains and assayed for PtdEtn-PLD activity. In
all three cases, PtdEtn-PLD levels were comparable to those
found in the wild-type strain (data not shown), indicating
that these ORFs do not encode PtdEtn-PLD nor any other
protein required for PtdEtn-PLD expression or activity.
Obviously, definitive verification of this conjecture must
await the identification of the yeast PtdEtn-PLD gene. Be
that as it may, the ability of PtdEtn-PLD to bind to
immobilized PtdEtn may facilitate the future use of a
PtdEtn-affinity matrix for purification of PtdEtn-PLD by
phospholipid-affinity chromatography.
In the second part of this work we aimed to study the
regulation of PtdEtn-PLD activity and, in particular, to
compare it with the other yeast PLD, Spo14p/Pld1p. We

have taken advantage of the fact that Spo14p/Pld1p and
PtdEtn-PLD activities can be measured independently in
the same samples. Spo14p/Pld1p is Ca
2+
-independent and
thus may be assayed in the presence of EGTA and
EDTA, conditions under which PtdEtn-PLD is inactive.
On the other hand, PtdEtn-PLD hydrolyses PtdEtn and
thus may be assayed with this phospholipid as substrate,
conditions under which Spo14p/Pld1p is inactive [15,16].
We have previously shown that initiation of yeast cell
proliferation upon transfer of stationary cultures to fresh
medium is associated with stimulation of Spo14p/Pld1p
activity [10]. Here we confirm these results and further
show that the activation of Spo14p/Pld1p is biphasic and
occurs, albeit with different kinetics, regardless of whether
the yeast cultures are initiated in YPD, YPG or YPA.
Interestingly, further analysis has shown that PtdEtn-PLD
activity is also stimulated upon exit of yeast cells from
stationary phase in either YPD, YPG or YPA. However,
the activation of PtdEtn-PLD was not as pronounced as
that of Spo14p/Pld1p, especially in YPG medium. The
fact that PtdEtn-PLD activation occurred in all tested
media, albeit to a different extent, suggests that it may
correlate with resumption of mitosis rather than with
glucose repression, induction of sporulation or the carbon
source being utilized. It may therefore be speculated that
PtdEtn-PLD shares a regulatory role with Spo14p/Pld1p
in one or more steps of the mitotic cell cycle. Alterna-
tively, the activation of the two yeast PLDs may reflect a

generalized stimulation of phospholipid metabolism asso-
ciated with new membrane synthesis coincident with
initiation of cell growth.
In mammals, PLD was strongly implicated in vesicular
trafficking, both in the Golgi (formation of nascent
exocytotic vesicles) and at the plasma membrane (endocy-
tosis) (reviewed in [7]). In yeast, recent work has indicated
that Spo14p/Pld1p plays a permissive role in SEC14-
independent secretion as evinced by abrogation of growth of
sec14
ts
-bypass mutants upon disruption of SPO14/PLD1
[13,14]. In addition, inactivation of sec14
ts
at its restrictive
temperature results in stimulation of Spo14p/Pld1p activity
[13,34,35]. Our data show that PtdEtn-PLD activity is
similarly stimulated upon inactivation of sec14
ts
by a 60-min
incubation at 37 °C. It is important to note that in this
experiment, as in all studies of PtdEtn-PLD regulation
described here, PLD activities were examined in vitro after
cell lysis and under optimal assay conditions. The in vitro
PtdEtn-PLD activity is therefore not likely to fully reflect
the activity in situ and probably underestimates the extent of
PtdEtn-PLD activation upon temperature-dependent inac-
tivation of sec14
ts
. The role played by Spo14p/Pld1p in

regulating normal Golgi transport activity is still not clear,
although several possibilities have been suggested. High
levels of PtdCho in the Golgi were hypothesized to interfere
with Golgi secretory activity [36–39]. Thus, under sec14
ts
-
bypass conditions, the activated Spo14p/Pld1p may act to
reduce Golgi PtdCho to levels that are compatible with
normal secretion and its ablation would result in Golgi
dysfunction. Another suggested function of Spo14p/Pld1p
might be to supply critical lipid metabolite(s) (diacylglycer-
ol, for example) that may be necessary for normal Golgi
activity [40]. How might PtdEtn-PLD fit in these schemes is
a matter of conjecture. PtdEtn-PLD is capable of hydro-
lysing PtdCho in vitro although not as efficiently as it
hydrolyses PtdEtn and PtdSer [16] and thus may perhaps
participate in regulating Golgi PtdCho levels and generating
lipid metabolites required in the Golgi. Such metabolites can
be produced also from PtdEtn and/or PtdSer. It is
noteworthy that defects in PtdEtn methylation effect
sec14
ts
-bypass when PtdCho synthesis via the CDP-choline
pathway is abrogated by eliminating uptake of free choline
[39]. Thus, activation of PtdEtn-PLD may support normal
Golgi function also by reducing the levels of the PtdCho
precursor PtdEtn. In this context, it may be supposed that
over-expression of PtdEtn-PLD should rescue the growth
defect of the sec14
ts

cki1 spo14D strain. Based on this
supposition we attempted to identify the PtdEtn-PLD gene
by multicopy suppression of the triple mutant with a yeast
genomic library. However, the only genes picked up in this
screen were SEC14 and SPO14/PLD1 (X. Tang & M. Lis-
covitch, unpublished data). Obviously, these negative results
do not rule out the possibility that, once identified, the
PtdEtn-PLD gene will be found as essential for SEC14-
independent secretion as was Spo14/Pld1p.
Numerous genes involved in yeast phospholipid biosyn-
thesis are repressed when inositol is present in the medium
[41–43]. The INO1 gene, whose product is inositol-1-
phosphate synthase [44], is the prototypic inositol-regulated
gene that gave its name to the entire INO1 regulon.
Repression by inositol is mediated by a repeated element,
UAS
INO
, found in the upstream promoter region of INO1
and other genes that are part of the INO1 regulon [45]. As
regulation of phospholipid biosynthesis and degradation are
likely to be coordinated, it was of interest to examine the
effect of inositol and choline on the level of PtdEtn-PLD
activity. Our results clearly show that PtdEtn-PLD activity
is down-regulated in cells grown in the presence of inositol.
Choline, which sometimes enhances the repressive effect of
inositol, had no influence on PtdEtn-PLD activity. In
3828 X. Tang et al. (Eur. J. Biochem. 269) Ó FEBS 2002
addition, the effect of inositol was seen in both wild-type
and spo14D cells. Future identification of the PtdEtn-PLD
gene and analysis of its promoter region will allow

examination of its transcriptional regulation by inositol
and the possible existence of UAS
INO
in its 5¢-untranslated
region.
Finally, in view of the potential role of mammalian
PLD in the oxidative stress response [26–29], we examined
the changes in PtdEtn-PLD activity upon exposure of
yeast to H
2
O
2
. The results were quite striking: Following
a rapid activation seen within 20 min of the oxidative
challenge, there was a gradual decline in activity that was
both time- and dose-dependent, reaching a maximal
decrease of almost 90% after exposure to 15 m
M
H
2
O
2
for 2 h. This result is most intriguing. Much additional
work is required to work out the mechanisms involved in
the down-regulation PtdEtn-PLD and its possible role in
the yeast oxidative stress response. Identification of the
gene encoding PtdEtn-PLD is an obvious key to progress
in understanding the structure, mechanism of action,
localization, regulation and function of this intriguing
enzyme.

ACKNOWLEDGEMENTS
We thank J. Gerst for providing sec mutants and for many helpful
discussions. We are grateful to Z. Elazar for his kind advice and interest
in this study. This work was supported in part by grants from the Israel
Science Foundation administered by the Israel Academy of Science and
Humanities (Jerusalem) and the Minerva Foundation (Munich). M. L.
is incumbent of the Harold L. Korda Professorial Chair in Biology.
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