(Received for publication, October 7, 1996, and in revised form, November 1, 1996)
From the Department of Biological Regulation and
§ Department of Molecular Genetics, Weizmann Institute of
Science, Rehovot 76100, Israel
We have previously reported the identification and partial characterization of a gene encoding a phospholipase D activity (PLD1) in the yeast, Saccharomyces cerevisiae. Here we report the existence of a second phospholipase D activity, designated PLD2, in yeast cells bearing disruption at the PLD1 locus. PLD2 is a Ca2+-dependent enzyme which preferentially utilizes phosphatidylethanolamine over phosphatidylcholine as a substrate. In contrast to PLD1, the activity of PLD2 is insensitive to phosphatidylinositol 4,5-bisphosphate, and the enzyme is incapable of catalyzing the transphosphatidylation reaction with short chain alcohols as acceptors. Subcellular fractionation shows that PLD2 localizes mainly to the cytosol, but could also be detected in the particulate fraction. Thus, the biochemical properties of PLD2 appear to be substantially different from those of PLD1. PLD2 activity is significantly and transiently elevated upon exit of wild type yeast cells from stationary phase, suggesting that it may play a role in the initiation of mitotic cell division in yeast. In view of the significantly different properties of PLD1 and PLD2, and because the yeast genome contains PLD1 as the sole member of the recently defined PLD gene family, it may be concluded that PLD2 is structurally unrelated to PLD1. Thus, the novel PLD2 activity described herein is likely to represent the first identified member of a new PLD gene family.
Phospholipase D catalyzes the hydrolysis of phospholipids at their distal phosphodiester bond to yield phosphatidic acid (PA)1 and a free polar head group (1). Most PLDs can utilize primary alcohols as acceptors of the phosphatidyl moiety, to yield the corresponding phosphatidylalcohols (1). In mammals, different PLD activities with various substrate specificities, activation requirements, and subcellular localization have been described (2). PLD activity can be rapidly activated upon receptor activation or other types of cell stimulation. Receptor activation of PLD is probably mediated by multiple factors including small GTP-binding proteins, heterotrimeric GTP-binding proteins, protein kinase C, phosphatidylinositol 4,5-bisphosphate (PIP2), tyrosine phosphorylation, and changes in the intracellular concentration of Ca2+ (3, 4). Phosphatidic acid, the product of the reaction catalyzed by PLD, is likely to serve as a lipid second messenger (5) and may mediate a variety of biological responses, including mitogenesis (6) and the respiratory burst (7). In addition, an ADP-ribosylation factor (ARF)-activated phosphatidylcholine (PC)-specific PLD which was discovered recently (8, 9) has been implicated in vesicular trafficking (10). The cloning of the first plant PC-specific PLD (11) enabled the identification of additional eukaryotic PC-specific PLDs, including a yeast PLD (12, 13, 14) and a human ARF-dependent PLD (15). The eukaryotic PC-PLD gene family shares a number of common homology domains (15, 16, 17). A short common motif is shared with certain phospholipid biosynthetic enzymes that catalyze a phosphatidyl transfer reaction, but are not PLDs (15, 16). In yeast, PLD1 has no direct homologs and is therefore the sole member of this family. This paper reports the identification of a second PLD activity in yeast and describes its biochemical properties, distribution in subcellular fractions, and activation during early growth phase.
1-Palmitoyl-2-[6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]caproyl-phosphatidylcholine (C6-NBD-PC), 1-acyl-2-[6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-phosphatidylethanolamine (C6-NBD-PE), 1-palmitoyl-2-[6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-phosphatidylglycerol (C6-NBD-PG), and 1-acyl-2-[6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-phosphatidic acid (C6-NBD-PA) were purchased from Avanti Polar Lipids. PIP2 was obtained from Sigma.
Yeast StrainsJC1 (Mat ade8 can1 his3 leu2 lys2
trp1 ura3) (18). PLD1
FS-1 (Mat
ade8 can1 leu2 lys2
trp1 ura3
pld1::HIS3) (13). W303-1B (Mata
ade2-1 his3-11, 15 leu2-3, 112 ura3-1
trp1-1) (19).
Wild-type yeast were maintained on synthetic complete
minimal medium (SC). PLD1FS-1 cells were maintained on SC drop-out medium, lacking histidine. Media were prepared essentially according to
Rose et al. (20).
Total cell lysates were prepared as described previously (13). C6-NBD-PC was dissolved in water. In solubilization of C6-NBD-PE, 1.5 mM Triton X-100 was added. The final concentration of Triton X-100 in assay reactions containing C6-NBD-PE was 0.25 mM. For determination of substrate specificity, all substrates were solubilized in 6 mM Triton X-100. The final concentration of the detergent in the reaction was 1.2 mM.
The hydrolysis of C6-NBD-phospholipids was monitored by the
production of C6-NBD-PA, essentially as described by Danin
et al. (21). For assaying PLD1 activity, we prepared cell
lysates from wild-type JC1 cells. The PLD1 reaction mixture contained 0.3 mg/ml protein, 35 mM HEPES, pH 7.2, 150 mM
NaCl, 400 µM C6-NBD-PC, 1 mM
EDTA, 5 mM EGTA, and 4 mol % PIP2. The surface
concentration of PIP2 is expressed as a percentage of the
total lipid concentration. For assaying PLD2 activity, we prepared cell
lysates from either wild type or PLD1FS-1 cells. The PLD2 reaction
mixture contained 0.3 mg/ml protein, 35 mM HEPES, pH 7.2, 150 mM NaCl, either 400 µM
C6-NBD-PC or 40 µM C6-NBD-PE, 1 mM EDTA, 5 mM EGTA, 7 mM
CaCl2, and no PIP2. The free Ca2+
concentration in the presence of EGTA and EDTA was calculated utilizing
the Calcon software (Version 4.0, for MS-DOS). For the transphosphatidylation assays, 300 mM concentrations of the
indicated primary alcohols were added as acceptors. The reaction
mixtures were incubated at 30 °C for 30 min at final volume of 120 µl. Termination, TLC separation, and quantification of the
fluorescent lipid products were conducted as described (13, 21).
Activity is expressed as the mean of two duplicate samples measured in arbitrary fluorescence units. Specific activity is expressed as the
PA-derived fluorescence units per µg of protein.
The identity of the fluorescent product of C6-NBD-PE hydrolysis as C6-NBD-PA was verified by TLC separation of the PLD2 reaction mixtures on Silica Gel 60 aluminum plates (Merck) with the following six solvent systems: (i) chloroform/methanol/acetic acid (50:25:8); (ii) butanol/acetic acid/water (6:1:1); (iii) acetone/chloroform/methanol/acetic acid/water (30:40:10:10:5); (iv) chloroform/methanol/water (65:25:4); (v) chloroform/methanol/ammonia (65:25:5); (vi) the upper phase of ethyl acetate/iso-octane:acetic acid:water (65:10:15:50). In all these systems, the fluorescent phospholipid product of the PLD2 enzymatic reaction co-migrated with an authentic C6-NBD-PA standard (data not shown).
We have previously reported that disruption of the PLD1
gene results in complete loss of PLD activity in pld1
cell lysates (13), when measured under assay conditions which included
EGTA, a Ca2+ chelator (Fig. 1,
left). In subsequent experiments designed to further
characterize PLD1 activity and in which EGTA was omitted from the
assay, we found elevated PLD activities in both wild type and
pld1 cell lysates (data not shown). These results
suggested that yeast cells express an additional,
Ca2+-dependent PLD activity, which is
independent of PLD1. Indeed, addition of CaCl2
at a concentration that allowed 1 mM free Ca2+
in the presence of EGTA and EDTA resulted in a substantial increase in
PLD activity which was evident in both wild type and
pld1 cell lysates (Fig. 1, right). Total PLD activity was
consistently lower in
pld1 cell lysates, reflecting the
loss of PLD activity contributed by the disrupted PLD1 gene
product. Therefore, the residual Ca2+-dependent
PLD activity observed in
pld1 cell lysates represents a
second PLD enzyme which we have designated PLD2. Thus, use of the yeast
system has allowed us to assay the activities of PLD1 and PLD2
separately: PLD1 activity could be assayed in wild type cell lysates in
the presence of EGTA (which abolishes PLD2 activity), whereas PLD2
activity could be assayed in the presence of Ca2+ in
pld1 cell lysates (in which PLD1 activity is abolished as a consequence of gene disruption).
The substrate specificity of PLD2 was next compared with that of PLD1.
This was accomplished by assaying both PLD activities with 400 µM C6-NBD-PC, C6-NBD-PG, and
C6-NBD-PE in the presence of 1.2 mM Triton
X-100 (Fig. 2). At a concentration of 0.5 mM, Triton X-100 inhibited PLD1 activity by 25% and
activated PLD2 activity using C6-NBD-PE by 30% (data not
shown). We found that the production of C6-NBD-PA was up to
75-fold higher with C6-NBD-PE as a substrate compared with
C6-NBD-PC. In subsequent experiments, when Triton X-100 was
used in a concentration that is below its critical micelle
concentration value, PLD2 activity with C6-NBD-PE was up to
10-fold higher than with C6-NBD-PC as a substrate (data not
shown). In contrast, PLD1 could not utilize C6-NBD-PE as a substrate. Similar results were reported previously for PLD1 with long
chain PE (12). Furthermore, compared with PLD1, PLD2 produced 4 and 11 times as much C6-NBD-PA from hydrolyzing
C6-NBD-PC and C6-NBD-PG, respectively. These
data suggest that a considerable difference exists in the catalytic
properties of PLD1 and PLD2. In addition, the results provide a tool by
which PLD1 and PLD2 activities could be discriminated in wild type cell
lysates, where C6-NBD-PE can be used as a substrate for
determination of PLD2 activity exclusively, without the interference of
PLD1. PLD2 activity increased when increasing concentrations of either
C6-NBD-PC or C6-NBD-PE were added to the
reaction mixture. Using C6-NBD-PC as a substrate, PLD2
activity reached saturation at 200 µM. In contrast,
saturation could not be reached with C6-NBD-PE
concentrations up to 400 µM (data not shown).
C6-NBD-PE was utilized routinely as a substrate in PLD2
reactions, as it is not used by PLD1 and because of the greater
activity that can be achieved with this substrate. Throughout the
remainder of PLD2 characterization assays, we used both
C6-NBD-PC and C6-NBD-PE. The concentration used
for C6-NBD-PC (400 µM) allowed enzyme
saturation, and the concentration used for C6-NBD-PE was
submaximal (40 µM) in the presence of 0.25 mM
Triton X-100.
There is evidence for PLD-mediated hydrolysis of PE in intact mammalian cells (22, 23). Furthermore, a cytosolic PLD activity identified in various bovine tissues exhibited some preference for PE over PC as substrate (24). The mammalian cytosolic PLD was stimulated by Ca2+ (24, 25). However, in contrast to yeast PLD2, the mammalian cytosolic PLD could efficiently catalyze a transphosphatidylation reaction (24, 25).
To examine the quantitative relationship between Ca2+
concentration and PLD2 activity, enzyme activity was determined in the presence of increasing free Ca2+ concentrations in the
presence of EGTA (Fig. 3). PLD2 activity was elevated
substantially and dose-dependently as a function of free
Ca2+. Significant activation was observed at free
Ca2+ concentrations above 0.1 µM while a
maximal effect was obtained at a Ca2+ concentration of 1 mM. The effect of Ca2+ was largely independent
of the substrate used. The mode of PLD2 activation by Ca2+
is not clear. It is known that intracellular Ca2+ can be
elevated in vivo to levels sufficient to activate PLD2 (26).
One possibility is that Ca2+ may be involved in mediating a
translocation of PLD2 from the cytosol to the membrane, as is the case
for conventional protein kinase C isoforms.
PIP2 was shown to act as a cofactor of two membranal PC-specific PLDs from brain (27, 28) and an activator of yeast PLD1 (13). The effect of increasing surface concentrations of PIP2 on yeast PLD1 and PLD2 was determined using either C6-NBD-PC or C6-NBD-PE as substrates. PLD2 activity was not affected by up to 10 mol % PIP2 (data not shown). In contrast, PLD1 activity was doubled in the presence of as little as 0.01 mol % PIP2. Thus, unlike the PC-specific PLD enzymes, PLD2 appears to be insensitive to PIP2. Consequently, PIP2 was omitted from the reaction mixture in PLD2 activity assays.
The production of phosphatidylalcohols by a transphosphatidylation
reaction is a unique property of PLDs which had been utilized extensively as a specific marker of PLD activity in vitro
and of PLD activation in situ (see Refs. 1 and 3).
Therefore, we have studied the efficiency by which PLD2 could use
primary alcohols as substrates for transphosphatidylation. Ethanol,
1-propanol, and 1-butanol were added to the PLD2 reaction mixture at a
concentration of 300 mM. Interestingly, PLD2 activity of
pld1 cell lysate produced no phosphatidylalcohols whether
C6-NBD-PC or C6-NBD-PE was utilized as
substrate (data not shown). Thus, PLD2 appears to be incapable of
transphosphatidylation when simple short chain primary alcohols are
added as acceptors. This contrasts sharply with the ability of yeast
PLD1 to catalyze transphosphatidylation (12, 13, 14) and with the efficient
transphosphatidylation reaction carried out by the mammalian
PC-hydrolyzing enzymes. Our data imply that certain forms of PLD lack
this property. This conclusion is supported by previous reports showing
that some PLD activities isolated from variant strains of the bacterium
Streptomyces chromofuscus exhibit a greatly reduced or lack
of ability to produce phosphatidylalcohols (29, 30, 31). Thus, whereas the
production of phosphatidylalcohols (with simple short chain alcohols)
remains a unique property of PLDs, not all PLDs can catalyze a
transphosphatidylation.
Mammalian cells contain both cytosolic (24, 25, 32, 33) and membrane-bound (34, 35) forms of PLD. To examine the subcellular localization of PLD2, its activity was determined in soluble and particulate fractions derived from yeast cell lysates (Table I). Subcellular fractionation shows that most of the PLD2 activity was soluble and found in the 100,000 × g supernatant. However, a significant amount of PLD2 activity was membrane-associated.
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It has already been demonstrated that PLD1 activity is required for
meiotic cell division in yeast (12, 13, 14). It was suggested that PLD1 may
inhibit mitotic cell division (36). We decided, therefore, to check
whether PLD2 activity is altered during mitotic cell division. PLD2
activity was determined at different time points after the dilution of
stationary-phase (G1-arrested) wild-type cells into fresh
medium (Fig. 4). It was found that PLD2 was transiently
activated upon culture dilution in fresh media, both before and during
the interval in which the first cell division took place. Two peaks of
activation could be seen. The first peak represents rapid, transient
2-fold activation of PLD2 measured within 20 min after the transfer to
fresh medium and before initiation of the first cell division. This
peak declines after 40 min. The second peak of activity is observed
2-4 h after the transfer and then declines. These results suggest a
biphasic response of PLD2 to induction of mitotic cell division in
G1-arrested cells and indicate that PLD2 is a regulated
enzyme. Interestingly, this activation of PLD2 was detected under
in vitro assay conditions. Hence, the stimulation of PLD2
activity during early growth phase may involve a persistent
modification of the enzyme. The rapid activation upon growth
stimulation suggests that PLD2 may mediate early growth signals and
play a role in the initiation of the mitotic cell cycle. In mammalian
cells, PLD is activated by growth factors and other mitogens,
suggesting a role in mediating G0-G1 transition
(6). In addition, there is evidence for a negative modulatory effect of
PLD also on G2-M phase transition (37). Presumably, the PA
produced by PLD2 may regulate one or more of the proteins involved in
cell cycle control.
The radically different catalytic properties of PLD1 and PLD2 indicate that these two enzymes are probably structurally unrelated. Indeed, an extensive analysis of the complete S. cerevisiae genome by the BLAST algorithm, using several highly conserved domains present in known PLD family members as well as the putative Ca2+-binding domain of plant PLD, has demonstrated that PLD1 has no direct homologs. Thus, in yeast, PLD1 is the sole member of the recently defined gene family (15, 16) that, in addition to PLD1, includes the plant PLDs, the human ARF-dependent PLD, and a putative Caenorhabditis elegans PLD gene. Consequently, we postulate that the gene encoding PLD2 does not belong to the PLD/phosphatidyltransferase gene family and, thus, may represent the first identified member of a novel PLD family. Ongoing work in our laboratory is aimed at identifying and cloning the PLD2 gene.