COMMUNICATION:
Identification of a Novel Ca2+-dependent, Phosphatidylethanolamine-hydrolyzing Phospholipase D in Yeast Bearing a Disruption in PLD1*

(Received for publication, October 7, 1996, and in revised form, November 1, 1996)

Michal Waksman Dagger , Xiaoqing Tang Dagger , Yona Eli Dagger , Jeffrey E. Gerst § and Mordechai Liscovitch Dagger par

From the Dagger  Department of Biological Regulation and § Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

Chemicals

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 Strains

JC1 (Matalpha ade8 can1 his3 leu2 lys2 trp1 ura3) (18). PLD1Delta FS-1 (Matalpha ade8 can1 leu2 lys2 trp1 ura3 Delta pld1::HIS3) (13). W303-1B (Mata ade2-1 his3-11, 15 leu2-3, 112 ura3-1 trp1-1) (19).

Media

Wild-type yeast were maintained on synthetic complete minimal medium (SC). PLD1Delta FS-1 cells were maintained on SC drop-out medium, lacking histidine. Media were prepared essentially according to Rose et al. (20).

Phospholipase D Assays

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 PLD1Delta FS-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).


RESULTS AND DISCUSSION

We have previously reported that disruption of the PLD1 gene results in complete loss of PLD activity in Delta 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 Delta 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 Delta pld1 cell lysates (Fig. 1, right). Total PLD activity was consistently lower in Delta 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 Delta 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 Delta pld1 cell lysates (in which PLD1 activity is abolished as a consequence of gene disruption).


Fig. 1. Effect of Ca2+ on total PLD activity in wild-type and Delta pld1 cells. Whole-cell lysates were prepared from midlog phase wild-type and Delta pld1 cultures as described under "Experimental Procedures." PLD activity was assayed using 400 µM C6-NBD-PC as a substrate. Where Ca2+ was added, the concentration of free Ca2+ ion was adjusted to 1 mM, taking into account the presence of EGTA and EDTA in the reaction. Total PLD activity is indicated with open bars for wild-type and with solid bars for Delta pld1 cell lysates.
[View Larger Version of this Image (15K GIF file)]


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.


Fig. 2. Substrate specificity of PLD1 and PLD2. PLD1 and PLD2 activities were assayed as described under "Experimental Procedures" except all substrates were used at 400 µM with 1.2 mM Triton X-100 and 4 mol % PIP2. Note the 10-fold difference in the scales of the y-axis used for C6-NBD-PE (solid bars; left panel) and for C6-NBD-PC and C6-NBD-PG (open and hatched bars, respectively; right panel). ND, not detectable.
[View Larger Version of this Image (21K GIF file)]


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.


Fig. 3. Effect of increasing free Ca2+ concentrations on PLD2 activity. Whole-cell lysates were prepared from mid-log phase Delta pld1 cultures as described under "Experimental Procedures." PLD2 was assayed in the presence of 4 mol % PIP2, with either C6-NBD-PC (open circles) or C6-NBD-PE (solid circles) as described under "Experimental Procedures." Increasing amounts of Ca2+ were added to standard assays to yield the indicated final concentrations of free Ca2+.
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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 Delta 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.

Table I.

Distribution of PLD2 activity in subcellular fractions

Stationary phase W303-1B wild-type cells were diluted and grown to midlog phase. Whole-cell lysates were centrifuged at 8,000 × g for 10 min. The supernatant was collected and pellet was washed by resuspension in lysis buffer for assay. The 8,000 × g supernatant was ultracentrifuged for 1 h at 100,000 × g. The supernatant was collected and the pellet was washed as above and resuspended in lysis buffer for assay. Fractions were assayed for PLD2 activity as described under "Experimental Procedures." Total activity is expressed in phosphatidic acid-derived fluorescence units at 520 nm. Specific activity is expressed as fluorescence units per µg of protein.
Fraction Total protein Specific activity Total activity

mg fluorescence units/ µg fluorescence units
Whole-cell lysate 50.5 6.8 341,170
8,000 × g pellet 3.7 0.01 52
8,000 × g supernatant 43.0 4.8 205,440
100,000 × g pellet 8.0 3.2 25,600
100,000 × g supernatant 22.5 4.4 98,490

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.


Fig. 4. Time course of PLD2 activation during exit from stationary phase. PLD2 activity was determined at different stages of growth in culture. A 12-h-old stationary phase culture of wild-type W303-1B cells was diluted in fresh YPD medium to 0.6 × 106 cells/ml and grown at 30 °C. Samples were taken at the indicated times for PLD2 activity assays (solid circles) and for cell density determination (open squares). PLD2 activity was assayed as described under "Experimental Procedures" except 20 µM C6-NBD-PE was used. The arrow indicates the approximate time of entry into the first cell division cycle.
[View Larger Version of this Image (13K GIF file)]


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.


FOOTNOTES

*   This work was supported in part by grants from the Israel Science Foundation administered by the Israel Academy of Science and Humanities (to M. L.), the Minerva Foundation, Munich, Germany (to M. L.), and the Leo and Julia Forchheimer Center for Molecular Genetics at the Weizmann Institute of Science (to J. E. G. and M. L.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Supported by an Yigal Allon Fellowship and the incumbent of the H. Kaplan Career Development Chair for Cancer Research.
par    Incumbent of the Harold L. Korda Professorial Chair in Biology. To whom reprint requests should be addressed. Tel.: 972-8-9342773; Fax: 972-8-9344116; E-mail: lhliscov{at}weizmann.weizmann.ac.il.
1    The abbreviations used are: PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PIP2, phosphatidylinositol 4,5-bisphosphate; ARF, ADP-ribosylation factor; C6-NBD, 2-[6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl; PLD, phospholipase D.

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