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: moti.liscovitch@weizmann.ac.il 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. 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Bacteriol. 175, 4235–4238. 3830 X. Tang et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . preferentially hydrolyses phosphatidylethanolamine (PtdEtn) and phos- phatidylserine (PtdSer); and its activity is not stimulated by PtdInsP 2 . In addition,. 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