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Phospholipase D1 is required for e¤cient mating projection formation in Saccharomyces cerevisiae Michelle L. Hair¢eld a , Amanda B. Ayers b , Joseph W. Dolan b

a;b;

*

a Molecular, Cellular Biology and Pathobiology Program, Medical University of South Carolina, Charleston, SC 29425, USA Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Ave., P.O. Box 250504, Charleston, SC 29425, USA

Received 17 January 2001; received in revised form 5 June 2001; accepted 6 June 2001 First published online 26 June 2001

Abstract Phospholipase D1 (PLD1) is an important enzyme involved in lipid signal transduction in eukaryotes. A role for PLD1 in signaling in Saccharomyces cerevisiae was examined. Pheromone response in yeast is controlled by a well-characterized protein kinase cascade. Loss of PLD1 activity was found to impair pheromone-induced changes in cellular morphology that result in formation of mating projections. The rate at which projections appeared following pheromone treatment was delayed, suggesting that PLD1 facilitates the execution of a ratelimiting step in morphogenesis. Mutants were found to be less sensitive to pheromone, again arguing that PLD1 is acting at a rate-limiting step. The fact that morphogenesis is most dramatically affected indicates that PLD1 functions primarily in the morphogenic branch of the pheromone response pathway. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Phospholipase D1; Saccharomyces cerevisiae ; Pheromone response

1. Introduction Saccharomyces cerevisiae has been a very useful model system for the study of signal transduction mechanisms in eukaryotes. The best characterized signaling pathway in S. cerevisiae is the pheromone response pathway which controls mating. Studies of the pheromone response pathway have provided signi¢cant insights into the action and regulation of receptor-coupled heterotrimeric G proteins and protein kinase signaling cascades [1]. The utility of S. cerevisiae for the analysis of lipid signal transduction pathways is only now being exploited. Phospholipases are important enzymes in lipid signaling pathways. Phospholipase D (PLD) preferentially hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) [2]. PA can be dephosphorylated to yield diacylglycerol (DAG). PLD has been found to play important roles in the response of mammalian cells to mitogens, growth factors and di¡erentiation factors [3]. A role for PLD in di¡erentiation has been conserved from mammals to yeasts [4^8]. In S. cerevisiae, PLD1, the product of the

* Corresponding author. Tel. : +1 (843) 792-1904; Fax: +1 (843) 792-2464. E-mail address: [email protected] (J.W. Dolan).

SPO14 gene, has been shown to be required for sporulation [6]; in Candida albicans, PLD1 has been shown to be important for morphogenesis [9]. The role for PLD1 in sporulation in S. cerevisiae has been well documented [6,10,11]. In contrast, the role for this enzyme in mitotic yeast cells has yet to be clearly de¢ned. PLD1 activity is essentially una¡ected by mating type with nearly identical levels of activity in a, K, and a/K cells [12]. Growth on secondary carbon sources has been shown to increase PLD1 activity; spo14v mutants are viable on secondary carbon sources but exhibit a slightly reduced growth rate relative to wild-type [12,13]. Mutants de¢cient in PLD1 show few obvious phenotypes other than a failure to sporulate. The constitutive expression of PLD1 in both haploid cells, which do not undergo meiosis and sporulation, and diploid cells, which are specialized for meiosis and sporulation, suggested that the enzyme had as yet undiscovered roles in mitotic cells. Pheromone response and mating [14] represented one physiological process in which PLD1 might play a role. Cells respond to pheromone by altering cell morphology and modifying the cell membrane at the tip of the mating projection. Both of these responses, especially the modi¢cation of membrane, are reasonable candidates for processes in which PLD1 may function. Mating is coordi-

1567-1356 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 1 3 5 6 ( 0 1 ) 0 0 0 2 7 - 7

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nated by mating pheromones that act through a G protein-coupled receptor. Activation of the receptor leads to dissociation of the heterotrimeric G protein and the activation of downstream components by the LQ subunits. The signal is propagated through a protein kinase cascade and terminates with the activation of the pheromone responsive transcriptional activator Ste12p. Cdc42p, a member of the Rho family of small GTP binding proteins, is also required for e¤cient pheromone response [15]. In mammalian systems, at least one form of PLD has been shown to be regulated by CDC42 [16]. In this report, evidence is presented that demonstrates the involvement of PLD1 in pheromone-induced morphogenesis. 2. Materials and methods 2.1. Strains, plasmids and growth conditions The strains used in this study are listed, with the relevant genotypes, in Table 1. Cells without plasmids were grown in YPD and those with plasmids were grown in Synthetic Complete (SC) missing the appropriate supplement [17]. Plasmid pGFP-SPO14 is a multicopy plasmid carrying a gene encoding a fusion protein composed of green £uorescent protein (GFP) fused near the N-terminus of PLD1 and expressed from the ADH1 promoter [11]. Plasmid pGS3: :TRP1 is the multicopy vector pBM150 carrying the STE12 gene expressed from the GAL1 promoter and the TRP1 gene inserted into the ApaI site within the URA3 gene; plasmid pGS3, which lacks the TRP1 insertion, was used in earlier studies [18]. Plasmid pSB234 is a single-copy plasmid carrying the pheromone-responsive reporter gene FUS1-lacZ [19]. 2.2. PLD1 assays PLD1 activity was assayed using a £uorescent in vitro assay as described previously [9]. The substrate for the assay is BODIPY-PC (Molecular Probes, Eugene, OR, USA) which is hydrolyzed to BODIPY-PA (BPA). BPA is dephosphorylated by lipid phosphate phosphatases to yield BODIPY-diglyceride. PLD1 activity has been nor-

Table 1 Yeast strains Strain

Genotype

EG123 246-1-1 W303-1A AB1 AB2 AB3 SF270 SF271

a can1-101 his4-519 leu2 trp1 ura3 K can1-101 his4-519 leu2 trp1 ura3 a ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 EG123 with pld1: :LEU2 246-1-1 with pld1: :LEU2 W303-1A with pld1: :LEU2 a lys1 K lys1

malized to the level in samples taken at time zero of each assay to yield the relative PLD1 activity. For cultures induced with K-factor, commercially prepared pheromone was added to a ¢nal concentration of 0.5 WM. For cultures induced with a-factor, the cell-free medium from a saturated culture of EG123 in YPD was used as a source of afactor. Induction by a-factor was initiated by adding a volume of a-factor-containing medium equal to the volume of the test culture. 2.3. Assays of pheromone responses To measure pheromone-induced cell cycle arrest, overnight cultures grown in YPD were diluted 1:20 into fresh YPD and incubated for 4 h at 30³C. Pheromone was added to 0.5 WM and samples were withdrawn at the indicated time points. The samples were sonicated brie£y to disrupt clumps and formaldehyde was added to 3.7%. Direct microscopic enumeration was used to determine the percentage of cells that had become arrested as unbudded cells. The same protocol was used to measure the formation of mating projection except that cells with projections were counted rather than unbudded cells. To measure pheromone-induced transcription, cells were cultured overnight in SC3Ura, diluted 1:20 into fresh SC3Ura medium, and grown for 4 h. The cultures were then split and K-factor was added to one set to a ¢nal concentration of 0.5 WM. Both sets of cultures were incubated at 30³C for 90 min and then assayed for L-galactosidase activity as described previously [18]. Pheromone sensitivity was measured by spreading cells suspended in YPD onto YPD agar with a sterile cotton-tipped applicator followed by spotting of K-factor (1 and 3.0 Wg) onto sterile circles of ¢lter paper on the lawn of cells. Plates were incubated at 30³C for 24 h and then photographed. 2.4. Mating assays Two-fold serial dilutions of mating reactions were prepared in 96-well microtiter plates. The initial samples contained 1U104 cells of each mating partner in a total volume of 100 Wl YPD. After dilution, 5 Wl of each diluted sample was spotted onto selective medium. For most of the mating reactions, cells were spotted onto Synthetic Minimal medium ; for mating reactions between two pld1: :LEU2 strains, cells were spotted onto SC3His medium. 2.5. Localization of actin and Ste2p Actin ¢laments were visualized by staining with Texas red-phalloidin (Molecular Probes) as per the manufacturer's instructions. Ste2p was visualized by indirect immuno£uorescent staining with antibody to Ste2p (Santa Cruz Biotechnology, Santa Cruz, CA, USA; catalog no. sc6780) and £uorescein-labeled rabbit anti-goat IgG anti-

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body (Sigma). Samples were prepared for immuno£uorescent staining using standard techniques [20]. 2.6. Over-expression of Ste12p For galactose-induced expression of Ste12p from plasmid pGS3: :TRP1, cells were cultured overnight in SC lacking tryptophan and containing sucrose as the carbon source (SS3Trp). The starter cultures were subcultured into fresh SS3Trp for 3 h, galactose was added to a ¢nal concentration of 3%, and the cultures were incubated for an additional 90 min. Samples were taken, sonicated to disrupt clumps, and formaldehyde was added to 3.7%. Samples were analyzed microscopically immediately or were stored at 4³C for analysis at a later time. 3. Results 3.1. PLD1 activity is responsive to pheromone As a ¢rst step in establishing a role for PLD1 in mating, PLD1 activity was measured in cells responding to pheromone. Activity was measured in cells of both haploid mating types that were responding to the appropriate pheromone. Fig. 1 shows that PLD1 activity was stimulated in both a (strain EG123) and K (strain 246-1-1) cells exposed to K-factor or a-factor, respectively. In both strains PLD1 activity increased approximately two-fold during response. This change in PLD1 activity was of a magnitude similar to that seen when cells are grown on secondary carbon sources and during sporulation, two

Fig. 2. Subcellular localization of PLD1 during mating. The subcellular localization of the GFP-PLD1 fusion protein was followed by £uorescent microscopy during a mating reaction between strain AB1 carrying pGFP-SPO14 and strain 246-1-1. A: Vegetative cells in the absence of mating partners. B: Typical cell forming a mating projection in response to K-factor produced by mating partners. C: Early zygote showing fused mating projections. D: First diploid daughter cell following successful mating.

other conditions that have been shown to stimulate PLD1 activity [12,13]. 3.2. PLD1 does not relocalize during pheromone response PLD1 has been shown previously to relocalize during sporulation, concentrating near the spindle pole body and developing prospore walls [11]. The subcellular distribution of a GFP-PLD1 fusion was followed during mating. Strain AB1 (a pld1: :LEU2) carrying pGFP-SPO14 was mated with strain 246-1-1 and £uorescence was monitored microscopically. As had been reported [11], PLD1 was observed to be generally distributed throughout the cytoplasm in vegetatively growing cells (Fig. 2A). This pattern did not change as the cells formed mating projections (Fig. 2B). Analysis of zygotes revealed that PLD1 was able to di¡use into the mating partner with no obvious impediment (Fig. 2C). By the time diploid daughter cells formed, GFP-PLD1 was found distributed throughout the parental and daughter cells (Fig. 2D). Thus, in contrast to sporulation, PLD1 does not alter its subcellular localization in response to pheromone. 3.3. Loss of PLD1 activity does not impair cell cycle arrest

Fig. 1. Pheromone induction of PLD1 activity. PLD1 activity was measured using a £uorescent in vitro assay. Samples were taken at the indicated time points after pheromone addition. The activity has been normalized such that the activity at time zero was equal to 1.00. Error bars indicate the standard error of the mean for at least three independent experiments.

One of the earliest responses to pheromone is arrest of progression through the cell cycle at the G1/S checkpoint designated START. Arrest at START was monitored by determining the percentage of unbudded G1-phase cells in pheromone-treated cultures. Wild-type (EG123) and pld1v: :LEU2 (AB1) cells exhibited identical kinetics of cell cycle arrest in response to treatment with K-factor (Fig. 3). Similar results were seen with K cells responding to a-factor (data not shown).

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Fig. 3. Pheromone-induced cell cycle arrest. Wild-type and pld1: :LEU2 cells were treated with 0.5 WM K-factor. Arrest was assessed by the increase in the percentage of unbudded, G1-arrested cells in the cultures. The error bars indicate the standard error of the mean for at least three independent experiments.

3.4. PLD1 activity is not required for induction of transcription Another response to pheromone is the induction of transcription of pheromone-responsive genes. The impact of the loss of PLD1 activity on pheromone-responsive genes was measured by quantitating the expression of a FUS1-lacZ reporter gene [19]. The level of L-galactosidase produced in response to treatment of a cells with K-factor increased by approximately the same extent regardless of the presence or absence of PLD1 activity (Fig. 4). 3.5. Formation of mating projections is defective When cells are exposed to pheromone, the cellular mor-

Fig. 4. Pheromone-induced transcription. Wild-type and pld1: :LEU2 cells carrying plasmid pSB234 were treated with 0.5 WM K-factor for 90 min. L-Galactosidase activity was measured and expressed in Miller units. The error bars indicate the standard error of the mean for at least two independent experiments.

Fig. 5. Mating projection formation. A: Kinetics of mating projection formation by wild-type (EG123) and pld1: :LEU2 (AB1) cells. Error bars indicate the standard error of the mean of at least three independent experiments. B: Mating projections formed in response to 90 min induction with 0.5 WM K-factor by wild-type (EG123) and pld1: :LEU2 (AB1) cells. Cells with mating projections are indicated with an arrowhead. Identical results were obtained with W303-1A and AB3 (data not shown).

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phology changes such that the normally ellipsoidal cell becomes pear-shaped due to the formation of a mating projection, or shmoo. The pld1: :LEU2 mutation resulted in a decrease in the number of cells that formed mating projections (Fig. 5A). Furthermore, the cells that did form projections did so with an extended delay in the appearance (Fig. 5B). By the time the pld1: :LEU2 mutant cells began to form projections, the cells were already beginning to recover from pheromone-induced cell cycle arrest. This conclusion was based on the kinetics of arrest and recovery from Fig. 3. This phenotype suggests a delay in the accumulation of a factor or complex critical for the change in morphology. Mating projection formation requires the remodeling of the actin cytoskeleton. In response to pheromone, actin relocalizes to form clusters at the tip of the mating projection [21]. Texas red-phalloidin staining of actin ¢laments revealed that, in the mutant cells that did form projections, actin relocalized as in wild-type cells with no gross defect associated with loss of PLD1 activity (data not shown). Many other proteins also relocalize to the mating projection. Ste2p, the K-factor receptor, migrates to the tip of the mating projection following treatment with pheromone [22]. Indirect immuno£uorescence staining of cells with anti-Ste2p antibody revealed no defect in the relocalization in the pld1: :LEU2 mutant (data not shown). In both wild-type and mutant cells, Ste2p was found to localize to the mating projection in the majority of pheromone-treated cells with no gross defect in localization associated with the loss of PLD1 activity. These results, taken together, indicate that the loss of PLD1 activity does not impact the ability of cells to reorient the cytoskeleton and to relocalize proteins to the mating projection. These results do not, however, preclude a defect in the relocalization of a speci¢c protein or set of proteins. 3.6. Loss of PLD1 activity renders cells less sensitive to pheromone When assayed at high pheromone concentrations (0.5 WM), pld1: :LEU2 cells exhibit wild-type arrest and transcriptional responses. In contrast, pld1: :LEU2 mutants were found to be less sensitive to pheromone at lower concentrations. A halo assay was used to measure pheromone sensitivity over a range of pheromone concentrations (Fig. 6). Pheromone (K-factor) was applied to ¢lter paper circles on a lawn of wild-type (EG123) and pld1: :LEU2 (AB1) cells, creating a gradient of pheromone concentration as the pheromone di¡used into the agar. The diameter of the clear halos is directly related to the sensitivity of the cells in the lawn to pheromone. The pld1: :LEU2 cells produced signi¢cantly smaller halos and growth resumed within the halos more readily, indicating that the strain was less sensitive to pheromone.

229

Fig. 6. Pheromone sensitivity. Zones of growth inhibition can be seen in lawns of both wild-type (EG123) and pld1: :LEU2 cells. The ¢gure is representative of several independent assays.

3.7. Loss of PLD1 activity does not result in sterility The formation of the mating projection has been shown to be important for e¤cient mating. While some mutations that alter the ability of the cell to form a projection or to modify the tip for fusion do not impair mating competence [23], other such mutations do reduce mating competence [24]. The ability of pld1: :LEU2 mutants to mate was measured with a semi-quantitative mating assay. Strain AB1 (a pld1: :LEU2), which is isogenic to EG123 (a PLD1) except for the disruption of PLD1, repeatedly exhibited poor mating competence when assayed with the K lys1 tester strain SF271. In contrast, strain AB3 (also a pld1: :LEU2), which is isogenic to W303-1A (a PLD1), mated e¤ciently with SF271. Strain AB2 (K pld1: :LEU2), which is isogenic to 246-1-1 (K PLD1) and EG123 (a PLD1) except for the disruption of PLD1, mated well with the a lys1 tester strain SF270. Interestingly, AB2 (K pld1: :LEU2) mated e¤ciently with AB1 (a PLD1) but not with AB3 (also a PLD1). These results, when taken together, suggest that the mating competence of pld1v strains is minimally a¡ected by the loss of PLD1 activity. The di¡erences in mating competence observed in these assays is more likely due to strain di¡erences independent of PLD1 activity. 3.8. PLD1 is required for Ste12p-induced morphogenesis Ste12p is the pheromone-responsive transcriptional activator [25]. High-level expression of Ste12p has been shown to elicit all of the standard responses to pheromone including increased transcription, cell cycle arrest at START, and mating projection formation [18]. To determine whether PLD1 activity was required for these Ste12pdependent events, strains W303-1A (wild-type) and AB3 (pld1: :LEU2) were transformed with plasmid pGS3: :TRP1, which is a 2W plasmid expressing Ste12p from a galactose-inducible promoter (Fig. 7). Plasmid

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4. Discussion

Fig. 7. Morphogenesis induced by overexpression of Ste12p. Wild-type W303-1A (top) and PLD1-de¢cient AB3 (bottom) cells transformed with pGS3: :TRP1 were induced to produce Ste12p at high levels by growth on galactose-containing medium for 90 min. Typical misshapen cells are seen in the wild-type culture and absent from the mutant culture.

pGS3: :TRP1 is a derivative of pGS3, which was used in the earlier study, with the TRP1 gene inserted into the URA3 gene. Growth of the wild-type W303-1A pGS3: :TRP1 cells on galactose-containing medium resulted in the formation of normal mating projections and, by 90 min, the formation of large misshapen cells that presumably arise due to unregulated mating projection formation. In contrast, growth of AB3 (pld1: :LEU2) pGS3: :TRP1 on galactose-containing medium resulted in the accumulation of small, round unbudded cells. These cells have arrested cell cycle progression like wild-type cells but have failed to initiate formation of mating projections; the percentage of cells exhibiting a misshapen morphology was less than 1%. These results indicate that the formation of mating projections in response to elevated Ste12p activity requires PLD1 activity while the arrest of cell cycle progression does not.

An enormous volume of literature details the importance of protein kinase signaling mechanisms in the control of mating and pheromone response in S. cerevisiae. In contrast, a role for lipid signaling mediators in pheromone response has never been reported. In the present study, PLD1 was shown to play an important but non-essential role in the response of yeast cells to mating pheromones. Consistent with its role in pheromone response, PLD1 activity was seen to increase in response to pheromone treatment. While most aspects of pheromone response were largely una¡ected by the loss of PLD1 activity, a pronounced e¡ect on morphogenesis was observed. The reduction and delay in the formation of mating projections suggest that PLD1 is required to facilitate completion of a rate-limiting step in morphogenesis. In the absence of PLD1 activity, the number of cells capable of completing the rate-limiting step before the cell becomes adapted to pheromone is reduced. There are several ways in which PLD1 could function to facilitate morphogenesis. PLD1 activity results in the production of PA and, following dephosphorylation by lipid phosphate phosphatase, DAG. Both of these molecules have well established functions as signal transduction mediators and as modi¢ers of the physicochemical properties of membranes. PA and DAG have been shown to enhance membrane curvature [26,27], which may be important for the tighter curves of the mating projection. By facilitating the budding of nascent secretory vesicles from the transGolgi network [26,28,29], PLD1 could enhance the directed transport of materials toward the tip of the growing mating projection. Acting as a signal mediator, PA might activate phosphatidylinositol 4-phosphate 5-kinase, encoded by the MSS4 gene. Mss4p has been shown to be stimulated by PA and mutants exhibit morphological defects [30,31]. Another potential target for PA is Ste20p, a member of the PAK family of protein kinases. A PAK from bovine brain has been shown to be stimulated by PA [32] and Ste20p is an important component of the bud-site selection/bud emergence machinery that is redirected to form mating projections [21]. Other, as yet unidenti¢ed, targets of PA and DAG are also possible. The inhibition of Ste12p-induced morphogenesis in the pld1v mutant is particularly noteworthy. Morphogenesis in response to Ste12p over-expression indicates that a transcriptionally regulated protein is required for mating projection formation. The failure of the pld1v mutant to form projections indicates that this transcriptionally regulated protein requires PLD1 activity in order to induce morphogenesis. High-level expression of Ste12p also has been shown to suppress the mating defect associated with ste2, ste7, and ste11 mutations but not a ste4 mutation [18]. The failure to suppress the loss of Ste4p, the L-subunit of the heterotrimeric G-protein, suggested the existence of a Ste4p-dependent, Ste12p-independent branch

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Fig. 8. Model for PLD1 in pheromone response. On the left, PLD1 activity enhances the signal from Ste20p leading to morphogenesis. On the right, the absence of PLD1 results in a weaker signal from Ste20p, leading to a delay in the formation of mating projections. Transcriptionally regulated factors are important, but not su¤cient, for formation of mating projections. In this ¢gure, the thickness of the arrows is intended to re£ect the relative intensity of the signal being transmitted at each step. (Further details are given in the text.)

of the pheromone response pathway. For mating to occur, low-level constitutive signaling by Ste4p must occur and induce an activity that cannot be mimicked by enhancing the transcription of pheromone-responsive genes. This type of low-level signal would be absent in the ste4v mutant but present in the ste2 receptor mutants; full Ste4pdependent signaling would occur in the ste7 and ste11 kinase mutants during mating reactions. Taken together the data suggest that one possible target of this Ste4p-dependent, Ste12p-independent signaling is PLD1 acting primarily in a morphogenic branch of the response pathway. The fact that mating projections do form at a reduced level and with delayed kinetics in pld1v mutants in response to pheromone suggests that PLD1 is acting to facilitate execution of one or more rate-limiting steps. A model incorporating this mechanism is shown in Fig. 8. Ste4p activity acting through Ste20p stimulates both transcriptional responses and morphogenesis. Activated PLD1 facilitates the successful completion of a rate-limiting step leading to the formation of mating projections. High-level expression of Ste12p coupled with the basal Ste4p activation of PLD1 is su¤cient to induce morphogenesis in the absence of pheromone. In the absence of PLD1, the basal level of Ste4p activation of this morphogenic branch of the pheromone response pathway is insuf¢cient to execute the rate-limiting step and mating projections fail to form. Acknowledgements We would like to thank Kevin Desrosiers and Beth Endsley for their technical assistance and Dr. Joanne Engebrecht for plasmid pGFP-SPO14. This work was supported by MUSC Institutional Funds for Research and by

the National Science Foundation Grant MCB-9728143 to J.W.D.

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