Phosphatidylglycerolphosphate Synthase Expression in Schizosaccharomyces pombe Is Regulated by the Phospholipid

Transcription

1 JOURNAL OF BACTRIOLOGY, OCt. 1991, p /91/ $2./ Copyright 1991, American Society for Microbiology Vol. 173, No. 19 Phosphatidylglycerolphosphate Synthase xpression in Schizosaccharomyces pombe Is Regulated by the Phospholipid Precursors Inositol and Choline ROXANN R. KARKHOFF-SCHWIZR, BTH L. KLLY, AND MIRIAM L. GRNBRG* Department ofbiological Chemistry, University of Michigan Medical School, 131 Catherine Road, Ann Arbor, Michigan Received 18 March 1991/Accepted 16 July 1991 The enzyme phosphatidylglycerolphosphate synthase (PGPS; CDP-diacylglycerol glycerol 3-phosphate 3-phosphatidyltransferase; C ) catalyzes the committed step in the cardiolipin biosynthetic pathway. To study the regulation of PGPS in Schizosacchlaromyces pombe, we characterized the enzyme biochemically. Maximum activity occurred in the presence of 6 mm Triton X-1 at ph 7.5. The apparent Km values for CDP-diacylglycerol and glycerol 3-phosphate were 13 and 26,M, respectively. Optimal activity was at 35 C, and enzyme activity was labile above 4 C. Thioreactive agents were inhibitory to PGPS activity. To determine whether S. pombe PGPS is regulated by phospholipid precursors, we examined the time-dependent expression of PGPS upon inositol and choline starvation. Starvation for inositol resulted in a threefold increase in PGPS expression in wild-type cells. In chol and cho2 mutants, which are blocked in phosphatidylcholine synthesis, starvation for choline resulted in transient derepression of PGPS expression. In choline auxotrophs starved for inositol, PGPS was derepressed 2.5- to 3-fold in the presence of choline and less or not at all in the absence of choline. This is the first description of PGPS regulation in S. pombe and the first demonstration of inositol-mediated regulation in the inositol-requiring yeast species. Inositol plays a crucial role in general phospholipid metabolism in the budding yeast Saccharomyces cerevisiae (3, 11, 15). In this organism, inositol can be synthesized from glucose 6-phosphate in a reaction catalyzed by inositol 1-phosphate synthase (5). Inositol is utilized in the synthesis of the membrane phospholipid phosphatidylinositol (PI) (Fig. 1) (8). In addition, inositol acts as a regulator of phospholipid synthesis by repressing levels of the PI and phosphatidylcholine (PC) branch enzymes inositol 1-phosphate synthase, CDP-diacylglycerol (CDP-DG) synthase, phosphatidylserine (PS) synthase, PS decarboxylase, and the phospholipid N-methyltransferases (3). Inositol regulation of these enzymes is dependent on PC synthesis as well as the interaction of three unlinked regulatory genes (IN2- IN4-OPII). Choline, which can be used to synthesize PC via the Kennedy pathway, represses inositol 1-phosphate synthase, PS synthase, and the N-methyltransferases, although to a much lesser extent than inositol does. Repression by choline is only apparent in cells grown in the presence of inositol (3, 13, 21, 22). We have made use of the knowledge gained from the genetic and molecular dissection of general phospholipid synthesis to understand how the mitochondrial membrane is synthesized. In S. cerevisiae, the phospholipid cardiolipin (CL) is found only in the mitochondrial membrane and is required for mitochondrial function (14, 19). Therefore, we can use this phospholipid as a marker for the study of mitochondrion-specific membrane biogenesis. The enzyme phosphatidylglycerolphosphate synthase (PGPS; CDP-DG sn-glycerol 3-phosphate 3-phosphatidyltransferase; C ) is located in the mitochondrial inner membrane (16) and catalyzes the committed step in the CL branch of * Corresponding author phospholipid synthesis. We have shown that this enzyme is repressed by inositol in S. cerevisiae (9). As in general phospholipid synthesis, inositol-mediated repression of PGPS is dependent on PC synthesis. Thus, in mutants blocked in de novo PC synthesis, inositol repression of PGPS is only apparent when the block is circumvented by the addition of choline. However, unlike its effects on general phospholipid biosynthetic enzymes, choline alone does not repress PGPS activity, and regulation is not mediated by the IN2-IN4-OPIJ regulatory circuit (9). We extended our studies of mitochondrial membrane synthesis to the fission yeast Schizosaccharomyces pombe. This is an interesting eucaryotic organism from the standpoint of phospholipid synthesis in that no de novo biosynthetic pathway exists for the synthesis of inositol (18). We initiated the characterization of the mitochondrial enzyme PGPS in this organism to understand how the mitochondrial CL pathway is regulated. In this report, we describe the enzymological properties of PGPS in S. pombe and we demonstrate that PGPS expression in this organism is regulated by the phospholipid precursors inositol and choline. This is the first demonstration of inositol-mediated regulation of a phospholipid biosynthetic enzyme in the inositolrequiring yeast S. pombe. MATRIALS AND MTHODS S. pombe strains. S. pombe wild-type 972 (h-) and the derivative mutants SKZ 121 (chol-6 leul-32 ade6-21) and SKZ 9 (cho2-3 ade6-21) were generously provided by Susan Henry. Growth media. Cultures were maintained in 15% glycerol at -8 C for long-term storage and on Y (5% yeast extract, 3% glucose, 2.3% agar, 1 mm choline, 5,uM inositol, 75 mg of adenine per liter) plates at 4 C for short-term storage. Vitamin-free yeast base used in synthetic

2 VOL. 173, 1991 PGPS XPRSSION IN S. POMB 6133 CMP Pi.1 PM5 PSD PNMT PNMT PNMT COP-DO PS - P -n sopmm - $ POM -o PC + *+ CMP G-3-P NP PS PGPas CLS LW PGP P G - ON- CL CDP-c3 CU FIG. 1. Pathway for de novo synthesis of phospholipids in the fission yeast S. pombe. Phosphatidylinositol (PI) is synthesized from exogenous inositol (I) and CDP-diacylglycerol (CDP-DG). The committed step in phosphatidylcholine (PC) synthesis is the conversion of CDP-DG and serine (S) to phosphatidylserine (PS). PS is decarboxylated to form phosphatidylethanolamine (P). P is methylated three times to form PC. The committed step in cardiolipin (CL) synthesis is the conversion of CDP-DG and glycerol 3-phosphate (G 3-P) to phosphatidylglycerolphosphate (PGP) by phosphatidylglycerolphosphate synthase (PGPS). PGP is rapidly dephosphorylated to phosphatidylglycerol (PG). CL is synthesized from CDP-DG and phosphatidylglycerol (PG). The chol and cho2 mutants blocked in the indicated steps have been characterized previously (12). PMM, phosphatidylmonomethylethanolamine; PDM, phosphatidyldimethylethanolamine; PNMT, phospholipid N-methyltransferase; PIS, phosphatidylinositol synthase; PSS, phosphatidylserine synthase; PSD, phosphatidylserine decarboxylase; PGPase, phosphatidylglycerolphosphatase; CLS, CL synthase. media consisted of ammonium sulfate (5 g/liter), boric acid (5 mg/liter), copper sulfate (4 mg/liter), potassium iodide (1 mg/liter), ferric chloride (2 mg/liter), manganese sulfate (4 mg/liter), sodium molybdate (2 mg/liter), zinc sulfate (4 mg/liter), potassium phosphate (1 g/liter), magnesium sulfate (5 g/liter), sodium chloride (1 g/liter), and calcium chloride (1 g/liter). Components of the complete synthetic medium were vitamin-free yeast base (67%), nicotinic acid (1 mg/liter), calcium pantothenate (1 mg/ liter), biotin (1 mg/liter), glucose (2%), adenine (75 mg/liter), uracil (7 mg/liter), arginine (6 mg/liter), histidine (6 mg/ liter), leucine (75 mg/liter), lysine (57.5 mg/liter), and methionine (5 mg/liter). When required, inositol and choline were added at final concentrations of 5,uM and 1 mm, respectively. Materials. All chemicals were reagent grade. Components of the growth media, Tris buffer, glycerol 3-phosphate, Triton X-1, P-mercaptoethanol, N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and bovine serum albumin were purchased from Sigma Chemical Co.; Ready Organic Scintillation fluid was purchased from Beckman Instruments; CDP-DG was obtained from Life Science Resources; and L-[2-3H(N)]glycerol 3-phosphate (5.8 Ci/mmol) was purchased from Dupont, NN Research Products. Growth conditions. Liquid cultures were inoculated from Y plates and grown overnight in complete synthetic medium (containing inositol and choline) at 3 C. For the enzyme characterization studies, experimental cultures were inoculated from an overnight culture and grown to the late exponential stage of growth. Cells were harvested by centrifugation at 4 C and washed once with buffer 1 containing 5 mm Tris-HCl (ph 7.5), 1 mm DTA, 3 mm sucrose, and 1 mm,-mercaptoethanol. Pellets were frozen at -8C. In the starvation studies, flasks with experimental medium were inoculated from a single overnight culture, grown to the mid-exponential stage (A55 = 5), and harvested by using sterile technique. The cells were washed twice with the appropriate unsupplemented synthetic medium by centrifugation at 15 C and reinoculated at an equivalent concentration into experimental medium. At specified intervals, cells were harvested, washed with buffer 1, and stored at -8 C. To determine the number of viable cells at the indicated times, aliquots from serial dilutions of cultures were spread onto Y plates and incubated for 4 days prior to counting. Preparation of mitochondrial extracts. Mitochondrial extracts were prepared as described by Greenberg et al. (9) with the following modification. After the initial isolation of the mitochondrial pellet, the mitochondria were washed twice in buffer 2, containing 5 mm Tris-HCl (ph 7.5), 2% glycerol (wt/vol), and 1 mm P-mercaptoethanol. The washed pellet was resuspended in buffer 2 at a concentration of 2.5 mg/,ul (wet weight) and stored at -8 C in 2-ml aliquots. Protein assay. Mitochondrial extracts were assayed for protein concentration by the method of Bradford (1) with bovine serum albumin as the standard. nzyme assay. PGPS activity was assayed at 35 C. Incorporation of 5 mm [2-3H]glycerol 3-phosphate (4,4 cpm/ nmol) into chloroform-soluble material was measured in the presence of 2 mm Tris-HCl (ph 7.5), 6 mm CDP-DG, 6 mm Triton X-1, and mitochondrial extract corresponding to 75,ug of protein in a total volume of 1 ml. The reaction was stopped at 2 min with 25 ml of 1 N HCl in methanol. One milliliter of chloroform and 1.5 ml of 1 M MgCl2 were added per assay tube. The tubes were vortexed, and the phases were separated by centrifugation for 3 min at low speed. A 5-ml aliquot of the chloroform phase was transferred into a scintillation vial and evaporated. Organic counting scintillant (5 ml) was added, and the radioactivity in each sample was determined with an LS-381 liquid scintillation counter (Beckman Instruments, Inc.). ach sample was assayed in triplicate, along with a blank consisting of a mitochondrial sample and assay components inactivated with HCI before the start of the reaction. Specific activity was defined as units per milligram of protein, where 1 U is the amount of enzyme catalyzing the formation of 1 nmol of product per min at 35 C. Phosphatidylglycerol was identified as the main product of the reaction by thin-layer chromatography, using the solvent system of Carman and Belunis (2). RSULTS nzymological properties of PGPS. PGPS activity was found associated with the mitochondrial fraction of S.

3 6134 KARKHOFF-SCHWIZR T AL. J. BACTRIOL a 5, 4 X 3 gn 2 1 _ 1.' : 8', 6- X 4- a 2- n ZU co n 1.' 8' 6' 4' 2' n n ph cation concentration (mm) Triton X-1 concentration (mm) FIG. 2. ffects of ph, cations, and Triton X-1 on PGPS activity. (A) PGPS was measured at ph 6 to 7 with 1 mm Tris-maleate and at ph 7.5 to 9.5 with 1 mm Tris-HCl. (B) PGPS was assayed in the presence of the indicated concentrations of MnCl2 (O) or MgCl2 (-). (C) PGPS was assayed with 6 mm CDP-DG and the indicated concentrations of Triton X-1 The specific activity of PGPS was measured as described in Materials and Methods. pombe. Activity was linear with time up to 3 min and with protein concentration from 25 to 1 jig under the standard assay conditions. The specific activity of the enzyme from numerous mitochondrial preparations was typically 5 to 9 U/mg. The main product of the reaction was identified as phosphatidylglycerol when compared with standards, suggesting that newly synthesized phosphatidylglycerolphosphate is rapidly dephosphorylated under the assay conditions described. The mitochondrial enzyme was stable at -8 C for at least three freeze-thaw cycles. PGPS activity was measured with Tris-maleate from ph 6. to 7. and with Tris-HCl from ph 7.5 to 9.5 (Fig. 2A). The ph profile suggested that maximum activity occurred at ph 7.5. Activity was also measured in the presence of divalent cations. Magnesium ions inhibited activity very slightly at concentrations greater than 1 mm. In contrast, manganese ions inhibited PGPS activity at all concentrations tested (Fig. 2B). The effect of Triton X-1 on PGPS activity was also measured (Fig. 2C). The addition of 6 mm Triton X-1 resulted in maximum stimulation of activity, while concentrations of Triton X-1 above 6 mm resulted in inhibition of activity. PGPS activity was measured in a controlled-temperature B C I> 3 X 2 cn a 1 IL _ 6 i 5- m >, U co cn 2- ( 1-3 I ~~~..I Temperature (C) 4 5 Temperature (C) FIG. 3. ffect of temperature on PGPS activity. (A) PGPS activity was assayed at the indicated temperatures in a controlledtemperature water bath. (B) Mitochondrial protein (75,ug) was incubated at the indicated temperatures for 2 min. After incubation, the samples were placed on ice and immediately assayed for PGPS activity at 35 C. The specific activity of PGPS was measured as described in the text. water bath from to 6 C under standard assay conditions (Fig. 3A). The temperature profile suggested that maximum activity was obtained at 35 C. An Arrhenius plot was constructed from the above data and used to calculate an activation energy for the reaction of 11 kcal/mol. PGPS was also examined for its thermal stability to temperatures ranging from 3 to 6 C (Fig. 3B). The enzyme was stable up to 4 C. However, approximately 9% of activity was lost upon heating at 5 C, and total inactivation of the enzyme was observed at 6 C. Kinetic experiments were performed with a mixed micelle substrate of Triton X-1 and CDP-DG at a molar ratio of 1:1. When CDP-DG was held constant at 1 mm and the L-glycerol 3-phosphate concentration varied, saturation kinetics were shown by PGPS (Fig. 4A). The apparent Km for glycerol 3-phosphate was determined to be 26,uM. Likewise, saturation kinetics were shown when glycerol 3-phosphate was held constant at 75 mm and the CDP-DG concentration varied (Fig. 4B). The apparent Km for CDP-DG was determined to be 13 p.m. PGPS was inhibited by various thioreactive compounds (data not shown). At a final concentration of 5 mm, p-chloromercuriphenylsulfonic acid and N-ethylmaleimide inhibited activity by 98 and 92%, respectively. The addition of,b-mercaptoethanol to the assay system containing the thioreactive agent reversed the inhibition, but it had no effect on a control assay mixture. These results suggest that a sulfhydryl group is essential for PGPS activity. In vitro regulation of PGPS activity by phospholipid precursors. To ascertain whether key phospholipid precursors regulate PGPS at the level of enzyme activity, we added 1 mm inositol, choline, or serine to a standard assay mixture. Compared with a control assay with no additive, no significant inhibition or stimulation of PGPS activity was observed with any of these precursors (data not shown). 6

4 VOL. 173, 1991 PGPS XPRSSION IN S. POMB A )._ /(G3P) cn ȧ /(CDP-DG) FIG. 4. Dependence of PGPS activity on the concentration of substrates CDP-DG and L-glycerol 3-phosphate. Data were plotted as 1/V (nanomoles per minute per milligram) versus the reciprocal of the L-glycerol 3-phosphate concentration (A) or the reciprocal of the CDP-DG concentration (B). PGPS activity was measured as described in the text. ffect of inositol starvation on PGPS expression in wild-type cells. To determine the role of inositol in the regulation of PGPS expression in S. pombe, wild-type cells were grown in the presence of inositol, washed, and transferred to inositolfree medium. Cells were harvested at intervals after the shift, and mitochondrial extracts were assayed for PGPS activity. As shown in Fig. SA, PGPS activity was derepressed after the shift to inositol-free medium. Derepression in inositol-depleted wild-type cultures was observed in both the presence and absence of exogenous choline. The derepression occurred in a time-dependent manner over 2 h, and the derepressed PGPS levels were approximately threefold greater than repressed levels. Cell viability was maintained throughout inositol starvation; however, the stationary growth phase was reached earlier in starved cells than in control cells grown in inositol-containing media (Fig. SB). Control cells shifted to medium containing inositol and choline showed no PGPS derepression. ffect of choline starvation on PGPS expression in choline auxotrophs. While choline represses some PI and PC branch enzymes in S. cerevisiae and S. pombe, the extent of repression differs in the two yeasts (4, 12, 2). In S. pombe, choline represses the phospholipid N-methyltransferases to a greater extent than that observed in S. cerevisiae (12). We have previously shown that the CL branch enzyme PGPS is not repressed by choline alone in S. cerevisiae (9). To ascertain whether S. pombe PGPS is affected by choline, we measured PGPS activity in cells depleted of choline. Wildtype cells can synthesize PC by the de novo pathway and do not require exogenous choline. It is not surprising, therefore, that choline starvation of wild-type cells resulted in only slight derepression in PGPS expression (Fig. 5A). In contrast, cells that are blocked in different methylation steps of de novo PC synthesis require choline for synthesis of PC by the Kennedy pathway. The cho2 mutant is blocked in the first methylation step of PC synthesis (Fig. 1) (12). Unlike wild-type cells, this mutant contains significantly reduced () ) FIG. 5. ffect of inositol starvation on PGPS activity (A) and on cell viability (B) in wild-type cells. Wild-type cells were grown for six to seven generations in complete synthetic medium (containing inositol and choline) to an A55 of about 5. The cells were then harvested, washed, and inoculated at time zero into the following experimental media: inositol-free and choline-free (d); inositol-free, with choline (A); choline-free, with inositol (L1); and containing both inositol and choline (A). At the indicated times after inoculation, cells were harvested and PGPS activity in mitochondrial extracts was determined as described in the text. The number of viable cells per milliliter of culture was determined by serial dilution and plating as described in the text. levels of all three methylated phospholipids when grown in the absence of choline (12). We measured PGPS activity in cho2 cells shifted from inositol- and choline-containing medium to medium with inositol and lacking choline as shown in Fig. 6A. Cell viability in choline-starved cells was similar to that of cells grown in the presence of choline during this 2-h interval (Fig. 6B). Transient derepression of PGPS activity was observed after the shift to choline-free medium. The extent of derepression was about twofold. Derepression peaked at 4 h after the shift, after which PGPS activity dropped slowly until starting levels were reached (at about 15 h). PGPS derepression was not observed in cho2 cells supplemented with choline. To determine whether PGPS derepression is unique to the cho2 mutant or is a property of cells blocked in the PC pathway, we measured PGPS levels in the chol mutant, which can synthesize phosphatidylmonomethylethanolamine but is unable to perform subsequent methylation steps (Fig. 1) (12). As observed in the cho2 mutant, transient derepression of PGPS activity occurred in chol cells after they were shifted to choline-free medium (Fig. 7A). In six independent experiments, the sharp increase in PGPS was maximal at 4 h after the shift to choline-free medium. However, PGPS activity dropped much more rapidly in the chol mutant than in the cho2 mutant. Control chol cells grown in the presence of choline did not exhibit PGPS derepression. Thus, mutants blocked in either of two steps in de novo PC synthesis derepress PGPS in response to chioline starvation. ffect of inositol starvation on PGPS expression in choline auxotrophs. We have shown previously that inositol-mediated regulation of PGPS in S. cerevisiae is dependent on a

5 6136 KARKHOFF-SCHWIZR T AL. J. BACTRIOL. 6 A 8- A _ C, _ (U FIG. 6. ffect of choline starvation on PGPS activity in the cho2 mutant. Cells were grown for six to seven generations in complete synthetic medium (containing inositol and choline) to an A55 of about 5. The cells were then harvested, washed, and inoculated at time zero into choline-free, inositol-containing () and inositol- and choline-containing (A) experimental media. At the indicated times after inoculation, cells were harvested and PGPS activity (A) in mitochondrial extracts was determined as described in the text. (B) Number of viable cells per milliliter of culture was determined by serial dilution and plating as described in the text. functioning PC pathway (9). In S. cerevisiae mutants blocked in PC synthesis, inositol repression of PGPS was observed only in cells grown in the presence of exogenous choline. To determine whether PGPS derepression during inositol depletion in S. pombe was affected by PC synthesis, we measured PGPS activity in cho2 cells starved for inositol. Figure 8A shows that inositol depletion of cho2 cells in choline-containing medium led to derepression of PGPS. Cells depleted of both inositol and choline did not appear to derepress PGPS expression. However, cells lost viability much more quickly in medium lacking inositol and choline than in medium lacking inositol but containing choline (Fig. 8B). PGPS derepression during inositol starvation of the chol mutant was similar to that observed for cho2 cells. The extent of derepression was two- to threefold after the shift to inositol-free, choline-containing medium, while derepression was less or absent during starvation for both inositol and choline (data not shown). DISCUSSION The enzyme PGPS catalyzes the committed step of the CL pathway. We have shown previously that in S. cerevisiae, this enzyme is repressed by inositol (9). However, in mutants blocked in the PC pathway, inositol repression of PGPS occurs only in cells grown in the presence of exogenous choline (9). Recent experiments have shown that the regulation of general phospholipid mnetabolism in S. pombe is considerably different from regulation in S. cerevisiae (12). First, the responses of the two yeasts to inositol starvation FIG. 7. ffect of choline starvation on PGPS activity in the chol mutant. Cells were grown for six to seven generations in complete synthetic medium (containing inositol and choline) to an A55 of about 5. The cells were then harvested, washed, and inoculated at time zero into choline-free, inositol-containing () and inositol- and choline-containing (A) experimental media. At the indicated times after inoculation, cells were harvested and PGPS activity (A) in mitochondrial extracts was determined as described in the text. (B) Number of viable cells per milliliter of culture was determined by serial dilution and plating as described in the text. are quite different. S. cerevisiae inositol auxotrophs starved for inositol undergo "inositolless death," in which cell viability decreases by 3 to 4 orders of magnitude within 24 h (6, 1). In contrast, the inositol-requiring yeast S. pombe exhibits a decrease in viability of less than 2 orders of magnitude after 12 h of inositol starvation (7). This increased resistance to inositol starvation may be due to several adaptations in S. pombe phospholipid metabolism. These include a greater increase in the PS/PI ratio in response to inositol starvation and the ability of this organism to recycle inositol (7). A second difference between the two yeasts relates to phospholipid regulation in response to choline. Choline represses methylation enzymes of the PC pathway to a much greater extent in S. pombe than in S. cerevisiae (12). In addition, S. pombe mutants defective in the methylation steps of the PC pathway are choline auxotrophs, whereas S. cerevisiae mutants with similar mutations are not (12). (Choline auxotrophs can grow initially in the absence of choline, as shown in Fig. 6B and 7B. However, after repeated dilution of cells in fresh cholinefree medium, chol and cho2 cells stop growing, in contrast to wild-type cells.) Analysis of the regulation of phospholipid metabolism in these two yeasts can provide a great deal of insight into membrane function and adaptation to environmental change (12). We initiated a comparison of mitochondrial phospholipid biosynthesis in the two yeasts to further our understanding of the role of cross-pathway control in the regulation of mitochondrial membrane biogenesis and function. In the work described here, we characterized the regulation of PGPS expression by inositol in the fission yeast S. pombe. From our results, we conclude the following. (i) Inositol is a

6 VOL. 173, 1991 (n V" A 4- / ) 1 o (a 156.; B 1 2 FIG. 8. ffect of inositol starvation on PGPS activity in the cho2 mutant. Cells were grown for six to seven generations in complete synthetic medium (containing inositol and choline) to an A55 of 5. The cells were then harvested, washed, and inoculated at time zero into the following experimental media: inositol-free, choline-free (U); inositol-free, choline-containing (A); and inositol and cholinecontaining (A). At the indicated times past inoculation, cells were harvested and PGPS activity (A) in mitochondrial extracts of cho2 cells was determined as described in the text. (B) Number of viable cho2 cells per milliliter of culture was determined by serial dilution and plating as described in the text. regulator of PGPS expression. Starvation for inositol results in derepression of PGPS. (ii) PC synthesis is involved in regulation of PGPS expression. Starvation of choline auxotrophs for choline in the presence of inositol leads to a transient derepression of PGPS. (iii) Starvation of choline auxotrophs for both inositol and choline simultaneously results in less PGPS derepression than does starvation for inositol alone and in significantly greater cell death than starvation for either precursor alone. To study the regulation of PGPS in the fission yeast S. pombe, it was necessary to characterize the biochemical properties of the enzyme in mitochondrial extracts, since reported assay conditions for this enzyme in other organisms were inadequate to measure activity in S. pombe. Biochemical characterization revealed significant differences when compared with the S. cerevisiae enzyme. The ph optimum (7.5), temperature optimum (35 C), and apparent Km for substrate CDP-DG (13,uM) (Fig. 2 to 4) were clearly different from the same parameters reported for the S. cerevisiae PGPS (ph 7, 3 C, 33 jim) (2). In wild-type S. pombe cells and in the cho2 and chol methylation mutants, PGPS was derepressed nearly threefold after inositol starvation for 2 h (Fig. SA and 8A and unpublished data). This is the first demonstration of regulation of phospholipid biosynthesis by inositol in S. pombe. In the cho2 and chol mutants, PGPS derepression in inositoland choline-free medium was less than that observed in inositol-free, choline-containing medium (Fig. 8A and unpublished data). This suggests that in the inositol-requiring yeasts as well as in S. cerevisiae, regulation by inositol PGPS XPRSSION IN S. POMB 6137 depends at least in part on the ability to synthesize PC. However, in both cho2 and chol strains, cell death in inositol- and choline-free medium was significantly greater than that in inositol-free, choline-containing medium. It is therefore difficult to compare the extent of derepression during these two conditions. Regulation by choline points out interesting differences both between the two yeasts and between mitochondrial and general phospholipid synthesis. In S. cerevisiae, choline represses the CHOI and INO1 genes and the phospholipid N-methyltransferases (12). However, choline does not measurably repress PGPS in wild-type cells or in opi3 cells (9). Therefore, in S. cerevisiae, mitochondrial and general phospholipid syntheses differ with respect to choline regulation. In S. pombe, choline represses the phospholipid N-methyltransferase enzymes (12). Furthermore, starvation of chol or cho2 cells (defective in the same steps as the S. cerevisiae mutants opi3 and cho2, respectively) for choline led to transient derepression of PGPS expression (Fig. 6A and 7A). Therefore, in this organism, mitochondrial and general phospholipid syntheses are both regulated by choline. It is not clear why PC synthesis is required for inositol regulation of phospholipid biosynthetic enzymes. In S. cerevisiae, the opi3 mutant, which is blocked in the ability to make phosphatidyldimethylethanolamine or PC, cannot carry out inositol-mediated repression of INOI, the gene encoding inositol 1-phosphate synthase. However, if the ability to make either phosphatidyldimethylethanolamine or PC is restored by the addition of exogenous dimethylethanolamine or choline, regulation of INOI is likewise restored (12, 17). Regulation of PGPS expression by inositol in the opi3 mutant is similar in that inositol-mediated repression of PGPS is restored by growth in exogenous choline (9). It is possible, therefore, that phosphatidyldimethylethanolamine, PC, or possibly some breakdown product of these lipids is sensed by the cell in the inositol regulatory circuit. While inositol and choline are involved in cross-pathway control of PGPS regulation in both S. pombe and S. cerevisiae, the mechanisms for mediating this regulation have not been elucidated. Understanding the mechanisms which regulate PGPS will be greatly expedited by the isolation and molecular analysis of the PGPS genes from each yeast, as well as regulatory genes which mediate control of PGPS expression. xperiments directed toward these goals are currently in progress. ACKNOWLDGMNTS We are grateful to Stacey Minskoff and Paulette Gaynor for useful discussions, John Hill and Dave ngelke for critical review of this manuscript, and Ruby Hogue for assistance and good humor in the preparation of the manuscript. This work was supported by Public Health Service grant GM from the National Institutes of Health. RoxAnn Karkhoff- Schweizer was supported in part by a Thumau Postdoctoral Fellowship in Molecular Genetics. Beth L. Kelly was supported in part by Public Health Service training grant T32 GM7315 from the National Institutes of Health. RFRNCS 1. Bradford, M. M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: Carman, G. M., and C. J. Belunis Phosphatidylglycerolphosphate synthase activity in Saccharomyces cerevisiae. Can. J. Microbiol. 29: Carman, G. M., and S. A. Henry Phospholipid biosynthesis in yeast. Annu. Rev. Biochem. 58:

7 6138 KARKHOFF-SCHWIZR T AL. 4. Carson, M. A., M. mala, P. Hogsten, and C. J. Waechter Coordinate regulation of- phosphatidylserine decarboxylase activity and phospholipid N-methylation in yeast. J. Biol. Chem. 259: Charalompous, F. C., and I. Chen Inositol-1-phosphate synthetase and inositol-1-phosphatase from yeast. Methods nzymol. 9: Culbertson, M. R., T. F. Donahue, and S. A. Henry Control of inositol biosynthesis in Saccharomyces cerevisiae: inositol-phosphate synthetase mutants. J. Bacteriol. 126: Fernandez, S., M. J. Homann, S. A. Henry, and G. M. Carman Metabolism of the phospholipid precursor inositol and its relationship to growth and viability in the natural auxotroph Schizosaccharomyces pombe. J. Bacteriol. 166: Fischl, A. S., M. J. Homann, M. A. Poole, and G. M. Carman Phosphatidylinositol synthase from Saccharomyces cerevisiae. Reconstitution, characterization and regulation of activity. J. Biol. Chem. 261: Greenberg, M. L., S. Hubbeli, and C. Lam Inositol regulates phosphatidylglycerolphosphate synthase expression in Saccharomyces cerevisiae. Mol. Cell. Biol. 8: Henry, S. A., K. D. Atkinson, A. I. Kolat, and M. R. Culbertson Growth and metabolism of inositol-starved cells of Saccharomyces cerevisiae. J. Bacteriol. 13: Henry, S. A., L. S. Klig, and B. S. Loewy The genetic regulation and coordination of biosynthetic pathways in yeast: amino acid and phospholipid synthesis. Annu. Rev. Genet. 18: Hill, J.., C. Chung, P. McGraw,. Summers, and S. A. Henry. 199 Biosynthesis and role of phospholipids in yeast membranes, p In P. J. Kuhn, A. P. J. Trinci, M. J. Jung, M. W. Goosey, and L. G. Copping (ed.), Biochemistry of cell walls and membranes of fungi. Springer-Verlag, New York. 13. Homann, M. J., S. A. Henry, and G. M. Carman J. BACTRIOL. Regulation of CDP-diacylglycerol synthase activity in Saccharomyces cerevisiae. J. Bacteriol. 163: Jakovcic, S., G. S. Getz, M. Rabinowitz, H. Jakob, and H. Swift Cardiolipin contents of wild type and mutant yeasts in relation to mitochondrial function and development. J. Cell Biol. 48: Keiley, M. J., A. M. Bails, S. A. Henry, and G. M. Carman Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. J. Biol. Chem. 263: Kuchler, K., G. Daum, and F. Paltauf Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae. J. Bacteriol. 165: McGraw, P., and S. A. Henry Mutations in the Saccharomyces opi3 gene: effects on phospholipid methylation, growth, and cross-pathway regulation of inositol synthesis. Genetics 122: McVeigh, I., and. Bracken The nutrition of Schizosaccharomyces pombe. Mycologia 47: Trivedi, A., A. V. Wearring, S. D. Kohlwein, F. Paltauf, and. R. Tustanoff Functional importance of mitochondrial cardiolipin in yeast cytochrome c oxidase activity, p In J. J. Lemasters, C. R. Hackenbrock, R. G. Thurman, and H. V. Weterhoff (ed.), Integration of mitochondrial function. Plenum Publishing Corp., New York. 2 Waechter, C. J., and R. L. Lester Differential regulation of the N-methyl transferases responsible for phosphatidylcholine synthesis in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 158: Yamashita, S., and A. Oshima. 198 Regulation of phosphatidylethanolamine methyltransferase level by myo-inositol in Saccharomyces cerevisiae. ur. J. Biochem. 14: Yamashita, S., A. Oshima, J. Kawa, and K. Hosaka Regulation of the phosphatidylethanolamine methylation pathway in Saccharomyces cerevisiae. ur. J. Biochem. 128: Downloaded from on November 7, 218 by guest