THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc

Vol. 259, No. 17, Issue of September 10, pp. 10857-10662,1984 Printed in U.S.A.

Phosphatidylserine Synthesis in Saccharomyces cerevisiae PURIFICATION AND CHARACTERIZATION OF MEMBRANE-ASSOCIATED PHOSPHATIDYLSERINE SYNTHASE*

(Received for publication, March 5, 1984)

Myong Suk Bae-Lee and George M. Carman4 From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers, The State University, New Brunswick, New Jersey 08903

Membrane-associated phosphatidylserine synthase (CDP-diacylglycero1:L-serine O-phosphatidyltransfer- ase, EC 2.7.8.8) was purified from the microsomal fraction of Saccharomyces cerevisiae strains S288C and VALBC(YEpCHO1). VALBC(YEpCHO1) contains a hybrid plasmid bearing the structural gene for phos- phatidylserine synthase and overproduces the enzyme 6-7-fold (Letts, V. A., Klig, L. S., Bae-Lee, M., Car- man, G. M., and Henry, s. A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7279-7283) compared to wild-type S288C. The purification procedure included Triton X- 100 extraction of the microsomal membranes, CDP- diacylglycerol-Sepharose affinity chromatography, and DE-53 chromatography. The procedure yielded a preparation from each strain containing a major pep- tide band (Mr = 23,000) upon sodium dodecyl sulfate- polyacrylamide gel electrophoresis. Phosphatidylser- ine synthase was dependent on manganese and Triton X-100 for maximum activity at pH 8.0. The apparent K,values for serine and CDP-diacylglycerol were 0.58 mM and 60 PM, respectively. Thioreactive agents inhib- ited enzyme activity. The enzyme was thermally labile above 40 “C. Results of isotopic exchange reactions between substrates and products suggest that the en- zyme catalyzes a sequential Bi Bi reaction.

Phosphatidylserine accounts for approximately 8% of the total membrane phospholipids in Saccharomyces cereuisiae and is important for growth (1). Atkinson et al. (1, 2) have shown that chol mutants of S. cereuisiae are defective in phosphatidylserine synthesis which results in abnormal pat- terns of phospholipid metabolism and physiological proper- ties. In addition, phosphatidylserine serves as a precursor to the major membrane phospholipids phosphatidylethanola- mine andphosphatidylcholine (3). The chol mutants however, synthesize phosphatidylethanolamine and phosphatidylcho- line by the CDP-choline- and CDP-ethanolamine-based path- ways described by Kennedy and Weiss (4) when supplemented with ethanolamine or choline (2). The CHOl locus has been identified as the structural gene for the regulated ( 5 ) , mem- brane-associated enzyme (3, 6), phosphatidylserine synthase

* This work was supported by state funds, U. S. Hatch Act Regional Research Funds, Public Health Service Grant GM-28140 from the National Institutes of Health, and the Charles and Johanna Busch Memorial Fund. This is New Jersey Agricultural Experiment Station Publication D-10108-1-84. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom reprint requests should be addressed.

(CDP-diacylglycero1:L-serine 0-phosphatidyltransferase, EC 2.7.8.8), by biochemical analysis of chol mutants (2) and by cloning the CHOl gene (7). Phosphatidylserine synthase is localized in both the mitochondrial and microsomal fractions of S. cereuisiae (6, 8).

Phosphatidylserine synthase catalyzes the formation of phosphatidylserine by displacing CMP from CDP-diacylglyc- erol with L-serine (9). Phosphatidylserine synthase was first identified from Escherichia coli (9), was purified to homoge- neity (10-12), and extensively characterized (10, 13). Unlike the membrane-associated phosphatidylserine synthase from yeast, the enzyme from E. coli (14), as well as other Gram- negative bacteria (15), is associated with ribosomes in cell- free extracts.

Phosphatidylserine synthase from yeast is a difficult en- zyme to purify due to its membrane-bound nature and low levels of activity. These obstacles have been overcome by the developments of a solubilization procedure to release phos- phatidylserine synthase from yeast membranes (5,6, 16), the application of CDP-diacylglycerol-Sepharose affinity chro- matography (17,18) for the partial purification of the enzyme (7), and the cloning of the CHOl gene on the YEpl3 plasmid which directs the 6-7-fold overproduction of the enzyme (7). In the present paper, we report the purification of phospha- tidylserine synthase from S. cereuisiae and our initial studies on the characterization of the enzyme. This is the first report of the purification of the membrane-associated form of phos- phatidylserine synthase from a eukaryotic organism. The differences in phosphatidylserine synthase between prokar- yotic organisms and yeast are discussed.

EXPERIMENTAL PROCEDURES

Materials

All chemicals were reagent grade. Phospholipids, L-serine, myo- inositol, sn-glycerol 3-phosphate, ethanolamine, cysteine, threonine, CTP, CMP, bovine serum albumin, N-ethylmaleimide, and p-chlo- romercuriphenylsulfonic acid were purchased from Sigma. Triton X- 100 (octylphenoxypolyethoxyethanol) is a product of Rohm and Haas Company. ~-[3-~H]Serine, my0-[2-~H]inositol, ~n-[2-~H]glycerol 3- phosphate, and [5-3H]CTP were purchased from New England Nu- clear. [5G3H]CMP was obtained from Schwarz/Mann. dCDP-diacyl- glycerol was purchased from Serdary Research Laboratories, Inc. Sepharose 4B and Sephadex G-25 were products of Pharmacia Fine Chemicals. DE-53 (DEAE-cellulose) was from Whatman. Precoated Silica Gel 60 analytical thin-layer Chromatography plates were pur- chased from E. Merck. Electrophoresis reagents and molecular weight standards were obtained from Bio-Rad Laboratories, Amino acids for yeast growth were from ICN Nutritional Biochemicals. Yeast Nitro- gen Base without amino acids, yeast extract, and peptone were purchased from Difco Laboratories.

1085 7

10858 Phosphatidylserine Synthase from S. cerevisiae

Preparation of Substrates

CDP-diacylglycerol derived from egg lecithin was prepared by the method of Carman and Fischl (19). [5-3H]CDP-diacylglycerol was prepared enzymatically from phosphatidic acid and [EP~H]CTP with yeast mitochondrial CDP-diacylglycerol synthase (20). [3-3H]Phos- phatidylserine was prepared from CDP-diacylglycerol and L - [ ~ - ~ H ] serine with purified phosphatidylserine synthase under standard as- say conditions. The labeled product was purified by thin-layer chro- matography (19) in the solvent system chloroform-methanol-water (65:25:4, v/v).

Preparation of CDP-diacylglycerol-Sepharose

The sodium periodate-oxidized derivative of CDP-diacylglycerol was covalently attached to Sepharose 4B via an adipic acid dihydra- zide spacer arm as described by Larson et al. (18) with the modifica- tions of Fischl and Carman (17).

Yeast Strains and Growth Conditions

Wild-type strain S288C (agal2) was grown in YEPD medium (1% yeast extract, 2% peptone, and 2% glucose). Strain VALZC(YEpCHOl), which overproduces phosphatidylserine syn- thase activity (7), was grown in synthetic complete medium (1) without leucine. Cultures were grown at 30 "C to stationary phase, harvested by centrifugation, and stored at -80 "C (17). Strain VALZC(YEpCHO1) was kindly provided by Susan A. Henry and Verity A. Letts (Albert Einstein College of Medicine, Bronx, NY).

Enzyme Assays

Phosphatidylserine synthase activity was measured at 30 "C for 20 min by following the incorporation of ~-[3-~H]serine (10,000 cpm/ nmol) into chloroform-soluble material or the release of water-soluble radioactive CMP from [5-3H]CDP-diacylglycerol (400 cpm/nmol) as described by Carman and Matas (6). The assay mixture contained 50 mM Tris-HC1 (pH 8.0), 0.6 mM MnCl,, 0.5 mM L-serine, 0.2 mM CDP-diacylglycerol, 4 mM Triton X-100, and enzyme protein in a total volume of 0.1 ml. Phosphatidylserine synthase activity was

when measured with either labeled substrate. The products of the linear with time and protein concentration under assay conditions

reaction, phosphatidylserine and CMP, were identified with stand- ards by thin-layer chromatography and paper chromatography, re- spectively (6). Phosphatidylinositol synthase activity was measured as previously described (17) using myo-[2-3H]inositol (10,000 cpm/ nmol). Phosphatidylglycerophosphate synthase activity was mea- sured as previously described (21) using ~n-[2-~H]glycerol 3-phos- phate (10,000 cpm/nmol). CDP-diacylglycerol synthase activity was measured as previously described (20) using [EJ-~HJCTP (l0,OOO cpm/ nmol). A unit of enzymatic activity was defined as the amount of enzyme that catalyzed the formation of 1 nmol of product/min under assay conditions. The specific activity was defined as the units/mg of protein.

Protein Determination

CDP-diacylglycerol was removed from enzyme preparations by a chloroform-methanol-water phase partition (19) and the protein was precipitated from the aqueous phase with 5% trichloroacetic acid. The protein pellet was resuspended in 50 mM Tris-HC1 buffer (pH 8.0) containing 0.5% sodium dodecyl sulfate and its concentration was determined by the method of Lowry et al. (22). Alternatively, the protein concentration was directly determined from enzyme prepa- rations by the method of Bradford (23). The presence of Triton X- 100 and CDP-diacylglycerol did not interfere with the Bradford (23) protein determination procedure, provided the blank contained final concentrations of detergent and CDP-diacylglycerol identical to that of the sample. Both protein determination methods gave essentially the same protein values with bovine serum albumin as the standard.

Electrophoresis

Anodic electrophoresis under nondenaturing conditions (24) was performed in 6% polyacrylamide gel tubes containing 0.5% Triton X- 100. Sodium dodecyl sulfate-polyacrylamide slab-gel electrophoresis (25) was performed with 10% gels as described by Fischl and Carman (17). Gels were stained in 0.125% Coomassie Blue and destained in a 7% acetic acid, 5% methanol solution.

Purification of Phosphatidylserine Synthase All steps were carried out at 5 "C. Step 1. Preparation of Microsomes-Cells were disrupted with glass

beads in 50 mM Tris-HC1 buffer (pH 7.5) containing 1 mM Na2- EDTA, 0.3 M sucrose, and 10 mM 2-mercaptoethanol (17). Micro- somes were collected by differential centrifugation as described by Fischl and Carman (17).

Step 2. Solubilization with Triton X-100-Phosphatidylserine syn- thase activity was extracted from microsomes with 50 mM Tris-HC1 buffer (pH 8.0) containing 2 mM MnCl,, 30 mM MgC12, 0.5 M KCI, 10 mM 2-mercaptoethanol, 20% glycerol (w/v), and 1% Triton X-100 (w/v) at a final protein concentration of 10 mg/ml as previously described (6).

Step 3. CDP-diacylglycerol-Sepharose Chromatography-A CDP- diacylglycerol-Sepharose column (0.9 X 4 cm) was equilibrated with 50 ml of chromatography buffer (50 mM Tris-HC1 (pH 8.0), 30 mM MgCl,, 0.6 mM MnC12, 20% glycerol, 10 mM 2-mercaptoethanol, and 0.5% Triton X-100). The Triton X-100 extract was applied to the affinity column in 1.5-ml aliquots. Each aliquot was incubated in the column for 10 min before the addition of the next aliquot to effect enzyme binding. After the entire Triton X-100 extract was applied, the column was washed with 30 ml of chromatography buffer con- taining 1 M NaCl. The column was then saturated with chromatog- raphy buffer containing 1 mM CDP-diacylglycerol and 1 M NaCl and incubated for 1 h. Phosphatidylserine synthase was then eluted from the column with this buffer at a flow rate of 1 ml/min. Fractions (3 ml) were assayed for phosphatidylserine synthase activity. Fractions containing activity were pooled and desalted on a Sephadex G-25 column equilibrated with 20 mM Tris-HC1 buffer (pH 8.0) containing 10 mM MgCl,, 0.3 mM MnCl,, 20% glycerol, 10 mM 2-mercaptoetha- nol, and 0.5% Triton X-100.

Step 4. DE-53 Chromatography-A DE-53 column (0.6 X 4 cm) was equilibrated with 20 ml of 20 mM Tris-HC1 buffer (pH 8.0) containing 10 mM Mg'&, 0.3 mM MnCl,, 20% glycerol, 10 mM 2- mercaptoethanol, and 0.5% Triton X-100. Desalted enzyme from the previous step was applied to the column at a flow rate of 1 ml/min. The column was washed with 10 ml of equilibration buffer followed by elution of the enzyme with equilibration buffer containing 1 M NaCl. Fractions containing activity were pooled and desalted by Sephadex G-25 chromatography as described above. The purified enzyme was stable for at least 4 months at -80 "C. After two cycles of freezing and thawing, approximately 50% of the original activity was retained.

RESULTS

Purification of Phosphatidylserine Synthase-The micro- somal fraction of s. cereuisiue strains VALZC (YEpCHO1) and wild-type S288C were used as the source for phosphati- dylserine synthase purification. By using the microsomal frac- tion for enzyme purification, we eliminated the CDP-diacyl- glycerol-dependent phosphatidylglycerophosphate synthase which is associated with the mitochondrial fraction of S. cerevisiae (8). A summary of a purification for the enzyme from each strain is presented in Table I. Solubilization of phosphatidylserine synthase from microsomes with Triton X- 100 was dependent on the presence of manganese ions in the solubilization buffer (6, 17). The major purification of phos- phatidylserine synthase from both strains was attained by CDP-diacylglycerol-Sepharose chromatography. Binding of phosphatidylserine synthase to the affinity column was de- pendent on Triton X-100 and manganese in the chromatog- raphy buffer. The elution of phosphatidylserine synthase from the affinity column was dependent on the substrate CDP- diacylglycerol and NaCl in the elution buffer. Further purifi- cation by chromatography on DE-53 resulted in only a slight increase in specific activity; however, the enzyme was concen- trated by the procedure. This concentrated enzyme contained approximately 0.5 mM CDP-diacylglycerol as determined pre- viously (19). The purification procedure yielded essentially a homogeneous protein preparation from the overproducing strain as evidenced by polyacrylamide gel electrophoresis under nondenaturing conditions (RF of 0.15) and in the pres-

Phosphatidylserine Synthase from S. cerevisiae 10859 TABLE I

Purification of phosphatidylserine synthase from wild-type (S288C) and strain VALZC(YEpCHO1) Wild type (S288C)” VAL2C(YEpCHOl)*

Purification step Total units Protein Specific

activitv Yield Purifi- cation Total units Protein Specific

activitv Yield Purifi- cat.ion

Microsomes Triton X-100

CDP-diacyl- extract

glycerol- Sepharose

DE-53 chroma- tomwhy

591 493 1.2 100 1 3550 350 10.2 100 579 241 2.4 98 2 2700 151 17.9 76 1.76

1

457 0.34 1340 77 1120 1620 0.76 2130 45 210

42 1 0.18 2300 71 1920 1510 0.40 3760 42 370

’ Data are based on starting with 75 g (wet weight) of yeast pellet. * Data are based on starting with 50 g (wet weight) of yeast pellet.

1 2 3 4

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electro- phoresis. Lane I , 20 pg of standards: phosphorylase B (M, = 92,500), bovine serum albumin (M, = 66,2001, ovalbumin (M, = 45,000), carbonic anhydrase (M, = 31,000), soybean trypsin inhibitor (M, = 21,500), and lysozyme (M, = 14,400). Lane 2, 5 pg of purified phos- phatidylserine synthase from S288C. Lane 3, 10 pg of purified phos- phatidylserine synthase from S288C. Lane 4, 8 pg of purified phos- phatidylserine synthase from VALZC(YEpCHO1).

ence of sodium dodecyl sulfate (Fig. 1). The purified enzyme preparation derived from the wild-type strain contained a minor peptide band just below the major peptide band (Fig. 1). This would indicate that the enzyme preparation from S288C was not as pure as the preparation derived from the overproducing strain. In addition, the specific activity of the

epzyme from the wild-type strain was about 1.6-fold lower than the specific activity of the enzyme from the overpro- ducing strain (Table 1). The apparent subunit molecular weight of phosphatidylserine synthase was estimated to be 23,000 (Fig. 1). The molecular weight of the native enzyme in Triton X-100 cannot be determined until the interaction with detergent has been quantitated (26). The enzyme preparation purified from each strain did not contain measurable phos- phatidylinositol synthase activity. Phosphatidylinositol syn- thase, which is solubilized from microsomes under the con- ditions used in this study (17), did not bind to the affinity resin under the conditions used to bind phosphatidylserine synthase. In addition, neither enzyme preparation contained the mitochondrial-associated enzymes phosphatidylglycero- phosphate synthase (21) nor CDP-diacylglycerol synthase (20).

Properties of Phosphatidylserine Synthase-The enzymo- logical properties of phosphatidylserine synthase purified from both strains were essentially the same. The pH optimum for the reaction was 8.0 (Fig. 2A) . Phosphatidylserine syn- thase activity was dependent on the addition of either man- ganese or magnesium ions (Fig. 2B). The maximum activity obtained with manganese (0.6 mM) was 2-fold greater than the maximum activity obtained with magnesium (20 mM). Calcium ions did not serve as a cofactor for the enzyme. The addition of Triton X-100 to the assay mixture stimulated phosphatidylserine synthase activity to a maximum a t a con- centration of 4 mM, (molar ratio of Triton X-100 to CDP- diacylglycerol of 201), followed by an apparent inhibition of activity a t higher detergent concentrations (Fig. 2C). These results resemble substrate dilution kinetics (13). Kinetic ex- periments were conducted using a uniform mixed micelle substrate of Triton X-100 and CDP-diacylglycerol (13) at a molar ratio of 201. Normal saturation kinetics were shown by phosphatidylserine synthase when the CDP-diacylglycerol concentration was held constant (1 mM) and L-serine ( K , = 0.58 mM) was varied (Fig. 3A). Saturation kinetics were also shown by the enzyme when the L-serine concentration was held constant (2 mM) and CDP-diacylglycerol (K, = 60 p ~ ) was varied (Fig. 3B). Phosphatidylserine synthase was inhib- ited by various thioreactive compounds (Table 11). The addi- tion of mercaptoethanol to the assay system reversed this inhibition. The addition of mercaptoethanol to the standard assay mixture slightly stimulated activity. These results sug- gest that a sulfhydryl group is essential for phosphatidylserine synthase activity. The ability of phosphatidylserine synthase to utilize substrates related to L-serine was tested under

10860 Phosphatidylserine Synthase from S. cereuisiae

Manganese or Magnesium, mM

I h C

' 0 5 10 15 20

Triton X-100, mM

FIG. 2. Effects of pH, manganese and magnesium, and Triton X-100 on phosphatidylserine (PS) synthase activity. A, phosphatidylserine synthase (0.01 unit) was measured at the indicated pH values with 50 mM Tris-HC1 (0) or 2-(N-morpholino)ethanesulfonic acid (MES)-HCl (0). B, phosphatidylserine synthase (0.016 unit) was assayed with the indicated concentrations of MnC12 (0) or MgCl2 (0). C, phosphatidylserine synthase (0.018 unit) was assayed with 0.2 mM CDP-diacylglycerol and the indicated concentrations of Triton X-100. Activity was measured by following the incomoration of ~-[3-~H]serine into chloroform-soluble product as described in the text. U, units.

-

I I 1

-5 0 10 2 0

- 1 2 ,mM Serine

-20 0 2 0 4 0 60

1 , mM CDP-diacylglycerol

FIG. 3. Dependence of phosphatidylserine synthase activity on the concentration of L-serine and CDP-diacylglycerol. A, the data are plotted as 1/V (nanomoles/min/ml) uersus the reciprocal of the L-serine concentration. B, the data are plotted as 1/V (nano- moles/min/ml) uersus the reciprocal of the CDP-diacylglycerol con- centration. The molar ratio of Triton X-100 to CDP-diacylglycerol was maintained at 201. Enzyme activity was measured with L - [ ~ - ~ H ] serine as described in the text. The curves drawn were a result of a least-squares analysis of the data.

standard assay conditions using 0.2 mM [E~-~H]CDP-diacyl- glycerol as the labeled substrate. At concentrations of 0.5, 1, and 10 mM, the compounds cysteine, ethanolamine, and thre-

TABLE I1 Effect of thioreactive compounds on phosphatidylserine synthase

activity Phosphatidylserine synthase (0.016 units) was assayed under

standard conditions in the presence of the indicated additions.

Component Relative activity

% Control 100

+ 1 mM HgC12 13 + 1 mM p-chloromercuri- 17

+ 1 mM N-ethylmaleimide 17 + 1 mM p-chloromercuri- 100

phenylsulfonic acid

phenylsulfonic acid + 10 mM 2-mercaptoethanol

+ 1 mM 2-mercaptoethanol 130

onine did not substitute for serine as a phosphatidyl acceptor in the reaction. When measured under standard assay condi- tions using ~-[3-~H]serine as the labeled substrate, these compounds (10 mM) did not inhibit phosphatidylserine syn- thase activity. dCDP-diacylglycerol(O.2 mM) could substitute for CDP-diacylglycerol as a phosphatidyl donor for L - [ ~ - ~ H ] serine under standard assay conditions. Belendiuk et al. (20) reported that CDP-diacylglycerol synthase from S. cereuisiae is unable to catalyze the formation of dCDP-diacylglycerol. Phosphatidylserine synthase was examined for its stability to temperatures ranging from 30-60 "C (Fig. 4). The enzyme was stable up to 40 "C. However, about 90% of the activity was lost on heating at 50 "C for 20 min with total inactivation at 60 "C.

Reactions Catalyzed by Phosphatidylserine Synthuse- Phosphatidylserine synthase was examined for its ability to catalyze a series of isotopic exchange reactions according to Cleland (27, Table 111). Phosphatidylserine synthase did not catalyze an exchange reaction between L-serine and phospha- tidylserine (Table 111, reactions 1 and 2) nor an exchange reaction between CMP and CDP-diacylglycerol (Table 111, reactions 3 and 4). The enzyme did not catalyze the hydrolysis of phosphatidylserine (Table 111, reaction 5 ) nor the hydrol- ysis of CDP-diacylglycerol (Table 111, reaction 6). The inabil- ity of phosphatidylserine synthase to catalyze these reactions indicate that the enzyme does not follow a ping-pong reaction mechanism (27). Phosphatidylserine synthase did not cata-

Phosphatidylserine Synthase from S. cerevis;ae 10861

I I I I

- - 1.6 - \ E 3

0 ln 0

- 1.2 -

g 0.8 - - U J

cn a 0.4 - -

0 30 40 50 60

Temperature, "C

FIG. 4. Thermal stability of phosphatidylserine synthase activity. Aliquots (10 pl, containing 0.018 unit) of phosphatidylser- ine synthase were incubated for 20 min at the indicated temperatures in a controlled-temperature water bath. After incubation, the aliquots of enzyme were placed on ice, assay components were added, and phosphatidylserine synthase activity was measured at 30 "C. U, units.

TABLE I11 Reactions catalyzed by phosphatidylserine synthase

Reactions were measured in the standard assay buffer described in the text. The assays were run for 1 h with 1 pg of purified enzyme and the indicated reaction components. Serine-phosphatidylserine exchange reactions were measured using either ~-[3-~H]serine (10,000 cpm/nmol) or [3-3H]phosphatidylserine (15,000 cpm/nmol) as sub- strates (10). CMP-CDP-diacylglycerol exchange reactions were mea- sured using either [5-3H]CMP (10,000 cpm/nmol) or [5-3H]CDP- diacylglycerol (400 cpm/nmol) as substrates (10). The release of water-soluble radioactive serine or CMP was measured after a chlo- roform-methanol-water phase partition (6). Chloroform-soluble ra- dioactive phosphatidylserine or CDP-diacylglycerol was measured after a chloroform-methanol-water Dhase Dartition (6).

~

Reaction Incorporated or released

1.

2.

3.

4.

5. 6.

7.

0.5 mM ~-[3-~H]serine + 0.5 mM phosphati-

0.5 mM L-serine + 0.5 mM [3-3H]phosphati-

0.5 m M [5-3H]CMP + 0.5 mM CDP-diacyl-

0.5 mM CMP + 0.5 mM [5-3H]CDP-diacyl-

0.5 mM [3-3H]phosphatidylserine hydrolysis 0.5 mM [5-3H]CDP-diacylglycerol hydroly-

5.0 mM CMP + 0.5 mM [5-3H]CDP-diacyl- sis

glycerol + 0.5 mM phosphatidvlserine

dylserine

dylserine

glycerol

glycerol

8. 0 . 5 m ~ [5-3H]CMP i 0 . 5 - m ~ CDP-diacyl- glycerol + 0.5 mM L-serine

9. 0.5 mM L-serine + 0.5 mM [3-3H]phosphati- dylserine + 0.5 mM CDP-diacylglycerol

10. 0.5 mM ~-[3-~H]serine + 0.5 mM phosphati- dylserine + 5.0 mM CMP

11. 0.5 mM CDP-diacylglycerol + 0.5 mM L-[3- 3H]serine

12. 0.5 mM CDP-diacylglycerol + 0.5 mM ~ - [ 3 - 3H)serine + 0.5 mM phosphatidylserine

13. 5.0 mM CMP + 0.5 mM [3-3H]phosphati- dylserine

14. 5.0 m M CMP + 0.5 mM [3-3H]phosphati- dylserine + 0.5 mM L-serine

15. 5.0 mM [5-3H]CMP + 0.5 mM phosphati- dylserine + 0.5 mM CDP-diacylglycerol

nmol <0.02

<0.02

<0.02

<0.02

<0.02 <0.02

<0.02

<0.02

1.30

3.90

42.30

43.90

0.75

4.49

<0.02

lyze exchange between CMP and CDP-diacylglycerol in the presence of either phosphatidylserine (Table 111, reaction 7) or L-serine (Table 111, reaction 8). On the other hand, phos- phatidylserine synthase did catalyze exchange between L- serine and phosphatidylserine in the presence of either CDP- diacylglycerol (Table 111, reaction 9) or CMP (Table 111, reaction 10). In addition, phosphatidylserine stimulated the incorporation of labeled L-serine into phosphatidylserine in the forward reaction (Table 111, reaction 12) and L-serine stimulated the release of labeled L-serine from phosphatidyl- serine in the reverse reaction (Table 111, reaction 14). Accord- ing to Cleland (27), the results in Table I11 would suggest that phosphatidylserine synthase in S. cereuisiae catalyzes a se- quential reaction mechanism with phosphatidylserine re- leased before CMP in the reaction sequence. In the forward reaction, phosphatidylserine synthase may bind to CDP-di- acylglycerol before L-serine in the reaction sequence as evi- denced by purification on CDP-diacylglycerol-Sepharose (Ta- ble I). The lack of exchange between CMP and CDP-diacyl- glycerol in the presence of phosphatidylserine (Table 111, reaction 7) might indicate that the binding of CDP-diacyl- glycerol in the forward reaction is ordered. However, this lack of exchange may also be due to the inhibition of the reverse reaction by CDP-diacylglycerol (Table 111, reaction 15).

The ability of the yeast phosphatidylserine synthase to catalyze the calcium-dependent phospholipid-base exchange reaction for the synthesis of phosphatidylserine was examined under the optimal assay conditions reported for the mamma- lian base exchange enzymes (28). Phosphatidylserine syn- thase did not catalyze the exchange of ~- [3-~H]ser ine for ethanolamine in phosphatidylethanolamine.

DISCUSSION

The study of phosphatidylserine synthase in yeast is im- portant because the enzyme plays a role in the regulation of phospholipid metabolism and yeast growth (1, 2). In this paper, we describe the purification and preliminary charac- terization of phosphatidylserine synthase from wild-type strain S288C and from strain VALBC(YEpCHO1) which con- tains the structural gene for phosphatidylserine synthase on the YEpl3 plasmid (7). This is the first report of the purifi- cation of the membrane-associated phosphatidylserine syn- thase from a eukaryotic organism. Successful purification of phosphatidylserine synthase from S. cereuisiae required the solubilization of the enzyme from the microsomal fraction with detergent, CDP-diacylglycerol-Sepharose affinity chro- matography, and ion-exchange chromatography with DE-53. The availability of strain VAL2C(YEpCHO1) facilitated the acquisition of larger amounts of pure enzyme. Phosphatidyl- serine synthase required a 370-fold purification from strain VAL2C(YEpCHOl) compared to approximately a 2000-fold purification from strain S288C to attain similar final specific activities of pure enzyme with a subunit molecular weight of 23,000. These results further support that phosphatidylserine synthase is overproduced, as opposed to being activated, in strain VAL2C(YEpCHOl).

The enzymological properties of phosphatidylserine syn- thase purified from S. cereuisiae were similar to those reported for pure phosphatidylserine synthase from E. coli with respect to pH optimum, the dependence on Triton X-100 for maxi- mum activity, and kinetic properties (10, 13). On the other hand, there are differences between phosphatidylserine syn- thase from S. cereuisiae and phosphatidylserine synthase from E. coli. The subunit molecular weight of the yeast enzyme was 23,000 while the subunit molecular weight of the E. coli enzyme is 54,000 (10). At 30 "C, the turnover number (90

10862 Phosphatidylserine Synthase from S. cerevisiae

min-’) for phosphatidylserine synthase from yeast was about 18-fold lower than the turnover number (1650 min”) for the enzyme from E. coli (10). Phosphatidylserine synthase puri- fied from yeast required the addition of divalent metal ions for in vitro activity. In contrast, there is no cation requirement for the assay of phosphatidylserine synthase from E. coli (10, 28). There is evidence which indicates a probable difference in the reaction mechanism between phosphatidylserine syn- thase derived from S. cereuisiae and the enzyme from E. coli. Based on a series of exchange reactions catalyzed by phos- phatidylserine synthase from S. cerevisiae, the enzyme ap- pears to follow a sequential Bi Bi reaction mechanism. On the other hand, the enzyme from E. coli follows a ping-pong reaction mechanism (10, 29). An additional indication of differing reaction mechanisms between both enzymes is that phosphatidylserine synthase from S. cerevisiae binds to CDP- diacylglycerol-Sepharose, while the enzyme from E. coli does not bind to the affinity resin (10). Yeast phosphatidylserine synthase is sensitive to thioreactive agents while E. coli phos- phatidylserine synthase (partially purified) is not affected by 4 mM N-ethylmaleimide (9). The enzyme from yeast is an integral membrane protein (3, 6 ) while the enzyme from E. coli is associated with ribosomes in cell extracts (14). Phos- phatidylserine synthase from E. coli is not associated with ribosomes during catalysis (30) and is believed to be a periph- eral membrane protein (30, 31). The differences between the enzymes from yeast and from E. coli indicate that two distinct forms of phosphatidylserine synthase catalyze the same over- all reaction. Furthermore, the cloned phosphatidylserine syn- thase structural gene from E. coli (11, 12) shows no homology to the cloned CHOl gene from S. cerevisiae.’ Phosphatidyl- serine synthase is an integral membrane protein in Gram- positive bacteria (32-35) and has been partially purified from Clostridium perfringens (36). The intracellular location and catalytic properties of phosphatidylserine synthase from Gram-positive bacteria more closely resembles those of phos- phatidylserine synthase from S. cerevisiae than those of E. coli.

Higher eukaryotic organisms do not have phosphatidylser- ine synthase activity. Instead they synthesize phosphatidyl- serine by an exchange reaction between phosphatidylethanol- amine and free serine (28). The purified phosphatidylserine synthase from S. cerevisiae did not catalyze this base exchange reaction. The evolutionary significance of the loss of phos- phatidylserine synthase in animals and plants is unclear.

In summary, we have purified the membrane-bound form of phosphatidylserine synthase from S. cereuisiae and have studied its basic enzymological properties. A liposome system containing CDP-diacylglycerol in the absence of Triton X- 100 is being developed to study the kinetic and physical properties of the enzyme.

Acknowledgments-We are grateful to Susan A. Henry and Verity A. Letts for providing us with S. cereuisiae strain VAL2C(YEpCHOl). We thank Michael Homann, Margaret Poole, and Ruth Werner for their technical assistance and Rose Ann Ullrich for typing the man- uscript.

V. A. Letts, personal communication.

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