JOURNAL OF BACTERIOLOGY, Apr. 1983, p. 304-311 Vol. 154, No. 10021-9193/83/040304-08$02.00/0Copyright C 1983, American Society for Microbiology

Phosphatidylinositol Biosynthesis in Saccharomycescerevisiae: Purification and Properties of Microsome-

Associated Phosphatidylinositol SynthasetANTHONY S. FISCHL AND GEORGE M. CARMAN*

Department ofFood Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers, The StateUniversity, New Brunswick, New Jersey 08903

Received 28 October 1982/Accepted 3 January 1983

The membrane-associated phospholipid biosynthetic enzyme phosphatidylino-sitol synthase (cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phos-phatidyltransferase, EC 2.7.8.11) was purified 1,000-fold from the microsomalfraction of Saccharomyces cerevisiae. The purification procedure included TritonX-100 solubilization of the microsomal membranes, CDPdiacylglycerol-Sephar-ose (Larson et al., Biochemistry 15:974-979, 1976) affinity chromatography, andchromatofocusing. The procedure resulted in the isolation of a nearly homoge-neous protein preparation with an apparent minimum subunit molecular weight of34,000, as determined by polyacrylamide gel electrophoresis in the presence ofsodium dodecyl sulfate. Phosphatidylinositol synthase was dependent on manga-nese and Triton X-100 for maximum activity. The pH optimum was 8.0.Thioreactive agents inhibited enzyme activity. The energy of activation was foundto be 35 kcal/mol (146,540 J/mol). The enzyme was reasonably stable at tempera-tures of up to 60°C.

Phosphatidylinositol (PI) has been shown to regulation and the kinetics of PI synthase inhave considerable importance in the growth of well-defined systems have been hampered bySaccharomyces cerevisiae. When inositol-re- difficulty in purifying this enzyme from yeastquiring strains of S. cerevisiae are deprived of membrane preparations. PI synthase has beeninositol, the cells undergo drastic changes in the solubilized with either the nonionic detergentmetabolism of lipids, carbohydrates, proteins, Renex 690 or Triton X-100 in reasonably highand nucleic acids, ultimately resulting in a loss yields with good stability (8). However, furtherof cell viability (1, 15). The requirement for purification of the enzyme by chromatographyinositol arises because membranes do not func- with hydroxyapatite, DEAE-cellulose, carboxy-tion correctly if they are deficient in inositol- methyl cellulose, or phosphocellulose was un-containing lipids (23). Besides being the third successful (8). A major problem in the purifica-major phospholipid component of yeast mem- tion of some membrane-associated enzymes isbranes (1), PI serves as a precursor in the that after solubilization, they tend to bind largesynthesis of other inositol-containing lipids, in- quantities of detergents. This binding masks thecluding di- and triphosphoinositides (21) and enzyme hydrodynamic and electrostatic proper-several sphingolipids (2). PI may also play a ties, the manifestation of which is essential fordirect role in the synthesis of yeast glycans (15). purification by classical techniques (16). AffinityThe enzyme responsible for the biosynthesis chromatography has proven useful for the purifi-

of PI is PI synthase (CDP-1,2-diacyl-sn-glycerol: cation of some membrane-associated enzymesmyo-inositol 3-phosphatidyltransferase, EC solubilized with nonionic detergents. The pres-2.7.8.11). PI synthase catalyzes the formation of ence of the nonionic detergents Used in solubiliz-PI and CMP from CDPdiacylglycerol and myo- ing these enzymes does not interfere with chro-inositol (25). PI synthase activity was first iden- matography since these detergents are requiredtified from the total particulate fraction of S. for enzymatic activity. A CDPdiacylglycerol-cerevisiae sedimenting between 1,500 and 'Sepharose affinity resin has been synthesized90,000 x g (26). The enzyme has been localized (20) and used to purify the CDPdiacylglycerol-in both mitochondrial and microsomal fractions dependent enzymes phosphatidylglycerophos-(8, 9) of the cell. Studies concerning the mode of phate synthase from Bacillus licheniformis (20)

and Escherichia coli (17) and phosphatidylserinet New Jersey Agricultural Experiment Station publication synthase from Clostridium perfringens (11) in

no. D-10517-1-82. the presence of Triton X-100. We were able to304

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PI SYNTHASE FROM S. CEREVISIAE 305

use the CDPdiacylglycerol-Sepharose affinityresin to purify a Triton X-100-solubilized prepa-ration of PI synthase from S. cerevisiae. Wereport details of the purification and describesome properties of yeast PI synthase.

MATERILS AND METHODSMaterials. All chemicals were reagent grade. Phos-

pholipids, myo-inositol, bovine serum albumin, and p-chloromercuriphenylsulfonic acid were purchasedfrom Sigma Chemical Co. Triton X-100 (octylphe-noxypolyethoxyethanol) was from Rohm and HaasCo. Radiochemicals were purchased from New En-gland Nuclear Corp. Sepharose 4B, Sephadex G-25,and chromotofocusing resin PBE 94 were purchasedfrom Pharmacia Fine Chemicals. Precoated silica gelanalytical thin-layer chromatography plates were ob-tained from E. Merck. Yeast extract and peptone werepurchased from Difco Laboratories. Electrophoresisreagents and molecular weight standards were ob-tained from Bio-Rad Laboratories.

Preparation of substmates. Soybean lecithin-derivedCDPdiacylglycerol was prepared by the method ofCarman and Fischl (7). CDPdiacylglycerol tritiated inthe cytidine moiety was prepared by reaction of soy-bean-derived phosphatidic acid (7) and [5-3H]CTPcatalyzed by yeast mitochondrial CTP:phosphatidicacid cytidylyltransferase as described by Belendiuk etal. (3).

Preparation of CDPdiacylglycerol-Sepharose affintyresin. The NaIO4-oxidized derivative of CDPdiacyl-glycerol was covalently attached to Sepharose 4B viaan adipic acid dihydrazide spacer arm as described byLarson et al (20). The procedure was modified byactivating Sepharose 4B with cyanogen bromide inacetonitrile as described by March et al (22). Inaddition, the incubation time for coupling the adipicacid spacer arm to activated Sepharose was increasedfrom 1 to 2 days, the time for the NaIO4 oxidation ofCDPdiacylglycerol was increased from 1 to 2 days,and the incubation time for coupling the oxidizedderivative of CDPdiacylglycerol to the adipic acid-Sepharose resin was increased from 1 to 3 days. Theseminor modifications of the Larson et al. (20) methodfor preparing the resin increased the concentration ofthe bound, oxidized CDPdiacylglycerol derivativefrom about 2 to 30 ,umol of ligand per ml of resin.Growth conditons. A culture of S. cerevisiae S288C

(a gal2) was grown at 28°C in 30 liters of mediumcontaining 2% peptone, 2% glucose, and 1% yeastextract in a 50-liter fermentor (New Brunswick Scien-tific Co.). Air at a rate of 15 liters/min was passedthrough the culture, which was stirred at 320 rpm. Theculture was grown to a stationary-phase-equivalentdensity of 40 mg (wet cell weight) per ml. Cells wereharvested by centrifugation in a Sharples AS-16 Super-centrifuge at 13,000 x g at a flow rate of 4 liters/min.The cell paste was frozen and stored at -80°C.

Purification of PI synthase. All steps for PI synthasepurification were carried out at 5°C.

(i) Preparation of microsomes. Frozen cells (100 g)were washed in 50 mM Tris-hydrochloride buffer (pH7.5) containing 1 mM Na2EDTA, 0.3 M sucrose, and10 mM 2-mercaptoethanol and were resuspended in100 ml of the same buffer. The cell suspension wasmixed with 300 g of prechilled glass beads (diameter,

0.3 to 0.5 mm) and disrupted by homogenization in aBead-Beater (Biospec Products) for five 1-min bursts,with a 4-min pause between bursts. The homogenatewas brought to a final volume of 300 ml and thencentrifuged at 1,500 x g for 10 min in an SS-34 rotor.The supernatant fraction was then centrifuged at27,000 x g for 10 min in an SS-34 rotor. The superna-tant fraction was saved. The pellet was resuspended in200 ml of the same buffer and recentrifuged at 27,000 xg for 10 min. The pellet was discarded, and thesupernatant was combined with the first 27,000 x gsupernatant. This material was then centrifuged at100,000 x g for 2 h in a 42.1 rotor to obtain themicrosomal fraction. The microsomes were washedwith 100 ml of 50 mM Tris-hydrochloride buffer (pH7.5) containing 10 mM 2-mercaptoethanol and 2Qoglycerol (wt/vol) and resuspended in 30 ml of the samebuffer.

(U) Preparation of Triton X-100 esact. The micro-somal fraction was suspended in solubilization buffer(50 mM Tris-hydrochloride buffer [pH 8.0], 30 mMMgCl2, 10 mM 2-mercaptoethanol, 20o glycerol [wt/vol]) plus 1% Triton X-100 (wtlvol) at a final proteinconcentration of 10 mg/ml. After incubation for 1 h at5°C, the suspension was centrifugated at 100,000 x gfor 2 h to obtain the solubilized (supernatant) fraction.

(iii) CDPdiacylglycerol-Sephre chromatography.CDPdiacylglycerol-Sepharose resin (25 ml) was equili-brated with 12 volumes of solubilization buffer con-taining 1% Triton X-100. The Triton X-100 extract(containing 1,500 U of PI synthase activity) was mixedin batches with the affinity resin and allowed to reactfor 1 h with shaking. This mixture was poured into acolumn (1.5 by 14 cm); the run-through material wasrecirculated through the column. The affinity columnwas washed with 20 column volumes of solubilizationbuffer containing 0.5% Triton X-100 and 0.5 M NH4Cl.The column was then saturated with solubilizationbuffer containing 0.5% Triton X-100, 0.65 mMCDPdiacylglycerol, and 0.8 M hydroxylamine-HCland allowed to react for 1 h. PI synthase was elutedfrom the resin with 2 column volumes of the buffer at aflow rate of 20 ml/h. Fractions (10 ml) were assayed forPI synthase activity. Fractions containing PI synthaseactivity were pooled and desalted on a Sephadex G-25column equilibrated with 20 mM Tris-hydrochloridebuffer (pH 7.4) containing 1 mM dithiothreitol, 20%oglycerol, and 0.5% Triton X-100.

(iv) Chromatofocusing. A column (0.9 by 24 cm) ofchromatofocusing resin (PBE 94) was equilibratedwith 20 mM Tris-hydrochloride buffer (pH 7.4) con-taining 1 mM dithiothreitol, 20% glycerol, and 0.5%Triton X-100. The Sephadex G-25 effluent (containing1,200 U of PI synthase activity) was applied to thechromatofocusing column at a flow rate of 20 ml/h.The column was then washed with 2 column volumesof equilibration buffer. Fractions (3.0 ml) were collect-ed and assayed for PI synthase activity. The enzymedid not bind to the resin and emerged in the run-through and wash fractions. Active fractions werepooled and concentrated with an Amicon ultrafiltra-tion device equipped with a PM 10 filter. Samples ofthe purified enzyme were stored at -80°C for laterstudies.Enzyme assay. PI synthase activity was measured at

30°C for 20 min, as described by Carman and Felder(6), by monitoring the incorporation of 0.5 mM myo-[2-

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3H]inositol (20,000 dpm/nmol) into chloroform-solublematerial in the presence of 50 mM Tris-hydrochloridebuffer (pH 8.0), 2 mM MnCl2, 0.2 mM CDPdiacylglyc-erol, 1.6 mM Triton X-100, and enzyme protein in atotal volume of 0.1 ml. Alternatively, PI synthaseactivity was measured by monitoring the release ofwater-soluble radioactive CMP from [5-3H]CDPdi-acylglycerol (1,200 dpm/nmol), as described by Raetzet al. (27), with nonradioactive inositol. A unit ofenzymatic activity was defined -as the amount ofenzyme that catalyzed the formation of 1 nmol ofproduct per min under the assay conditions describedabove. The specific activity was defined as the unitsper milligram of protein. The phospholipid product ofthe reaction, PI, was identified by chromatography onactivated (110°C, 30 min) silica gel thin-layer plates.Radioactive profiles on thin-layer plates were deter-mined by counting 1-cm strips in scintillation fluid asdescribed by Carman and Felder (6). Standard PI wasvisualized with phosphate spray reagent (12). Theradioactive product cochromatographed with standardPI had an Rf of 0.12 in a solvent system containingchloroform-acetone-methanol-glacial acetic acid-wa-ter (50:20:10:10:5, vol/vol) and an Rf of 0.49 in asolvent system containing chloroform-methanol-gla-cial acetic acid-water (50:28:4:8, vol/vol). Alternative-ly, the reaction product CMP was identified by paperchromatography on Whatman no. 1 paper. Radioac-tive profiles on Whatman no. 1 paper were determinedby counting 1-cm strips in scintillation fluid as de-scribed by Raetz et al (27). Standard CMP was locatedby its absorbance under UV light. The radioactiveproduct cochromatographed with standard CMP hadan Rf of 0.33 in a solvent system containing 95%ethanol-1 M ammonium acetate (pH 7.4) (7:3, vol/vol)and an Rf of 0.90 in a solvent system containingsaturated ammonium sulfate in 0.1 M potassium phos-phate (pH 6.8)-isopropanol (100:2, vol/vol). PI syn-thase activity was linear with time and protein concen-tration under the assay conditions when measured bymonitoring the incorporation of myo-[2-3H]inositolinto radioactive PI or the release of radioactive CMPfrom [5-3H]CDPdiacylglycerol. The activity of PI syn-thase measured by either method was essentially iden-tical. Activity was routinely measured by monitoringthe incorporation of radioactive inositol into chloro-form-soluble product.

SDS-polyacrylamide gel electrophoresis. Electropho-resis in the presence of sodium dodecyl sulfate (SDS)was carried out as described by Laemmli (18) with anacrylamide/N,N'-methylenebisacrylamide ratio of36.5:1. Samples for electrophoresis were first treatedat 100°C for 90 s in buffer containing 62.5 mM Tris-hydrochloride (pH 6.8), 2% SDS, 10%o glycerol, 5% 2-mercaptoethanol, and 8 M urea. Electrophoresis wascarried out in a buffer consisting of 0.25 M Tris-hydrochloride (pH 8.3), 0.192 M glycine, and 0.1%SDS at 30 mA for 4 h with a model SE-600 vertical slabgel electrophoresis unit with a PS-500 power supply(Hoefer Scientific Instruments, Inc.). Gels werestained in 0.125% Coomassie blue and destained in a7% acetic acid-5% methanol solution. Protein molecu-lar weight standards were phosphorylase B (92,500),bovine serum albumin (66,200), ovalbumin (45,000),carbonic anhydrase (31,000), soybean trypsin inhibitor(21,500), and lysozyme (14,400).

Protein determination. Protein was determined bythe Coomassie blue dye binding method of Bradford(4), with bovine serum albumin as the standard.Buffers which were identical to those containing theprotein samples were used as blanks. The presence ofTriton X-100 did not interfere with the protein determi-nation, provided the blank contained a final concentra-tion of detergent identical to that of the sample.

RESULTSPurification of PI synthase. The CDPdiacyl-

glycerol-dependent enzymes identified in S.cerevisiae are PI synthase, phosphatidylserinesynthase, and phosphatidylglycerophosphatesynthase (26). PI synthase and phosphatidylser-ine synthase are localized in both the mitochon-drial and the microsomal fractions (8, 9), where-as phosphatidylglycerophosphate synthase islocalized in the mitochondrial fraction (9). Toeliminate phosphatidylglycerophosphate syn-thase during the purification of PI synthase, weused microsomes for the starting material. Themicrosomal fraction was treated with solubiliza-tion buffer containing 1% Triton X-100 to releasePI synthase activity from the membranes. Theyield of solubilized enzyme was 75%, with anincrease in specific activity of 1.5-fold. Underthe solubilization conditions reported, the mi-crosome-associated phosphatidylserine syn-thase was not solubilized. However, if 2 mMMnCl2 was substituted for MgCl2 in the solubili-zation buffer, both PI synthase and phosphati-dylserine synthase activities were released fromthe microsomal membranes (8). The presence ofmanganese in the solubilization buffer is alsorequired for the release of phosphatidylserinesynthase from the cell envelope fraction of C.perfringens (10). The major purification of PIsynthase was achieved by affinity chromatogra-phy with the CDPdiacylglycerol-Sepharose resindeveloped by Larson et al. (20). Binding of thePI synthase to the resin was dependent on thepresence of the enzyme cofactor (magnesium ormanganese) and the presence of at least 0.5 to1% Triton X-100. It was necessary to allow theenzyme to interact with the resin for at least 30min to achieve binding at 5°C. Under theseconditions, 100% of the applied units of PIsynthase activity in the Triton X-100 extractbound to the CDPdiacylglycerol-Sepharose res-in, whereas over 99% of the applied protein wasrecovered in the run-through and wash frac-tions. A typical elution profile for PI synthasefrom an affinity resin is shown in Fig. 1. Elutionof the enzyme from the resin was dependent onboth the substrate CDPdiacylglycerol and thehydroxylamine-HCl in the elution buffer. In theabsence of either of these components, enzymedissociation from the column was negligible.Salts, such as NaCl, at an equivalent ionic

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PI SYNTHASE FROM S. CEREVISIAE 307

275

c

0

I-

.C00

100

75

50

25

Fraction Number

FIG. 1. Elution profile of PI synthase fromCDPdiacylglycerol-Sepharose. Fractions (10 ml) werecollected and assayed for PI synthase activity asdescribed in the text. , Point at which elution bufferwas applied.

strength could substitute for hydroxylamine-HCl in the elution buffer. Under the elutionconditions reported here, 80%o of the bound PIsynthase activity was recovered from the resinwith elution buffer. Fractions containing PI syn-thase activity were pooled and passed throughSephadex G-25 to remove salt and to equilibratethe enzyme preparation for chromatofocusing.The affinity chromatography step resulted in a400-fold increase in specific activity over theactivity in the Triton X-100 extract. The enzymepreparation after the affinity chromatographystep showed one major protein band and severalminor protein bands after SDS-polyacrylamidegel electrophoresis. Most of the minor proteinswere removed from the preparation by passingthe eznyme through the chromatofocusing col-umn at pH 7.4. PI synthase did not bind to theresin, whereas the minor proteins apparently

did. The overall purification of PI synthase overthe microsomal fraction was 1,000-fold, with anactivity yield of 60%. A summary of the purifica-tion of the enzyme is presented in Table 1.The PI synthase preparation did not containCDPdiacylglycerol-dependent phosphatidylser-ine synthase nor phosphatidylglycerophosphatesynthase activities, which bind to the CDPdia-cylglycerol-Sepharose resin (M. Lee-Bae and G.M. Carman, unpublished data). When a sampleof the PI synthase preparation was subjected toSDS-polyacrylamide gel electrophoresis, onemajor band was evident with an apparent mini-mum subunit molecular weight of 34,000 (Fig.2). Purified PI synthase activity, as well as itsassociated protein, did not enter 5% polyacryl-amide gels under nondenaturing conditions. Thiswas probably due to the association of theenzyme with a Triton X-100 micelle, resulting ina high size-to-charge ratio for the mixed micellecomplex (17). In addition, when a sample of thepurified enzyme was applied to a Sepharose 4Bgel filtration column, the enzyme emerged in thevoid volume, presumably associated with amixed micelle containing Triton X-100. The mo-lecular weight of the native enzyme in Triton X-100 cannot be determined until the interactionwith detergent has been quantitated (29). Wecannot unequivocally state that the major pro-tein purified with the subunit molecular weightof 34,000 is PI synthase. Unequivocal evidencewould come from the identification of an in vitrotranslation product from the structural gene ofPI synthase with PI synthase activity which hasa subunit molecular weight of 34,000. Suchevidence awaits the cloning of the PI synthasegene.

Properties of PI synthase. PI synthase activitywas measured as a function of pH. 2-(N-Mor-pholino)ethanesulfonic acid buffer was used forpH 5.0 to 7.0, and Tris buffer was used for pH7.0 to 10.0. Optimal activity of the enzyme wasobtained at pH 8.0.

PI synthase activity was measured in theabsence and presence of divalent cations. Theenzyme had an absolute requirement for a metal

TABLE 1. Purification of PI synthasea

Total Total Sp act (nmol/min Yield PurificationPurification step units protein per mg of protein) (%) (fold)(mg)

(i) Microsomes 2,000 2,500 0.8 100 1(ii) Triton X-100 extract 1,500 1,250 1.2 75 1.5(iii) CDP diacylglycerol- 1,200 2.5 480 60 600

Sepharose(iv) Chromatofocusing 1,200 1.5 800 60 1,000

run-through

a Data are based on starting with 100 g (wet weight) of yeast paste.

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308 FISCHL AND CARMAN

1 2E0

0

92.5 K _U

66.2 K .... ...

-4.

45.Oi K

31.0 K

21.5K -

14.4 KFIG. 2. SDS-polyacrylamide gel electrophoresis.

Twenty micrograms of standard-molecular-weightproteins (lane 1) and 4 ,ug of the chromatofocusingeluant (lane 2) were applied to the stacking gel andsubjected to electrophoresis as described in the text.

Manganese or Magnesium, mMFIG. 3. Effect of manganese and magnesium on PI

synthase activity. A preparation of PI synthase wasdesalted by Sephadex G-25 chromatography. The en-zyme (0.015 U) was assayed with the indicated con-centrations of MnCl2 (0) or MgCl2 (0). Activity wasmeasured as described in the text.

chloromecuriphenylsulfonic acid. Zn2+, Cd2+,and Hg2+ inhibited PI synthase activity from 95to 97%. The concentration of p-chloromecuri-phenylsulfonic acid required to inhibit 50% ofthe PI synthase activity was 10 p.M. Theseresults suggest that a sulphydryl group is essen-tial for activity.

PI synthase activity was measured from 5 to60° for 20 min in a controlled-temperature waterbath (Fig. 5A). Maximum activity was observedwhen the enzyme was assayed at 35°C. AnArrhenius plot for PI synthase (Fig. SB) wasconstructed using the values in Fig. 5A from 5 to35°C. The activation energy was 35 kcal/mol(146,540 J/mol). PI synthase was examined for

cation (either Mn2+ or Mg2+) for activity (Fig.3). The maximum activity obtained with MnCl2(2 mM) showed a 5.4-fold-greater stimulation ofactivity over that obtained with MgCl2 (20 mM).The effect of the nonionic detergent Triton X-

100 on PI synthase activity is shown in Fig. 4.The addition of the detergent to the assay stimu-lated PI synthase activity to a maximum at 2.4mM followed by a gradual decrease in activity athigher concentrations. The maximum activityobtained corresponded to a molar ratio of TritonX-100-to-CDPdiacylglycerol of 12:1. The appar-ent inhibition of PI synthase activity is presum-ably due to the dilution of the lipid substrate inthe mixed micelle of Triton X-100 and CDPdi-acylglycerol (5).

PI synthase activity was measured under stan-dard assay conditions in the presence of thio-reactive agents. At a concentration of 1 mM, p-

4

0

40

2

1

5 10 15 20 25

Triton X100, mMFIG. 4. Effect-ofTriton X-100 on PI synthase activ-

ity. PI synthase activity (0.035 U) was measured with0.2 mM CDPdiacylglycerol at the indicated concentra-tions of Triton X-100. Activity was measured as de-scribed in the text.

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PI SYNTHASE FROM S. CEREVISIAE 309

1.6E

4d

0

CL

1.21

0.81

0.4

ec 0

0 -10

0

L -2.0o0

-i

I I I I I IA

I I I I I

10 30 50Temperature, °C

BEa= 35 Kcal / mole

I I a I

3.3 3.4 3.5 3.6

. X 10

FIG. 5. Effect of temperature on PI synthase activ-ity. (A) PI synthase activity (0.019 U) was measured atthe indicated temperatures for 20 min under standardassay conditions in a controlled-temperature waterbath. (B) The Arrhenius plot was constructed from thevalues in (A) from 5 to 35C. Ea, Energy of activation.

its stability to temperature. Samples of PI syn-thase (containing 0.02 U) were incubated for 10min at 5 to 70°C in a controlled-temperaturewater bath. After incubation, the samples wereplaced on ice, assay components were added,and PI synthase activity was measured at 30°C.The enzyme was essentially 1O0o stable for atleast 10 min at temperatures ranging from 5 to50°C, with an inactivation of about 35% at 60°Cand 100%o at 70°C. The purified enzyme wascompletely stable for at least 6 months at -80°C.Activity was also stable for at least four cyclesof freezing and thawing.

DISCUSSIONPI synthase catalyzes the formation of PI, a

membrane phospholipid essential for the growthof S. cerevisiae (1) and other eukaryotic organ-isms (23). The phospholipid biosynthetic en-zyme PI synthase is difficult to purify, owing toits membrane-bound nature and the presence ofits activity in low levels. Successful purificationof PI synthase from S. cerevisiae requires thesolubilization of the enzyme from the mem-

branes with detergent and a purification proce-dure not complicated by the presence of adetergent. Purification of the Triton X-100-solu-bilized PI synthase is facilitated by the use of theCDPdiacylglycerol-Sepharose affinity resin de-veloped by Larson et al. (20) for the purificationof phosphatidylglycerophosphate synthase frombacteria. Binding of PI synthase to the CDPdia-cylglycerol-Sepharose resin is dependent on thepresence of Triton X-100 and cofactors in thechromatography buffer. Triton X-100 and mag-nesium or manganese are required for in vitroactivity of PI synthase (8), and their presence inthe buffer presumably increases enzyme bindingto the ligand of the affinity resin. The affinityresin was initially prepared as described byLarson et al. (20). PI synthase would bind to theresin and could be eluted from the resin with thechromatography buffer containing CDPdiadyl-glycerol in a broad peak of activity (data notshown). When the procedure to prepare theresin was modified to increase the binding ca-pacity of the resin (see above), the PI synthasecould no longer be eluted from the affinitycolumn with CDPdiacylglycerol in chromatogra-phy buffer. Hirabayashi et al. (17) also foundthat the phosphatidylglycerophosphate synthaseof E. coli could not be eluted from a CDPdia-cylglycerol-Sepharose resin with CDPdiacylgly-cerol in chromatography buffer. Elution of theE. coli enzyme from the affinity resin wasachieved with chromatography buffer containing0.8 M hydroxylamine-HCl. On the other hand,the E. coli phosphatidylglycerophosphate syn-thase could be eluted from the resin with chro-matography buffer containing CDPdiacylgly-cerol, if the affinity resin were reduced withNaBH4 as a last step in the preparation of theresin (17). It is possible that the NaBH4 treat-ment reduces the binding-effectiveness of theresin by destroying sonie CDPdiacylglycerolbinding sites, resulting in a lower-capacity resinequivalent to the resin we initially prepared asdescribed by Larson et al. (20). Larson et al. (20)were able to elute the B. licheniformis phospha-tidylglycerophosphate synthase from a lower-capacity affinity resin with buffer containingCDPdiacylglycerol. We found that the yeast PIsynthase could be eluted from the higher-capaci-ty affinity resin in a sharp peak of activity withbuffer containing both CDPdiacylglycerol and0.8 M hydroxylamine-HCl.The CDPdiacylglycerol-Sepharose affinity

resin (20) has been a valuable tool for thepurification of CDPdiacylglycerol-dependent en-zymes in the presence of the nonionic detergentTriton X-100. These enzymes include phosphati-dylglycerophosphate synthase from B. licheni-formis (20) and E. coli (17), phosphatidylserinesynthase from C. perfringens (11) and B. licheni-

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formis (A. Dutt and W. Dowhan, personal com-munication), and now PI synthase from S.cerevisiae.The only other phospholipid biosynthetic en-

zyme that has been purified from S. cerevisiae isthe mitochondrial-associated CTP:phosphatidicacid cytidylyltransferase (3). This enzyme waspurified about 100-fold over mitochondrial mem-branes and 500-fold over the cell extract. Thespecific activity of the purified CTP:phosphati-dic acid cytidylyltransferase was 450 nmol/minper mg. This value is similar to the specificactivity of 800 nmol/min per mg for purified PIsynthase from yeast microsomes. The specificactivities of the purified phospholipid biosyn-thetic enzymes from S. cerevisiae are about 15-to 100-fold lower than the specific activities ofpurified phospholipid biosynthetic enzymesfrom E. coli (13, 14, 17, 19). In addition, about4000- to 6000-fold purifications were required topurify the enzymes from wild-type E. coli tonear homogeneity (13, 17, 19). Purification ofmilligram quantities of phospholipid biosynthet-ic enzymes from E. coli has been facilitated bythe cloning of genes on plasmids directing theoverproduction of enzyme activities (14, 24, 28).It is clear that the purification of large quantitiesof yeast PI synthase will require the cloning ofthe enzyme structural gene on a replicatingplasmid which directs enzyme overproduction.The basic enzymological properties of yeast

PI synthase are similar to those previously re-ported for the solubilized preparation of theenzyme (8).The availability of the purified preparation of

PI synthase will allow the study of the enzymeunder well-defined conditions. We are develop-ing a liposome system containing CDPdiacylgly-cerol in the absence of detergent to study kineticand physical properties of the enzyme.

ACKNOWLEDMENTISThis work was supported by state funds, U.S. Hatch Act

R.R.F., Public Health Service grant GM-28140 from theNational Institutes of Health, Universal Foods, and theCharles and Johanna Busch Memorial Fund.We thank William Dowhan for his helpful discussions. We

also thank Rose Ann Ullrich for typing the manuscript.

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