The reconstituted enzyme was used as a model system to study the regulation of activity. .... 1 nmol of product/min under the assay conditions described. The specific activity ... conditions of the experiment, the vesicles maintained their integrity (no .... bind to CDP-DG before inositol in the forward reaction, since the enzyme ...
THE JOURNAL OP BIOLOCK!AL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.
Phosphatidylinositol RECONSTITUTION,
Vol. 261, No. 7, Issue of March 5, pp. 317%3183,1986 Printed in U.S.A.
Synthase from Succharomyces
CHARACTERIZATION,
AND
REGULATION
cerevisiae
OF ACTIVITY* (Received for publication,
June 10, 1985)
Anthony S. FischlS, Michael J. Homann, Margaret A. Poole, and George M. Carmane From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08903
Purified membrane-associated phosphatidylinositol synthase (CDPdiacylglycerol:myo-inositol3-phosphatidyltransferase, EC 2.7.8.11) from Saccharomyces cerevisiae was reconstituted into unilamellar phospholipid vesicles. Reconstitution of the enzyme was performed by removing detergent from an octylglucosidej phospholipid/Triton X- loo/enzyme mixed micelle mixture by Sephadex G-50 superfine column chromatography. The average diameter of the vesicles was 40 nm and chymotrypsin treatment of intact vesicles indicated that over 90% of the reconstituted enzyme had its active site facing outward. The enzymological properties and reaction mechanism of reconstituted phosphatidylinositol synthase were determined in the absence of detergent. The reconstituted enzyme was used as a model system to study the regulation of activity. Phosphatidylinositol synthase was constitutive in wil,d type cells grown in the presence of water-soluble phospholipid precursors as determined by. enzyme activity and immunoblotting. Reconstituted enzyme was not effected by water-soluble phospholipid precursors or nucleotides. Maximum activity was found when the enzyme was reconstituted into phosphatidylcholine: phosphatidylethanolamine:phosphatidylinositol:phosphatidylserine vesicles. Phosphatidylserine stimulated reconstituted activity, suggesting that the local phospholipid environment may regulate phosphatidylinositol synthase activity. Phosphatidylinositol (PI’) is the third major phospholipid in Saccharomyces cereuisiue membranes (1) and is essential for the growth and metabolism of the organism (2-4). PI is the precursor of the polyphosphoinositides, PI 4-phosphate and PI 4,5-diphosphate (5), a precursor of sphingolipids (6), and is required for the synthesis of cell wall glycans (7). The enzyme responsible for the synthesis of PI is PI synthase
(CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11). PI synthase catalyzes the formation of PI and CMP from CDPdiacylglycerol (CDP-DG) and myo-inositol (8). We have purified PI synthase to near homogeneity from S. cereuisiae (9) using CDP-DG-Sepharose affinity chromatography (10). The enzyme has an apparent minimum molecular weight of 34,000 as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and electroblotting (9, 11). PI is an essential membrane phospholipid in S. cereuisiae and the levels of this phospholipid may fluctuate in various strains (1, 12). When wild type strains are grown in synthetic medium supplemented with inositol, the level of PI increases about l&fold, whereas the level of phosphatidylserine (PS) decreases l&fold (13, 14). The relative composition of the other major phospholipids do not change significantly when wild type cells are grown in the presence of inositol (13, 14). In this study we examined whether the fluctuation in PI synthesis was the result of an alteration in enzyme synthesis or a regulation of enzyme activity. Enzyme formation was analyzed using antiserum directed against purified PI synthase, whereas enzyme activity was studied using pure enzyme reconstituted into phospholipid vesicles. EXPERIMENTAL
Materials-All serine,
3178
ethanolamine,
choline,
CDP-ethanolamine,
CDP-choline,
L-
nu-
cleotides, bovine serum albumin, chymotrypsin, egg phosphatidylcholine (PC), egg phosphatidylethanolamine (PE), soybean PI, PI 4phosphate, PI 4,5-diphosphate, bovine brain PS, and dipalmitoylphosphatidic acid (PA) were purchased from Sigma. Triton X-100 (octylphenoxypolyethoxyethanol) was a gift from Rohm and Haas Co. mvo-12-3H11nositol. L-lmethvl-3H1PC. and L-3-DhosuhatidvllZ[3H]Triton X-i00 were.pu&hased from New England Nuclear. iir-‘[l“C]Octyl-/3-n-glucopyranoside was purchased from Research Products International Corp. ]5-3H]CMP was purchased from Schwarz/
Mann. Cctyl-P-n-glucopyranoaide (octylglucoside), 5,5’-dithiobis-(2nitrobenzoic acid) (DTNB). elutathione. and egg lecithin were purchased
*This work was supported by New Jersey State 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-10531-285. 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 thjs fact. $ Present address: Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115. $ To whom reprint requests should be addressed at: the Dept. of Food Science, Cook College, Rutgers University, P. 0. Box 231, New Brunswick, NJ 08903. 1 The abbreviations used are: PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PA, dipalmitoylphosphatidic acid, DTNB, 5,5’-dithiobis-(2nitrobenzoic acid); octylglucoside, octyl-p-n-glycopyranoside; MES, 4-morpholineethanesulfonic acid.
PROCEDURES
chemicals were reagent grade. myo-Inositol,
from
Calbiochem-Behring.
Sephadex
G-50
phacryl S-1000 were purchased from Pharmacia.
superfine
Molecular
and Se-
weight
standards and electrophoresis and immunoblotting reagents were obtained from Bio-Rad. Unmodified nitrocellulose paper was obtained from Schleicher & Schuell. Eag lecithin-derived CDP-DG was prepared by the method of Carman and Fischl(15). CDP-DG tritiated in the cytidine moiety was prepared by reaction of egg-derived PA (15) and [5-3H]CTP catalyzed by yeast mitochondrial CDP-DG synthase (16). Yeast Strains and Growth Conditions-Wild type strain S288C (crgal2) was used for the purification of PI synthase. Cells were grown in YEPD medium (1% yeast extract, 2% peptone, and 2% glucose) at 28 “C to late exponential phase, harvested by centrifugation, and stored at -80 “C (9). Strain ode&, which shows normal regulation of phospholipid biosynthesis (13, 14), was used in growth studies. Cells were grown at 28 “C in 100 ml of complete synthetic medium (14) containing inositol (50 PM), ethanolamine (1 mM), and choline (1 mM) where indicated. Cells were grown to late exponential phase and harvested by centrifugation (14).
PhosphatidylinositolSynthase from S. cerevisiae Preparation of Cell-free Extracts and Purification of PI 8 y n t h e Cells were disrupted with glass beads in 50 mM Tris-HC1 buffer (pH 7.5) containing 1 mM disodium EDTA, 0.3 M sucrose, and 10 mM 2mercaptoethanol (14). Glass beads and unbroken cells were removed by centrifugation a t 1500 X g for 5 min. The supernatant (cell-free extract) was used for enzyme assays and immunoblotting. PI synthase was purified to near homogeneity from microsomes by Triton X-100 extraction, CDP-DG-Sepharose affinity chromatography, and chromatofocusing as described by Fischl and Carman (9). Preparation of Antibody against PI Synthase-Anti-PI synthase was raised in New Zealand White rabbits immunized with purified PI synthase (150 pg/injection) according to standard procedures in the laboratory of Susan A. Henry (Albert Einstein College of Medicine, Bronx, NY). Triton X-100 was removed from the purified enzyme with Bio-Beads SM-2 (17) prior to injection with adjuvant. The titer of antiserum was determined by measuring PI synthase activity remaining after treating Triton X-100-solubilized extracts (18) with immunizedrabbit antiserumand protein A (19). Preimmune serum was used as a controlfor nonspecific effects during the enzyme assay and immunoprecipitation. EZectrophoresis and Zmmunoblotting-Samples of cell-free extract (100 pg) and pure PI synthase were subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis with 10% gels (20). Molecular weights were calculated from the mobilities of the following standards: phosphorylase b (Mr= 92,500),bovine serum albumin (M, = 66,200), ovalbumin ( M , = 45,000), carbonic anhydrase ( M , = 31,000), soybean trypsin inhibitor (M, = 21,500), and lysozyme (Mr = 14,400). Proteins were then transferred electrophoretically to 0.2 pm unmodified nitrocellulose paper as described by Burnette (21) with the modifications of Haid and Suissa (22). The transfer was run for 1 h at 100 V using a Hoefer T E 52 Transphor unit. The nitrocellulose paper was then probed with a 1:50 dilution of PI synthase antiserum as described by Burnette (21) using non-fat dry milk as a blocking agent (23). The paper was then incubated with goat antirabbit IgG conjugated to horseradish peroxidase followed by development with chloronaphthol and Hz02 (24). Reconstitution of PI Synthase-PI synthase was reconstituted in phospholipid vesicles by a method modeled after those described by Mimms et al. (25) and Green and Bell (26). Highly purified phospholipids (25 mg) in chloroform/methanol (9:1, v/v) were transferred to a test tube and solvent was evaporated under a stream of nitrogen. Residual solvent was removed in uucuo for 2 h. Dried phospholipids were suspended in 1 ml of 750 mM octylglucoside. A 0.10-ml sample of purified P I synthase (specific activity of 800-900 units/mg) in 50 mM Tris-HC1 (pH 8.0), 7.8 mM Triton X-100, 10 mM 2-mercaptoethanol, and 10% glycerolwasmixed with 0.1 ml of octylglucosidesolubilized phospholipids and 0.25 pmol of CDP-DG. The mixture was then diluted to a final volume of 0.5 ml with 50 mM Tris-HC1 (pH 7.5). The final concentrations of Triton X-100, octylglucoside, and phospholipids in this reconstitution mixture were 2.0mM, 150 mM, and 5.5 mM, respectively. The final molar ratios of octylglucoside to Triton X-100 and octylglucoside to phospholipids were 75:l and 27:1, respectively. A small portion of the reconstitution mixture was assayed for PI synthase activity in the absence of added detergent. The remainder was immediately passed through a Sephadex G-50 superfine column (1.5 X 32 cm) equilibrated with reconstitution buffer (50 mM Tris-HC1 (pH 7.5), 1mM MnCl2, 10 mM 2-mercaptoethanol, 250 mM NaC1, and 10% glycerol) a t a flow rate of20 ml/h at 8 "C. Vesicles and PI synthase activity emerged from the column in the voidvolume. PI synthase was reconstituted into vesicles having various phospholipid compositions. The molar ratio of phospholipids to CDP-DG in the various vesicles was 1O:l. Vesicle size was determined by gel filtration using Sephacryl s-1000 chromatography (27) and electron microscopy. Vesicles werenegatively stained with uranyl acetate as previously described (26). Vesicle integrity was determined by the method of Ganong and Bell (28) using DTNB. Enzyme Assays-PI synthase activity was measured at 30 "C by following the incorporation of myo-[2-3H]Inositol(10,000 cpm/nmol) into PI or the release of CMP from [5-3H]CDP-DG (500 cpm/nmol) as previously described (9). The assay mixture contained 50 mM TrisHC1 (pH 8.0), 2.5 mM MnC12,0.5 mM myo-inositol, 0.2mM CDP-DG, 2.4 mM Triton X-100, and enzyme protein in a total volume of 0.1 ml. Reconstituted PI synthase activity was measured in the absence of Triton X-100 and added CDP-DG. A unit of enzymatic activity was defined as the amount of enzyme.that catalyzed the formation of 1 nmol of product/min under the assay conditions described. The specific activity was defined as the units/mg of protein. Protein was
3179
determined by the method of Bradford (29), with bovine serum albumin as the standard.
RESULTS
Reconstitution of PI Synthase-Since PI synthase was purified in the presence of Triton X-100 (9) it was necessary to reconstitute PI synthase into vesicles directly from mixed micelles containingpure enzyme, phospholipid, and detergent. Owing to the low critical micelle concentration of Triton X100, the removal of this detergent by dialysis or gel filtration is not easily accomplished (30). Successful reconstitution of PI synthase was achieved by passing an octylglucoside/phospholipid/Triton X-lOO/enzyme mixed micelle througha Sephadex G-50 superfine column. Fig. 1 shows a typical elution profile from a Sephadex G-50 superfine column of PI synthase activity, phospholipid, Triton X-100, and octylglucoside. The phospholipid composition of the reconstitution mixture contained PC:PE:PI:PS (3:2:2:1).This is the approximate composition of the major phospholipids of S. cerevisiae grown in complex medium containing inositol (1).Enzyme activity coeluted with phospholipid near the void volume of the column and was well separated from Triton X-100 and octylglucoside peaks. A molar ratio of octylglucoside to Triton X-100 of 60:l or greater was necessary for the reconstitution of PI synthase activity into vesicles that were separated from the Triton X-100 peak. A ratio of 75:l was routinely used since it resuited in the reduction of Triton X-100 concentration to 6 p ~well , below the critical micelle concentration for this detergent. PI synthase was also reconstituted intovesicles with various phospholipid compositions (see below). The elution profiles of vesicles, PI synthase activity, and detergents from Sephadex G-50 superfine columns for all reconstitution mixtures were identical to thatshown in Fig. 1. Analysis of negatively stained vesiclescomposed of the variou! phospholipid compositions by electron microscopy revealed a population of unilamellar vesicles with sizes ranging from 30 to 100 nm in diameter. These vesicles had an average diameter of 40 nm and were similar to the estimates of vesicle size using Sephacryl S-1000 chromatography (27). These results are in good agreement with the vesicle sizes obtained bygel filtration of octylglucoside/phospholipid mixed micelles having a molar ratio of 251 (28). Vesicle integrity was determined by the release of DTNB (a non-penetrating, colorimetric, sulfhydryl reagent), that was incorporated into the vesicles during the reconstitution procedure (28). There was no DTNB leakage from the vesicles when incubated at 30 "C (assay temperature) over a 4-h period or when incubated at 8 "C over a 3-day period. The topography of the active site of PI synthase within the transverse plane of the vesicle bilayer was examined by measuring the activity remaining after treatingvesicles with chymotrypsin (26). Over 90% of the PIsynthase activity that was present in the control vesicles was inactivated after protease treatment. Under the conditions of the experiment, the vesicles maintained their integrity (no DTNB leakage), and chymotrypsin was unable to penetrate the vesicles (26). These results indicate that the enzyme was reconstituted asymmetrically into the vesicle. Our results do not rule out the presence of transmembrane segments that could have been hydrolyzed by chymotrypsin, resulting in a loss of enzyme activity. In addition, reconstitution at low protein-to-lipid ratios results in the formation of vesicles containing one or zero proteins oriented asymmetrically in the phospholipid bilayer (31). When 5.5 pmol of phospholipid and 0.4 nmol of protein were reconstituted, our calculations estimated that a 40-nm vesicle wouldbe expected to contain about 1molecule of PI synthase. These calculations
3 180
FIG. 1. Sephadex G-50 superfine chromatography of the reconstitution mixture. Fractions (1.1 ml) were collected and assayed for phospholipid (PL, e),octylglucoside (O), and Triton X-100 (0)by scintillation counting. PI synthase activity (W) was measured with [2-3H]inositol as described in the text. The figure is a composite of four separate experiments.
FRACTION NUMBER
were based on the assumption that thehydrocarbon thickness -E 1.2 qf the vesicle is 40 A and thateach phospholipid occupies 70 A' (26). 3 u- 0.8 Properties of Reconstituted PI Synthase-All studies were u) conducted with freshly prepared vesicles. Under standard 4 r assay conditions, the activity of the reconstituted enzyme was 0.4 > linear for, up to 1 h and the enzyme was 100% stable for at u) -n least 3 days of storage at 8 "C. The effects of pH, cofactors, and inositol on reconstituted PI synthase activity are shown 6 7 B 9 10 1 1 in Fig. 2. The pH optimum for the reaction was 8.0. Reconstituted PI synthase activity was dependent on the addition of either manganese or magnesium ions. The maximum activity obtained with manganese ions (2.5 mM) was1.9-fold greater than themaximum activity obtained with magnesium a ions (20 mM). Normal saturation kinetics were shown by reconstituted PI synthase when the CDP-DG concentration in thevesicles washeld constant andinositol (K, = 0.08 mM) was varied. The enzyme did not follow saturation kinetics when the CDP-DG concentration was varied in the vesicles. The enzymological properties of reconstituted PI synthase were similar to those previously reported for solubilized (18) and purified (9) preparations of the enzyme assayed in the presence of Triton X-100. Therefore, these properties have Manganese or Magnesium, mM not changed by reconstituting the enzyme. The reaction mechanism for PI synthasehasnot been previously reported. We were unable to conduct the detailed kinetic measurements for the determination of the reaction mechanism for PI synthase as described by Cleland (32). However we have examined the ability of reconstituted PI synthase to catalyze isotopic exchange reactions according to Cleland (32). Our studies were novel in that the.enzyme was reconstituted into vesicles containing the appropriate phospholipid mixtures to carry outthe various exchange reactions. Reconstituted PI synthase did not catalyze an exchange reaction between inositol and PI in PC:PE:PI vesicles (Table I, Reaction 1) nor an exchange reaction between CMPand CDP-DG in PC:PE:CDP-DG vesicles (Table I, Reaction 2). The reconstituted enzyme did not catalyze the hydrolysis of 1 .mM PI in PC:PE:PI vesicles (Table I, Reaction 3), nor the hyInositol drolysis of CDP-DG in. PC:PE:CDP-DG vesicles (Table I, FIG. 2. Effects of pH, manganese and magnesium, and inoReaction 4). The inability of PI synthase'to catalyze these sitol on reconstituted PI synthase activity. In A, PI synthase reactions indicates that the enzyme does not follow a ping- (0.02 unit) was measured at the indicated pH values with 50 mM pong reaction mechanism (32). PI synthase did not catalyze Tris-HC1 (0)or 50 mM MES-HCl (e).In B, PI synthase (0.03 unit) was assayed with the indicated concentrations of MnCL (e)or MgCL an exchange reaction between CMPand CDP-DG in the (0).In C, the data areplotted as 1/V (units/ml) uersm the reciprocal presence of inositol in PC:PE:CDP-DG vesicles (Table I, of the inositol concentration. The curue drawn was a result of a leastReaction 5), nor in the presence of PI in PC:PE:PI:CDP-DG squares analysis of the data. Activity was measured with my0-[2-~H] vesicles (Table I, Reaction 6).The reconstituted enzyme did, inositol as described in the text.. \
Phosphatidylinositol Synthase TABLE I Reactions catalyzed by reconstituted PI synthase Reactions were measured in standard assay buffer as described in the text. The assays were run for 1h with 0.06-0.10 unit of reconstituted enzyme and theindicated reaction components and vesicle type. Inositol-PI exchange reactions were measured using either [2-3H] inositol (11,500 cpm/nmol) or [2-3H]PI (8,500 cpm/nmol) as substrates.CMP-CDP-DG exchange reactions were measured using either [5-3H]CMP (8,000 cpm/nmol) or [5-3H]CDP-DG (500 cpm/ nmol) as substrates. The release of water-soluble radioactive inositol or CMP was measured aftera chloroform/methanol/water phase partition and paper chromatography (9). Chloroform-soluble radioactive PI or CDP-DG was measured after a chloroform/methanol/ water phase partition and thin-layer chromatography (9). Reaction
Vesicle composition (molar ratio)
Incorporated or released
from S. cerevisiae
TABLE I1 PI synthase activity in cell-free extracts from strain ade5a grown in the presenceof phospholipid precursors Cells were grown in complete synthetic medium with 50 p~ inositol, 1mM ethanolamine, and 1mM choline where indicated. The specific activities (units/mg) of PI synthase were calculated from triplicate determinations from a minimum of two independent growth studies. Growth condition
+ [3H]PI 2. [3H]CMPPC:PECDP-DG + CDP-DG 3. [3H]PI + H20 4. [3H]CDP-DG+ Hz0 5. [3H]CMP + CDP-DG + inositol 6. CMP + [3H]CDP-DG + PI 7. [3H]Inositol + PI + CMP 8. CDP-DG + [3H]inositol 9. CDP-DG + [3H]inositol + PI 10. CMP + [3H]PI 11. CMP + [3H]PI + 1. Inositol
~~
inositol
PC:PE:PI
0.01
(3:2:2)
0.01 (3:2:0.5)
PC:PE:PI
%
unitslmg
+
~
~ ~ ~ ~ ~ ~ t ' y "
~~
100 97 97 94
100 90
All values are the mean k S.D.
1
2
3
4
5
6
7
0.01
(3:2:2)
PCPECDP-DG
Specific activity
Complete synthetic medium 0.30 f 0.01" + Ethanolamine 0.29 f 0.03 + Choline 0.29 f 0.03 Inositol 0.28 ? 0.02 + Inositol + ethanolamine 0.30 f 0.03 + Inositol + choline . ~ ~ . ~ ~ . . .0.27 - .- . + - 0.04 -. ~
nmol
3181
0.02
" " " L
(3:2:0.5)
0.01 (3:2:0.5)
PC:PE:PI:CDP-DG
0.02
(3:2:2:0.7)
PC:PEPI
0.54
(3:2:2)
PC:PECDP-DG
3.60
(3:2:0.5)
PC:PE:PI:CDP-DG
4.90
(3:2:2:0.7)
PC:PE:PI
0.10
(3:2:2)
PC:PE:PI
0.30
(3:2:2)
however, catalyze the exchange between inositol and PI in the presence of CMP inPC:PE:PI vesicles (Table I, Reaction 7). In addition, PI stimulated the incorporation of labeled inositol into PI in the forward reaction in PC:PE:PI:CDPDG vesicles (Table I, Reaction 9), and inositol stimulated the release of labeled inositol from PI in the reverse reaction in PC:PE:PI vesicles (Table I, Reaction 11).These results suggest that PI synthase in S. cerevisiae catalyzes a sequential reaction mechanism (32). In sequential reaction mechanisms involving two substrates andtwo products, both substrates or both productsmust bind to the enzyme before the first product or first substrate is released. The presence of one of the products in the reaction can enhance an exchange reaction between that product and one of the reactantsin the presence of the other reactant (32). Whichever product shows exchange into reactant in the absence of the other product is the first product released inthe reaction (32). The results of the various exchange reactions suggest that PI synthasebinds to CDP-DG before inositol and PI is released prior to CMP in the reaction sequence. It is also known that PI synthasecan bind to CDP-DG before inositol in theforward reaction, since the enzyme was purified by CDP-DG-Sepharose affinity chromatography in the absence of inositol (9). PI Synthase from S. cerevisiae Grown in Medium Supplemented with Phospholipid Precursors-PI synthase activity was measured from cell-free extracts of cells grown in complete synthetic medium in the absence or presence of watersoluble phospholipid precursors (Table 11). 'The level of PI synthase specific activity did not change significantly when cells were grown under the various conditions. These results
FIG.3. Immunoblotting of PI synthase from cell-free ex-
ade5a cells grown under various conditions. The figure is a portion of an immunoblot showing the M,= 34,000 subunit of PI synthase from cells grown in complete synthetic medium with no addition (lane I ) , with 1 mM ethanolamine (lune 2), with 1 mM choline (lane 3 ) ,with 50 p M inositol ( l u n e 4 ) , with 50 PM inositol and 1mM ethanolamine ( l u n e 5), and with 50 p M inositol and 1 mM choline ( l u n e 6 ) . Lane 7 is an immunoblot of purified PI synthase standard. Electrophoresis and immunoblotting were performed as described in the text.
tracts of strain
are consistent with those previously reported for cells grown in the presence of inositol and choline (14). The effects of ethanolamine alone and in combination with inositol on the expression of PI synthase activity have not been previously reported. When the cell-free extracts were probed with antiserumdirectedagainst purified PI synthase, the level of enzyme subunit did not change significantly (Fig. 3). Effect of Water-solubleCompounds on Reconstituted PI Synthase Activity-Reconstituted PI synthase was assayed in the presence of0.1-10 mM of the following water soluble compounds: ethanolamine, choline, CDP-ethanolamine, CDP-choline, glycerol 3-phosphate, serine, and the mono-, di-, and triphosphorylated derivatives of adenosine, cytidine, guanosine, and uridine. The effects of water-soluble phospholipid precursors were studied because they have been shown to influence phospholipid metabolism in growing cells (12). We examined what effect various nucleotides had on activity because the phospholipid substrate for PI synthase is a liponucleotide. These compounds did not significantly effect reconstituted PI synthase activity (data notshown). Effect of Phospholipids on Reconstituted PI Synthase Actiuity-PI synthase was reconstituted into a varietyof phospholipid vesicles to examine the effect of phospholipid composition on enzyme activity (Table 111). The enzyme was first reconstituted into PC:PE:PI:PS vesicles at a molar ratio of 3:2:2:1. These vesicles represent the approximate composition of the major phospholipids of wild type cells grown in complex medium containing inositol (1)and were used as a control. When the content of PS was increased in the PC:PE:PI:PS vesicles, a 1.4-fold increase in reconstituted PI synthase activity was obtained. There was a 1.26-fold increase in activity when PI synthase was reconstituted into PC:PS vesicles compared to the control vesicles. When PI synthase was reconstituted into PC, PC:PE, PC:PA, PC:PI, PC:PI:PI 4-phos-
Phosphatidylinositol Synthase from S. cerevisiae
3182
TABLEI11 Effect of vesicle phospholipid composition on PI synthase activity PI synthase was reconstituted intothe indicated phospholipid vesicles. The relative activity (%) was calculated by normalizing the activity obtained in PC:PE:PIPS (3:2:2:1) vesicles (2.2 units/ml) against the activity obtained in the other vesicles. The final phospholipid concentration of all vesicles was the same and the molar ratio of phospholipid to CDP-DG was 1O:l. PIP, PI4-phosphate; PIP2, PI 4,Ei-diphosphate. Vesicle composition (molar ratio)
Relative activity %
126 36 47
PC:PE:PIPS (3:2:2:1) PC:PE:PI:PS (3:2:2:3) PC:PS (3:2) PC PC:PE (3:2) PC:PA (3:l) PC:PI (3:2) 46 PC:PI:PIP (3:2:0.2) PC:PIPIP2 42 (3:2:0.2)
100 142 45 35
phate, and PC:PI:PI 4,5-diphosphate vesicles, over a 2-fold reduction in activation was obtained. DISCUSSION
In order to gain insight into the regulation of PI synthase activity, we have constructed a model membrane system in which purified enzyme was inserted into unilamellar phospholipid vesicles. This system closely mimics the in vivo environment of an intact membrane. This is the first report of PI synthase from any organism being reconstituted into phospholipid vesicles and characterized in the absence of detergent. The four major phospholipids in S. cerevisiae are PC, PE, PI, and PS (1).The synthesis of PC through the reaction sequence PA + CDP-DG + PS + P E + PC appears to be coordinately regulated to the synthesis of inositol (12, 33). It has been suggested that thebiological role of this regulation is to control the netcharge of the membrane (2). The biosynthetic enzymes leading to theformation of PC, namely CDPDG synthase (34), PS synthase (14, 35), PS decarboxylase (36), andthe phospholipid N-methyltransferases (37,38), are all repressed when wild type cells are grown in medium containing inositol and choline. CDP-DG synthase (34) and PS synthase2activities are also repressed when wild type cells are grown in medium containing inositol and ethanolamine. In the absence of inositol, choline or ethanolamine have no effect on CDP-DG synthase (34), PS synthase (14); and the phospholipid N-methyltransferase (38) activities. The studies presented in thispaper showed that, unlike the enzymes leading to PC formation, PI synthase activity was not regulated incells grown in thepresence of water-soluble phospholipid precursors. In addition, immunoblot analysis of extracts from cells grown under the various conditions showed'that the level of PI synthase subunit protein was expressed in a constitutive fashion. Since PI synthase activity was constitutive under growth conditions which cause fluctuations in PI content of growing cells, the levels of PI in themembranes must be controlled by regulating the activity of PI synthase. To examine direct regulation of PI synthase activity by water-soluble phospholipid precursors, studies with reconstituted enzyme were performed. PI synthase activity was not effected by various water-soluble phospholipid precursors or by nucleotides. These data suggest that the regulation of PI 2 M . A. Poole, M. J. Homann, M. Bae-Lee, and G.M. Carman, manuscript submitted to J. Bwl. Chem. for publication.
synthesis in growing cells does not occur as a direct result of enzyme activity modulation by phospholipid precursors. Wild type cells growing in the absence of inositol rely on endogenous inositol synthesis (19) and have elevated PS content (13, 14). When such cells are exposed to exogenous inositol, the content of PI increases about 1.5-fold and PS decreases about 1.5-fold (13,14). Thelevels of PE and PC do not change significantly when cells are grown in thepresence of inositol (13,14). PI synthase was reconstituted into vesicles with various phospholipid compositions to examine the effect of phospholipids on enzyme activity. Maximal activity was found when the enzyme was reconstituted into PC:PE:PIPS vesicles. An increase in the PScontent of phospholipid vesicles stimulated PI synthase activity about 1.4-fold. Vesicles containing elevated PS may reflect the in vivo membrane environment of cells grown in medium without inositol. Under these conditions, PS synthase would not be repressed in growing cells (14) and could compete fully with PI synthase for the common substrate CDP-DG. In this manner, PI synthesis and PC synthesis are ultimately coordinated through the synthesis of PS. However, in the presence of inositol, PS synthase is repressed 1.6-fold,' resulting in lower PS content in the membrane (13, 14). This in turn may result in the removal of stimulation of PI synthase activity by PS. On the other hand,the available pool of the substrate CDP-DG would increase due to the reduced level of PS synthase. Therefore PI synthase activity may increase as a result of mass action given increased pools of CDP-DG and readily available exogenous inositol. The reason for such an increase in PI synthase activity may be due to the cells' efforts to balance the membrane net charge under conditions of reduced PS synthesis. Thus the finding reported here may provide evidence about the phy8iologically important regulatory mechanism of PI synthesis. Acknowledgments-We thank Robert M. Bell, Phillip R. Green, Charles E. Martin, and Susan A. Henry for their many helpful discussions. We are grateful to Susan A. Henry for the preparation of the PI synthase antiserum. Beverly E. Maleeff and Susan Jones are gratefully acknowledged for performing the electron microscopy. REFERENCES 1. Henry, S. A. (1982) in The Molecular Biology of the Yeast Saccharomyces: Metabolismand Gene Expression (Strathern, J. N., Jones, E. W., and Broach, J. R., eds) pp. 101-158, Cold Spring Harbor Laboratory, Cold Spring Harbor,NY 2. Becker, G. W., and Lester, R. L. (1977)J. Biol. Chem. 252,86848691 3. Hanson, B. A., and Lester, R. L. (1980) J. Bacteriol. 142,79-89 4. Henry, S. A., Atkinson, K. D., Kolat, A. I., and Culbertson, M. R. (1977) J.Bacteriol. 130,472-484 5. Lester, R. L., and Steiner, M. R. (1968) J. Bwl. Chem. 243, 4889-4893 6. Becker, G. W., and Lester, R. L. (1980) J. Bacteriol. 142, 747754 7. Hanson, B. A. (1984) J. Bacteriol. 159,837-842 8. Paulus, H., andKennedy, E. P. (1960) J. Biol. Chem. 235,13031311 9. Fischl, A. S., and Carman, G. M., (1983) J. Bacteriol. 154, 304311 10. Larson, T. J., Hirabayashi, T., and Dowhan, W. (1976) Biochemistry 1 5 , 974-979 11. Poole, M. A., Fischl, A. S., and Carman, G. M. (1985)J. Bucterwl. 161, 772-774 12. Henry, S. A., Klig, L. S., and Loewy, B. S. (1984) Annu. Rev. Genet. 18,207-231 13. Greenberg, M. L., Klig, L. S., Letts, V. A., Loewy, B. S., and Henry, S. A. (1983) J. Bacteriol. 153,791-799 14. Klig, L. S., Homann, M. J., Carman, G. M., and Henry, S. A. (1985) J. Bacteriol. 162, 1135-1141
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