298
SYNTHASES
[35]
although phosphatidylethanolamine is an essential phospholipid under normal laboratory growth conditions, the nonspecific nature of the substitution by several divalent metal ions for this major membrane phospholipid suggests a primarily structural role for phosphatidylethanolamine. One possibility may be its role as an inert membrane matrix since this phospholipid carries no net charge; divalent metal ions could be acting to neutralize the high negative charge density of the remaining phospholipids. A second possibility may be the need for a phospholipid which can form nonbilayer structures possibly necessary in such processes as membrane fusion and translocation of macromolecules across membranes. This property of phosphatidylethanolamine 25could be substituted for by cardiolipin, which is known to display nonbilayer structures 26 in the presence of divalent metal ions with the same order of effectiveness as the three divalent metal ions which suppress the growth phenotype of the pss :: kan allele. 25 p. R. Cullis and B. de Kruijff, Biochim. Biophys. Acta 513, 31 (1978). 26 I. Vasilenko, B. de Kruijff, and A. J. Verkleij, Biochim. Biophys. Acta 684, 282 (1982).
[35] P h o s p h a t i d y l s e r i n e S y n t h a s e f r o m Y e a s t By GEORGE M. CARMAN and MYONGSUK BAE-LEE
Introduction Phosphatidylserine synthase (CDPdiacylglycerol-L-serine O-phosphatidyltransferase, EC 2.7.8.8) catalyzes the incorporation of serine into CDPdiacylglycerol + serine ~ phosphatidylserine + C M P
phosphatidylserine.l The enzyme plays an important role in the regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae. 2 Phosphatidylserine synthase activity is associated with the mitochondrial and microsomal fractions of S. cerevisiae) '4 Phosphatidylserine synthase expression is regulated by inositol alone and in concert with serine, etha-
I j. N. Kanfer and E. P. K e n n e d y , J. Biol. Chem. 239, 1720 (1964). 2 G. M. C a r m a n and S. A. Henry, Annu. Rev. Biochem. 58, 635 (1989). 3 G. S. Cobon, P. D. Crowfoot, and A. W. Linnane, Biochem. J. 144, 265 (1974). 4 K. Kuchler, G. D a u m , a n d F. Paltauf, J. Bacteriol. 16S, 901 (1986).
METHODS IN ENZYMOLOGY.VOL. 209
Copyright © 1992by AcademicPress. Inc. All rights of reproduction in any form reserved.
[35]
PHOSPHATIDYLSERINE SYNTHASE FROM YEAST
299
nolamine, and choline) -8 Microsomal-associated phosphatidylserine synthase has been purified to near homogeneity using CDPdiacylglycerolSepharose affinity chromatography. 9 We describe here the purification, reconstitution, and properties of the enzyme. Preparation of CDPdiacylglycerol and CDPdiacylglycerol-Sepharose CDPdiacylglycerol is prepared from phosphatidic acid and CMPmorpholidate by the method of Agranoff and Suomi ~° with the modifications of Carman and Fischl) l The CDPdiacylglycerol-Sepharose affinity resin of Dowhan and co-workers 12:3 is synthesized as described by Fischl and Carman 14with the following modifications. The incubation times for coupling the adipic acid spacer arm to activated Sepharose 4B, NaIO 4 oxidation of CDPdiacylglycerol, and coupling the oxidized derivative of CDPdiacylglycerol to the adipic acid-Sepharose resin are 1 day, 1 day, and 1 day, respectively. These incubation times result in a low-capacity affinity resin) 4 The preparations of CDPdiacylglycerol and CDPdiacylglycerolSepharose are described elsewhere in this volume, is Assay Method Phosphatidylserine synthase activity is routinely measured by following the incorporation of 0.5 mM L-[3-aH]serine [10,000 counts/min (cpm)/ nmol] into phosphatidylserine in 50 mM Tris-HCl buffer (pH 8.0) containing 0.6 mM MnCl 2 , 0.2 mM CDPdiacylglycerol, 4 mM Triton X-100, and enzyme protein in a total volume of 0.1 ml at 30°.9,16Purified phosphatidylserine synthase reconstituted in phospholipid vesicles is measured in the absence of Triton X-100 and added CDPdiacylglycerol. The reaction is terminated by the addition of 0.5 ml of 0. l N HCI in methanol. Chloroform (1 ml) and 1 M M g C I 2 (1.5 ml) are added, the system is mixed, and s L. S. Klig, M. J. Homann, G. M. Carman, and S. A. Henry, J. Bacteriol. 162, 1135 (1985). 6 M. J. Homann, A. M. Bailis, S. A. Henry, and G. M. Carman, J. Bacteriol. 169, 3276 (1987). 7 M. A. Poole, M. J. Homann, M. Bae-Lee, and G. M. Carman, J. Bacteriol. 168, 668 (1986). 8 A. M. Bailis, M. A. Poole, G. M. Carman, and S. A. Henry, Mol. Cell. Biol. 7, 167 (1987). 9 M. Bae-Lee and G. M. Carman, J. Biol. Chem. 259, 10857 (1984). 10 B. W. Agranoff and W. D. Suomi, Biochem. Prep. 10, 47 (1963). H G. M. Carman and A. S. Fischl, J. FoodBiochem. 4, 53 (1980). 12 T. J. Larson, T. Hirabayashi, and W. Dowhan, Biochemistry 15, 974 (1976). 13 W. Dowhan and T. Hirabayashi, this series, Vol. 71, p. 555. 14 A. S. Fischl and G. M. Carman, J. Bacteriol. 154, 304 (1983). ~5G. M. Carman and A. S. Fischl, this volume [36]. i6 G. M. Carman and J. Matas, Can. J. Microbiol. 27, 1140 (1981).
300
SYNTHASES
[35]
the phases are separated by a 2-min centrifugation at 100 g. A 0.5-ml sample of the chloroform phase is removed and taken to dryness on an 80° water bath. Betafluor (4 ml) is added to the sample, and radioactivity is determined by scintillation counting. The chloroform-soluble phospholipid product of the reaction phosphatidylserine is analyzed by thin-layer chromatography with standard phosphatidylserine.9,16 Alternatively, activity is measured by following the release of radiolabeled CMP from radiolabeled CDPdiacylglycerol. 16 One unit of enzymatic activity is defined as the amount of enzyme that catalyzes the formation of 1 nmol of product per minute. Protein is determined by the method of Bradford.17 Buffers which are identical to those containing the protein samples are used as blanks. The presence of Triton X-100 does not interfere with the protein determination, provided the blank contains a final concentration of detergent identical to that of the sample. 9 Electroblotting of Phosphatidylserine Synthase Activity 18 The procedure for the electroblotting of phosphatidylserine synthase is similar to that used for the electroblotting of phosphatidylinositol synthase) 5 Briefly, the enzyme is dialyzed for 3 hr at 8° against sodium dodecyl sulfate-polyacrylamide gel electrophoresis treatment buffer. The enzyme is subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Enzyme on the nitrocellulose paper is renatured in 50 mM Tris-HCl (pH 8.0), 0.6 mM MnC12, 30 mM MgCI2, 1 mM CDPdiacylglycerol, 0.5% Triton X-100, 10 mM 2mercaptoethanol, 20% glycerol, 3% bovine serum albumin for 1 hr at 8°. The presence of CDPdiacylglycerol and cofactors in the renaturation buffer stabilizes phosphatidylserine synthase activity. The nitrocellulose paper is used for enzyme assays. The recovery of phosphatidylserine synthase activity is about 10%. This procedure allows the confirmation of the molecular weight of phosphatidylserine synthase. Growth of Yeast
Wild-type S. cerevisiae strain $288C (a gal2) is used for enzyme purification. Cells are grown in 1% yeast extract, 2% peptone, and 2% glucose at 28° to late exponential phase, harvested by centrifugation, and stored at - 80°. 14,15 17 M. M. Bradford, Anal. Biochem. 72, 248 (1976). 18 M. A. Poole, A. S. Fischl, and G. M. Carman, J. Bacteriol. 161, 772 (1985).
[35]
PHOSPHATIDYLSERINE SYNTHASE FROM YEAST
301
Purification Procedure All steps are performed at 5° .
Step 1: Preparation of Microsomes. Cells (75 g, wet weight) are disrupted with glass beads in 50 mM Tris-HCl buffer (pH 7.5) containing I mM Na2EDTA, 0.3 M sucrose, and 10 mM 2-mercaptoethanol.14'15 Glass beads and unbroken cells are removed by centrifugation at 1500 g for 10 min to obtain the cell extract. Microsomes are collected from the cell extract by differential centrifugation.14'15 Microsomes are washed with and resuspended in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM 2mercaptoethanol and 20% glycerol (w/v). Step 2: Preparation of Triton X-IO0 Extract. The microsome fraction is suspended in 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM MnCI2, 30 mM MgCI 2, 10 mM 2-mercaptoethanol, 0.5 M KC1, 20% glycerol (w/v), and 1% Triton X-100 at a final protein concentration of 10 mg/ml. After incubation for 1 hr at 5°, the suspension is centrifuged at 100,000 g for 2 hr to obtain the solubilized (supernatant) fraction. Solubilization of the enzyme from microsomes with 1% Triton X-100 is dependent on the presence of MnCI 2 in the solubilization buffer. 14'16 Step 3: CDPdiacylglycerol-Sepharose Chromatography. A CDPdiacylglycerol-Sepharose column (0.9 x 4 cm) is equilibrated with 50 ml of 50 mM Tris-HC1 buffer (pH 8.0) containing 0.6 mM MnC12, 30 mM MgC12, 20% glycerol, 10 mM 2-mercaptoethanol, and 0.5% Triton X-100. The Triton X-100 extract is applied to the affinity column in 1.5-ml samples. To effect enzyme binding, each sample is incubated in the column for 10 min before the addition of the next sample. After the entire Triton X-100 extract is applied, the column is washed with 30 ml of equilibration buffer containing 1 M NaCI. The column is then saturated with equilibration buffer containing 1 mM CDPdiacylglycerol and 1 M NaCI and incubated for 1 hr. Phosphatidylserine synthase is eluted from the column with this buffer at a flow rate of I ml/min. Fractions containing activity are pooled and desalted by dialysis or Sephadex G-25 chromatography using 20 mM Tris-HCl buffer (pH 8.0) containing 0.3 mM MnC12, 10 mM MgCI 2 , 20% glycerol, 10 mM 2-mercaptoethanol, and 0.5% Triton X-100. Step 4:DE-53 Chromatography. A DE-53 (Whatman, Clifton, N J) column (0.6 × 4 cm) is equilibrated with 20 ml of 20 mM Tris-HCl buffer (pH 8.0) containing 0.3 mM MnCI 2 , 10 mM MgC12 , 20% glycerol, 10 mM 2-mercaptoethanol, and 0.5% Triton X-100. Desalted enzyme from the previous step is applied to the column at a flow rate of 1 ml/min. The column is washed with 10 ml of equilibration buffer followed by elution of the enzyme with equilibration buffer containing 1 M NaCl. Fractions
302
SYNTHASES
[35]
TABLE I PURIFICATION OF PHOSPHATIDYLSERINE SYNTHASE FROM S a c c h a r o m y c e s c e r e v i s i a e a
Purification step 1. Microsomes 2. Triton X-100 extract 3. CDPdiacylglycerolSepharose 4. DE-53 chromatography
Total units (nmol/min)
Protein (mg)
Specific activity (units/mg)
Purification (-fold)
Yield (%)
591 579 457
493 241 0.34
1.2 2.4 1340
1 2 1120
100 98 77
421
0.18
2300
1920
71
a Data are from Bae-Lee and Carman. 9
containing activity are pooled and desalted as described above. The purified enzyme is 100% stable for at least 6 months when stored at - 8 0 °. Enzyme Purity. The four-step purification scheme summarized in Table I results in a nearly homogeneous preparation of phosphatidylserine synthase as evidenced by native 19and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 9 Native polyacrylamide gels contain 0.5% Triton X100. The enzyme preparation does not contain the CDPdiacylglyceroldependent enzymes phosphatidylinositol synthase, CDPdiacylglycerol synthase, nor phosphatidylglycerophosphate synthase. The subunit molecular weight of the purified enzyme is 2 3 , 0 0 0 . 9'18 When the enzyme is partially purified in the presence of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF), a protein with a molecular weight of 30,000 as well as the 23,000 subunit is isolated. 2°The 23,000 subunit ofphosphatidylserine synthase is a proteolytic cleavage product of the 30,000 subunit of phosphatidylserine synthase. 2° The enzyme is purified about 2000-fold over the microsomal fraction and about 5000-fold relative to the activity in cell extracts. Phosphatidylserine synthase can also be purified from strain VAL2C(YEpCH01), 9 which bears a hybrid plasmid that directs overproduction of the enzyme. 21 The phosphatidylserine synthase-overproducing strain facilitates the acquisition of larger amounts of pure enzyme. 9
19 M. A. Poole, Ph.D. Thesis, Rutgers University, New Brunswick, New Jersey (1986). 2o K. Kiyono, K. Miura, Y. Kushima, T. Hikiji, M. Fukushima, I. Shibuya, and A. Ohta, J. Biochem. (Tokyo) 102, 1089 (1987). 21 V. A. Letts, L. S. Klig, M. Bae-Lee, G. M. Carman, and S. A. Henry, Proc. Natl. Acad. Sci. U.S.A. 80, 7279 (1983).
[35]
PHOSPHATIDYLSERINE SYNTHASE FROM YEAST
303
Reconstitution of Phosphatidylserine Synthase Pure phosphatidylserine synthase has been reconstituted into unilamellar phospholipid vesicles containing its substrate CDPdiacylglycerol.22 The procedure is similar to that used for the reconstitution of phosphatidylinositol synthase 23 with some modifications. The reconstitution procedure is described elsewhere in this volume.15 Pure phosphatidylserine synthase is reconstituted into vesicles directly from mixed micelles containing pure enzyme, phospholipid, Triton X-100, and octylglucoside. The final concentrations of Triton X-100, octylglucoside, and phospholipids in the reconstitution mixture are 2.0, 150, and 10.6 mM, respectively. The final molar ratios of octylglucoside to Triton X-100, octylglucoside to phospholipids, and phospholipids to CDPdiacylglycerol are 75 : 1, 14 : l, and 16 : l, respectively. Octylglucoside inhibits phosphatidylserine synthase, and activity cannot be measured in the reconstitution mixture. The reconstitution mixture is immediately passed through a Sephadex G-50 superfine column (1.5 x 32 cm) equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM MnCI2, I0 mM 2-mercaptoethanol, 250 mM NaCl, and 10% glycerol at a flow rate of 20 ml/hr at 5°. The column is pretreated with a 1 mg/ml sonicated suspension of phospholipids (phosphatidylcholine-phosphatidylethanolamine-phosphatidylinositol-phosphatidylserine, 3 : 2 : 2 : l, v/v), which increases the yields of reconstituted activity. Reconstituted phosphatidylserine synthase elutes from the column in the void volume. The average size of vesicles containing phosphatidylserine synthase is 90 nm in diameter. 22 Each vesicle contains about 1 molecule of enzyme, which is reconstituted asymmetrically with 80-86% of its active site facing outward. 22 Maximum reconstituted phosphatidylserine synthase activity is obtained with vesicles containing phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine, 22the four major phospholipids in wild-type S. cerevisiae. 5'24 Properties of Phosphatidylserine Synthase Optimum phosphatidylserine activity requires either MnCI2 (0.6 mM) or MgCI2 (20 mM) at the pH optimum of 8.0 using a mixed micelle assay of Triton X-100 and CDPdiacylglycerol.9 The manganese requirement of 22 j. M. Hromy and G. M. Carman, J. Biol. Chem. 261, 15572 (1986). 23 A. S. Fischl, M. J. Homann, M. A. Poole, and G. M. Carman, J. Biol. Chem. 261, 3178 (1986). 24 M. L. Greenberg, L. S. Klig, V. A. Letts, B. S. Loewy, and S. A. Henry, J. Bacteriol. 153, 791 (1983).
304
SYNTHASES
[35]
the enzyme reconstituted into phospholipid vesicles is 5 m M . 22 The K m value for serine is 0.83 mM as determined with a mixed micelle substrate of Triton X-100 and CDPdiacylglycerol.25 Phosphatidylserine synthase activity is dependent on the bulk (83 /zM) 25 and surface (2.9 mol %)26 concentrations of CDPdiacylglycerol, characteristic of surface dilution kinetics. 27 Based on the results of kinetic experiments, 25 the ability of the enzyme to catalyze isotopic exchange reactions between substrates and products, 23 and a stereochemical analysis of the reaction using 3~p nuclear magnetic resonance spectroscopy 2~ the enzyme catalyzes a Bi-Bi sequential reaction mechanism. Phosphatidylserine synthase binds to CDPdiacylglycerol before serine, and phosphatidylserine is released prior to CMP in the reaction sequence. 9 Phosphatidylserine synthase uses dCDPdiacylglycerol as well as CDPdiacylglycerol as substrate. 9 Phosphatidylserine synthase is labile above 40 ° and is inactivated by thioreactive agents. 9 Inositol (K i 65 ~M) is a noncompetitive inhibitor of phosphatidylserine synthase, 25 whereas cardiolipin (K i 0.7 mol %) and diacylglycerol (7 mol %) are competitive inhibitors of the enzyme. 26 Phosphatidate (Ka 0.033 mol %), phosphatidylcholine (Ka 3.4 mol %), and phosphatidylinositol (Ka 3.2 mol %) are activators of phosphatidylserine synthase activity. 26 Phosphatidylserine synthase (23,000 subunit 29and 30,000 subunit) is phosphorylated by cAMP-dependent protein kinase. 29 The enzyme has one phosphorylation site, a serine residue, which results in a 60-70% reduction in enzyme activity. 29The phosphorylation of phosphatidylserine synthase results in a decrease in the rate phosphatidylserine synthesis in o i v o . 3°
Synthetic and Analytical Uses
Pure phosphatidylserine synthase can be used to synthesize radiolabeled phosphatidylserine from CDPdiacylglycerol and labeled serine.9 The enzyme can be used to synthesize various fatty acyl derivatives of phosphatidylserine using the appropriate fatty acyl derivatives of CDPdiacylglycerol as substrate. Pure phosphatidylserine synthase can also be used 25 M. J. Kelley, A. M. Bailis, S. A. Henry, and G. M. Carman, J. Biol. Chem. 263, 18078 (1988). 26 M. Bae-Lee and G. M. Carman, J. Biol. Chem. 26.5, 7221 (1990). 27 R. A. Deems, B. R. Eaton, and E. A. Dennis, J. Biol. Chem. 250, 9013 (1975). C. R. H. Raetz, G. M. Carman, W. Dowhan, R.-T. Jiang, W. Waszkuc, W. Loffredo, and M.-D. Tsai, Biochemistry 26, 4022 0987). z9 A. J. Kinney and G. M. Carman, Proc. Natl. Acad. Sci. U.S.A. 85, 7962 (1988). 30 A. J. Kinney, M. Bae-Lee, S. Singh Panghaal, M. J. Kelley, P. M. Gaynor, and G. M. Carman, J. Bacteriol. 172, 1133 (1990).
[36]
PHOSPHATIDYLINOSITOL
SYNTHASE
FROM
YEAST
305
to determine quantitatively the serine concentration b y following the formation of radiolabeled C M P f r o m radiolabeled CDPdiacylglycerol. Acknowledgments This work was supported by U.S. Public Health Service Grant GM-28140 from the National Institutes of Health, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund.
[36] P h o s p h a t i d y l i n o s i t o l
Synthase
from Yeast
B y GEORGE M. CARMAN and ANTHONY S. FISCHL
Introduction Phosphatidylinositol synthase (CDPdiacylglycerol-myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) catalyzes the incorporation of CDP-diacylglycerol + inositol--~ phosphatidylinositol + CMP inositol into phosphatidylinositol.~ The e n z y m e 2 and its product phosphatidylinositol 3,4 are essential to the growth of the yeast S a c c h a r o m y c e s cerevisiae. Phosphatidylinositol synthase activity is associated with the mitochondrial, 5'6 m i c r o s o m a l : ,6 and p l a s m a m e m b r a n e 7 fractions of S. cereoisiae. Microsome-associated phosphatidylinositol synthase has been purified to near homogeneity, primarily b y CDPdiacylglycerol-Sepharose affinity c h r o m a t o g r a p h y : We describe here the purification, reconstitution, and properties of the e n z y m e . Preparation of CDPdiacylglycerol CDPdiacylglycerol is p r e p a r e d from phosphatidic acid and C M P m o r pholidate by the method of Agranoff and Suomi 9 with the modifications of t H. Paulus and E. P. Kennedy, J. Biol. Chem. 235, 1303 (1960). 2 j. Nikawa, T. Kodaki, and S. Yamashita, J. Biol. Chem. 262, 4876 (1987). 3 S. A. Henry, K. D. Atkinson, A. J. Kolat, and M. R. Culbertson, J. Bacteriol. 130, 472 (1977). 4 G. W. Becker and R. L. Lester, J. Biol. Chem. 252, 8684 (1977). 5 G. S. Cobon, P. D. Crowfoot, and A. W. Linnane, Biochem. J. 144, 265 (1974). 6 K. Kuchler, G. Daum, and F. Paltauf, J. Bacteriol. 165, 901 (1986). 7 A. J. Kinney and G. M. Carman, J. Bacteriol. 172, 4115 (1990). 8 A. S. Fischl and G. M. Carman, J. Bacteriol. 154, 304 (1983). 9 B. W. Agranoff and W. D. Suomi, Biochem. Prep. 10, 47 (1963).
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
SYNTHASES
[35]
although phosphatidylethanolamine is an essential phospholipid under normal laboratory growth conditions, the nonspecific nature of the substitution by several divalent metal ions for this major membrane phospholipid suggests a primarily structural role for phosphatidylethanolamine. One possibility may be its role as an inert membrane matrix since this phospholipid carries no net charge; divalent metal ions could be acting to neutralize the high negative charge density of the remaining phospholipids. A second possibility may be the need for a phospholipid which can form nonbilayer structures possibly necessary in such processes as membrane fusion and translocation of macromolecules across membranes. This property of phosphatidylethanolamine 25could be substituted for by cardiolipin, which is known to display nonbilayer structures 26 in the presence of divalent metal ions with the same order of effectiveness as the three divalent metal ions which suppress the growth phenotype of the pss :: kan allele. 25 p. R. Cullis and B. de Kruijff, Biochim. Biophys. Acta 513, 31 (1978). 26 I. Vasilenko, B. de Kruijff, and A. J. Verkleij, Biochim. Biophys. Acta 684, 282 (1982).
[35] P h o s p h a t i d y l s e r i n e S y n t h a s e f r o m Y e a s t By GEORGE M. CARMAN and MYONGSUK BAE-LEE
Introduction Phosphatidylserine synthase (CDPdiacylglycerol-L-serine O-phosphatidyltransferase, EC 2.7.8.8) catalyzes the incorporation of serine into CDPdiacylglycerol + serine ~ phosphatidylserine + C M P
phosphatidylserine.l The enzyme plays an important role in the regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae. 2 Phosphatidylserine synthase activity is associated with the mitochondrial and microsomal fractions of S. cerevisiae) '4 Phosphatidylserine synthase expression is regulated by inositol alone and in concert with serine, etha-
I j. N. Kanfer and E. P. K e n n e d y , J. Biol. Chem. 239, 1720 (1964). 2 G. M. C a r m a n and S. A. Henry, Annu. Rev. Biochem. 58, 635 (1989). 3 G. S. Cobon, P. D. Crowfoot, and A. W. Linnane, Biochem. J. 144, 265 (1974). 4 K. Kuchler, G. D a u m , a n d F. Paltauf, J. Bacteriol. 16S, 901 (1986).
METHODS IN ENZYMOLOGY.VOL. 209
Copyright © 1992by AcademicPress. Inc. All rights of reproduction in any form reserved.
[35]
PHOSPHATIDYLSERINE SYNTHASE FROM YEAST
299
nolamine, and choline) -8 Microsomal-associated phosphatidylserine synthase has been purified to near homogeneity using CDPdiacylglycerolSepharose affinity chromatography. 9 We describe here the purification, reconstitution, and properties of the enzyme. Preparation of CDPdiacylglycerol and CDPdiacylglycerol-Sepharose CDPdiacylglycerol is prepared from phosphatidic acid and CMPmorpholidate by the method of Agranoff and Suomi ~° with the modifications of Carman and Fischl) l The CDPdiacylglycerol-Sepharose affinity resin of Dowhan and co-workers 12:3 is synthesized as described by Fischl and Carman 14with the following modifications. The incubation times for coupling the adipic acid spacer arm to activated Sepharose 4B, NaIO 4 oxidation of CDPdiacylglycerol, and coupling the oxidized derivative of CDPdiacylglycerol to the adipic acid-Sepharose resin are 1 day, 1 day, and 1 day, respectively. These incubation times result in a low-capacity affinity resin) 4 The preparations of CDPdiacylglycerol and CDPdiacylglycerolSepharose are described elsewhere in this volume, is Assay Method Phosphatidylserine synthase activity is routinely measured by following the incorporation of 0.5 mM L-[3-aH]serine [10,000 counts/min (cpm)/ nmol] into phosphatidylserine in 50 mM Tris-HCl buffer (pH 8.0) containing 0.6 mM MnCl 2 , 0.2 mM CDPdiacylglycerol, 4 mM Triton X-100, and enzyme protein in a total volume of 0.1 ml at 30°.9,16Purified phosphatidylserine synthase reconstituted in phospholipid vesicles is measured in the absence of Triton X-100 and added CDPdiacylglycerol. The reaction is terminated by the addition of 0.5 ml of 0. l N HCI in methanol. Chloroform (1 ml) and 1 M M g C I 2 (1.5 ml) are added, the system is mixed, and s L. S. Klig, M. J. Homann, G. M. Carman, and S. A. Henry, J. Bacteriol. 162, 1135 (1985). 6 M. J. Homann, A. M. Bailis, S. A. Henry, and G. M. Carman, J. Bacteriol. 169, 3276 (1987). 7 M. A. Poole, M. J. Homann, M. Bae-Lee, and G. M. Carman, J. Bacteriol. 168, 668 (1986). 8 A. M. Bailis, M. A. Poole, G. M. Carman, and S. A. Henry, Mol. Cell. Biol. 7, 167 (1987). 9 M. Bae-Lee and G. M. Carman, J. Biol. Chem. 259, 10857 (1984). 10 B. W. Agranoff and W. D. Suomi, Biochem. Prep. 10, 47 (1963). H G. M. Carman and A. S. Fischl, J. FoodBiochem. 4, 53 (1980). 12 T. J. Larson, T. Hirabayashi, and W. Dowhan, Biochemistry 15, 974 (1976). 13 W. Dowhan and T. Hirabayashi, this series, Vol. 71, p. 555. 14 A. S. Fischl and G. M. Carman, J. Bacteriol. 154, 304 (1983). ~5G. M. Carman and A. S. Fischl, this volume [36]. i6 G. M. Carman and J. Matas, Can. J. Microbiol. 27, 1140 (1981).
300
SYNTHASES
[35]
the phases are separated by a 2-min centrifugation at 100 g. A 0.5-ml sample of the chloroform phase is removed and taken to dryness on an 80° water bath. Betafluor (4 ml) is added to the sample, and radioactivity is determined by scintillation counting. The chloroform-soluble phospholipid product of the reaction phosphatidylserine is analyzed by thin-layer chromatography with standard phosphatidylserine.9,16 Alternatively, activity is measured by following the release of radiolabeled CMP from radiolabeled CDPdiacylglycerol. 16 One unit of enzymatic activity is defined as the amount of enzyme that catalyzes the formation of 1 nmol of product per minute. Protein is determined by the method of Bradford.17 Buffers which are identical to those containing the protein samples are used as blanks. The presence of Triton X-100 does not interfere with the protein determination, provided the blank contains a final concentration of detergent identical to that of the sample. 9 Electroblotting of Phosphatidylserine Synthase Activity 18 The procedure for the electroblotting of phosphatidylserine synthase is similar to that used for the electroblotting of phosphatidylinositol synthase) 5 Briefly, the enzyme is dialyzed for 3 hr at 8° against sodium dodecyl sulfate-polyacrylamide gel electrophoresis treatment buffer. The enzyme is subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Enzyme on the nitrocellulose paper is renatured in 50 mM Tris-HCl (pH 8.0), 0.6 mM MnC12, 30 mM MgCI2, 1 mM CDPdiacylglycerol, 0.5% Triton X-100, 10 mM 2mercaptoethanol, 20% glycerol, 3% bovine serum albumin for 1 hr at 8°. The presence of CDPdiacylglycerol and cofactors in the renaturation buffer stabilizes phosphatidylserine synthase activity. The nitrocellulose paper is used for enzyme assays. The recovery of phosphatidylserine synthase activity is about 10%. This procedure allows the confirmation of the molecular weight of phosphatidylserine synthase. Growth of Yeast
Wild-type S. cerevisiae strain $288C (a gal2) is used for enzyme purification. Cells are grown in 1% yeast extract, 2% peptone, and 2% glucose at 28° to late exponential phase, harvested by centrifugation, and stored at - 80°. 14,15 17 M. M. Bradford, Anal. Biochem. 72, 248 (1976). 18 M. A. Poole, A. S. Fischl, and G. M. Carman, J. Bacteriol. 161, 772 (1985).
[35]
PHOSPHATIDYLSERINE SYNTHASE FROM YEAST
301
Purification Procedure All steps are performed at 5° .
Step 1: Preparation of Microsomes. Cells (75 g, wet weight) are disrupted with glass beads in 50 mM Tris-HCl buffer (pH 7.5) containing I mM Na2EDTA, 0.3 M sucrose, and 10 mM 2-mercaptoethanol.14'15 Glass beads and unbroken cells are removed by centrifugation at 1500 g for 10 min to obtain the cell extract. Microsomes are collected from the cell extract by differential centrifugation.14'15 Microsomes are washed with and resuspended in 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM 2mercaptoethanol and 20% glycerol (w/v). Step 2: Preparation of Triton X-IO0 Extract. The microsome fraction is suspended in 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM MnCI2, 30 mM MgCI 2, 10 mM 2-mercaptoethanol, 0.5 M KC1, 20% glycerol (w/v), and 1% Triton X-100 at a final protein concentration of 10 mg/ml. After incubation for 1 hr at 5°, the suspension is centrifuged at 100,000 g for 2 hr to obtain the solubilized (supernatant) fraction. Solubilization of the enzyme from microsomes with 1% Triton X-100 is dependent on the presence of MnCI 2 in the solubilization buffer. 14'16 Step 3: CDPdiacylglycerol-Sepharose Chromatography. A CDPdiacylglycerol-Sepharose column (0.9 x 4 cm) is equilibrated with 50 ml of 50 mM Tris-HC1 buffer (pH 8.0) containing 0.6 mM MnC12, 30 mM MgC12, 20% glycerol, 10 mM 2-mercaptoethanol, and 0.5% Triton X-100. The Triton X-100 extract is applied to the affinity column in 1.5-ml samples. To effect enzyme binding, each sample is incubated in the column for 10 min before the addition of the next sample. After the entire Triton X-100 extract is applied, the column is washed with 30 ml of equilibration buffer containing 1 M NaCI. The column is then saturated with equilibration buffer containing 1 mM CDPdiacylglycerol and 1 M NaCI and incubated for 1 hr. Phosphatidylserine synthase is eluted from the column with this buffer at a flow rate of I ml/min. Fractions containing activity are pooled and desalted by dialysis or Sephadex G-25 chromatography using 20 mM Tris-HCl buffer (pH 8.0) containing 0.3 mM MnC12, 10 mM MgCI 2 , 20% glycerol, 10 mM 2-mercaptoethanol, and 0.5% Triton X-100. Step 4:DE-53 Chromatography. A DE-53 (Whatman, Clifton, N J) column (0.6 × 4 cm) is equilibrated with 20 ml of 20 mM Tris-HCl buffer (pH 8.0) containing 0.3 mM MnCI 2 , 10 mM MgC12 , 20% glycerol, 10 mM 2-mercaptoethanol, and 0.5% Triton X-100. Desalted enzyme from the previous step is applied to the column at a flow rate of 1 ml/min. The column is washed with 10 ml of equilibration buffer followed by elution of the enzyme with equilibration buffer containing 1 M NaCl. Fractions
302
SYNTHASES
[35]
TABLE I PURIFICATION OF PHOSPHATIDYLSERINE SYNTHASE FROM S a c c h a r o m y c e s c e r e v i s i a e a
Purification step 1. Microsomes 2. Triton X-100 extract 3. CDPdiacylglycerolSepharose 4. DE-53 chromatography
Total units (nmol/min)
Protein (mg)
Specific activity (units/mg)
Purification (-fold)
Yield (%)
591 579 457
493 241 0.34
1.2 2.4 1340
1 2 1120
100 98 77
421
0.18
2300
1920
71
a Data are from Bae-Lee and Carman. 9
containing activity are pooled and desalted as described above. The purified enzyme is 100% stable for at least 6 months when stored at - 8 0 °. Enzyme Purity. The four-step purification scheme summarized in Table I results in a nearly homogeneous preparation of phosphatidylserine synthase as evidenced by native 19and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 9 Native polyacrylamide gels contain 0.5% Triton X100. The enzyme preparation does not contain the CDPdiacylglyceroldependent enzymes phosphatidylinositol synthase, CDPdiacylglycerol synthase, nor phosphatidylglycerophosphate synthase. The subunit molecular weight of the purified enzyme is 2 3 , 0 0 0 . 9'18 When the enzyme is partially purified in the presence of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF), a protein with a molecular weight of 30,000 as well as the 23,000 subunit is isolated. 2°The 23,000 subunit ofphosphatidylserine synthase is a proteolytic cleavage product of the 30,000 subunit of phosphatidylserine synthase. 2° The enzyme is purified about 2000-fold over the microsomal fraction and about 5000-fold relative to the activity in cell extracts. Phosphatidylserine synthase can also be purified from strain VAL2C(YEpCH01), 9 which bears a hybrid plasmid that directs overproduction of the enzyme. 21 The phosphatidylserine synthase-overproducing strain facilitates the acquisition of larger amounts of pure enzyme. 9
19 M. A. Poole, Ph.D. Thesis, Rutgers University, New Brunswick, New Jersey (1986). 2o K. Kiyono, K. Miura, Y. Kushima, T. Hikiji, M. Fukushima, I. Shibuya, and A. Ohta, J. Biochem. (Tokyo) 102, 1089 (1987). 21 V. A. Letts, L. S. Klig, M. Bae-Lee, G. M. Carman, and S. A. Henry, Proc. Natl. Acad. Sci. U.S.A. 80, 7279 (1983).
[35]
PHOSPHATIDYLSERINE SYNTHASE FROM YEAST
303
Reconstitution of Phosphatidylserine Synthase Pure phosphatidylserine synthase has been reconstituted into unilamellar phospholipid vesicles containing its substrate CDPdiacylglycerol.22 The procedure is similar to that used for the reconstitution of phosphatidylinositol synthase 23 with some modifications. The reconstitution procedure is described elsewhere in this volume.15 Pure phosphatidylserine synthase is reconstituted into vesicles directly from mixed micelles containing pure enzyme, phospholipid, Triton X-100, and octylglucoside. The final concentrations of Triton X-100, octylglucoside, and phospholipids in the reconstitution mixture are 2.0, 150, and 10.6 mM, respectively. The final molar ratios of octylglucoside to Triton X-100, octylglucoside to phospholipids, and phospholipids to CDPdiacylglycerol are 75 : 1, 14 : l, and 16 : l, respectively. Octylglucoside inhibits phosphatidylserine synthase, and activity cannot be measured in the reconstitution mixture. The reconstitution mixture is immediately passed through a Sephadex G-50 superfine column (1.5 x 32 cm) equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM MnCI2, I0 mM 2-mercaptoethanol, 250 mM NaCl, and 10% glycerol at a flow rate of 20 ml/hr at 5°. The column is pretreated with a 1 mg/ml sonicated suspension of phospholipids (phosphatidylcholine-phosphatidylethanolamine-phosphatidylinositol-phosphatidylserine, 3 : 2 : 2 : l, v/v), which increases the yields of reconstituted activity. Reconstituted phosphatidylserine synthase elutes from the column in the void volume. The average size of vesicles containing phosphatidylserine synthase is 90 nm in diameter. 22 Each vesicle contains about 1 molecule of enzyme, which is reconstituted asymmetrically with 80-86% of its active site facing outward. 22 Maximum reconstituted phosphatidylserine synthase activity is obtained with vesicles containing phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine, 22the four major phospholipids in wild-type S. cerevisiae. 5'24 Properties of Phosphatidylserine Synthase Optimum phosphatidylserine activity requires either MnCI2 (0.6 mM) or MgCI2 (20 mM) at the pH optimum of 8.0 using a mixed micelle assay of Triton X-100 and CDPdiacylglycerol.9 The manganese requirement of 22 j. M. Hromy and G. M. Carman, J. Biol. Chem. 261, 15572 (1986). 23 A. S. Fischl, M. J. Homann, M. A. Poole, and G. M. Carman, J. Biol. Chem. 261, 3178 (1986). 24 M. L. Greenberg, L. S. Klig, V. A. Letts, B. S. Loewy, and S. A. Henry, J. Bacteriol. 153, 791 (1983).
304
SYNTHASES
[35]
the enzyme reconstituted into phospholipid vesicles is 5 m M . 22 The K m value for serine is 0.83 mM as determined with a mixed micelle substrate of Triton X-100 and CDPdiacylglycerol.25 Phosphatidylserine synthase activity is dependent on the bulk (83 /zM) 25 and surface (2.9 mol %)26 concentrations of CDPdiacylglycerol, characteristic of surface dilution kinetics. 27 Based on the results of kinetic experiments, 25 the ability of the enzyme to catalyze isotopic exchange reactions between substrates and products, 23 and a stereochemical analysis of the reaction using 3~p nuclear magnetic resonance spectroscopy 2~ the enzyme catalyzes a Bi-Bi sequential reaction mechanism. Phosphatidylserine synthase binds to CDPdiacylglycerol before serine, and phosphatidylserine is released prior to CMP in the reaction sequence. 9 Phosphatidylserine synthase uses dCDPdiacylglycerol as well as CDPdiacylglycerol as substrate. 9 Phosphatidylserine synthase is labile above 40 ° and is inactivated by thioreactive agents. 9 Inositol (K i 65 ~M) is a noncompetitive inhibitor of phosphatidylserine synthase, 25 whereas cardiolipin (K i 0.7 mol %) and diacylglycerol (7 mol %) are competitive inhibitors of the enzyme. 26 Phosphatidate (Ka 0.033 mol %), phosphatidylcholine (Ka 3.4 mol %), and phosphatidylinositol (Ka 3.2 mol %) are activators of phosphatidylserine synthase activity. 26 Phosphatidylserine synthase (23,000 subunit 29and 30,000 subunit) is phosphorylated by cAMP-dependent protein kinase. 29 The enzyme has one phosphorylation site, a serine residue, which results in a 60-70% reduction in enzyme activity. 29The phosphorylation of phosphatidylserine synthase results in a decrease in the rate phosphatidylserine synthesis in o i v o . 3°
Synthetic and Analytical Uses
Pure phosphatidylserine synthase can be used to synthesize radiolabeled phosphatidylserine from CDPdiacylglycerol and labeled serine.9 The enzyme can be used to synthesize various fatty acyl derivatives of phosphatidylserine using the appropriate fatty acyl derivatives of CDPdiacylglycerol as substrate. Pure phosphatidylserine synthase can also be used 25 M. J. Kelley, A. M. Bailis, S. A. Henry, and G. M. Carman, J. Biol. Chem. 263, 18078 (1988). 26 M. Bae-Lee and G. M. Carman, J. Biol. Chem. 26.5, 7221 (1990). 27 R. A. Deems, B. R. Eaton, and E. A. Dennis, J. Biol. Chem. 250, 9013 (1975). C. R. H. Raetz, G. M. Carman, W. Dowhan, R.-T. Jiang, W. Waszkuc, W. Loffredo, and M.-D. Tsai, Biochemistry 26, 4022 0987). z9 A. J. Kinney and G. M. Carman, Proc. Natl. Acad. Sci. U.S.A. 85, 7962 (1988). 30 A. J. Kinney, M. Bae-Lee, S. Singh Panghaal, M. J. Kelley, P. M. Gaynor, and G. M. Carman, J. Bacteriol. 172, 1133 (1990).
[36]
PHOSPHATIDYLINOSITOL
SYNTHASE
FROM
YEAST
305
to determine quantitatively the serine concentration b y following the formation of radiolabeled C M P f r o m radiolabeled CDPdiacylglycerol. Acknowledgments This work was supported by U.S. Public Health Service Grant GM-28140 from the National Institutes of Health, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund.
[36] P h o s p h a t i d y l i n o s i t o l
Synthase
from Yeast
B y GEORGE M. CARMAN and ANTHONY S. FISCHL
Introduction Phosphatidylinositol synthase (CDPdiacylglycerol-myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) catalyzes the incorporation of CDP-diacylglycerol + inositol--~ phosphatidylinositol + CMP inositol into phosphatidylinositol.~ The e n z y m e 2 and its product phosphatidylinositol 3,4 are essential to the growth of the yeast S a c c h a r o m y c e s cerevisiae. Phosphatidylinositol synthase activity is associated with the mitochondrial, 5'6 m i c r o s o m a l : ,6 and p l a s m a m e m b r a n e 7 fractions of S. cereoisiae. Microsome-associated phosphatidylinositol synthase has been purified to near homogeneity, primarily b y CDPdiacylglycerol-Sepharose affinity c h r o m a t o g r a p h y : We describe here the purification, reconstitution, and properties of the e n z y m e . Preparation of CDPdiacylglycerol CDPdiacylglycerol is p r e p a r e d from phosphatidic acid and C M P m o r pholidate by the method of Agranoff and Suomi 9 with the modifications of t H. Paulus and E. P. Kennedy, J. Biol. Chem. 235, 1303 (1960). 2 j. Nikawa, T. Kodaki, and S. Yamashita, J. Biol. Chem. 262, 4876 (1987). 3 S. A. Henry, K. D. Atkinson, A. J. Kolat, and M. R. Culbertson, J. Bacteriol. 130, 472 (1977). 4 G. W. Becker and R. L. Lester, J. Biol. Chem. 252, 8684 (1977). 5 G. S. Cobon, P. D. Crowfoot, and A. W. Linnane, Biochem. J. 144, 265 (1974). 6 K. Kuchler, G. Daum, and F. Paltauf, J. Bacteriol. 165, 901 (1986). 7 A. J. Kinney and G. M. Carman, J. Bacteriol. 172, 4115 (1990). 8 A. S. Fischl and G. M. Carman, J. Bacteriol. 154, 304 (1983). 9 B. W. Agranoff and W. D. Suomi, Biochem. Prep. 10, 47 (1963).
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
