Purification and Characterization of Phosphatidate Phosphatase from ...

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Dec 12, 1988 - Yi-Ping Lin and George M. Carman$. From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, ...
Vol. 264,, No. 15, Issue of May 25. pp. 8641-8645,1989 Printed in U.S.A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification and Characterization of Phosphatidate Phosphatase from Saccharomyces cerevisiae* (Received for publication, December 12, 1988)

Yi-Ping Lin and GeorgeM. Carman$ From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers Uniuersity, New Brunswick, New Jersey08903

Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae. The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53,AffiGel Blue, hydroxylapatite, Mono Q, and Superose 12. The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidate phosphatase activity wag associated with the purified 91,000 subunit. The molecular weight of thenative enzyme was estimated tobe 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 value for phosphatidate was 50 pM, at pH 7. The K,,, and the V,, was 30 pmol/min/mg. The turnover number (molecular activity) for the enzyme was 2.7 X 10’ min“ at pH 7 and 30 “C.The activation energy for the reaction was 11.9kcal/mol, and the enzyme was labile above 30 “C. Phosphatidate phosphatase activity was sensitive tothioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.

PA‘ is an important intermediate of lipid metabolism in the unicellular eucaryote Saccharomyces cerevisiae.The major phospholipids are derived from PA by the reaction sequence PA + CDP-diacylglycerol+phosphatidylserine + phosphatidylethanolamine -+ phosphatidylcholine (1). Triacylglycerols are derived from PA by the reaction sequence P A + diacylglycerol -+ triacylglycerol (1). An auxiliary pathway exists inS. cerevisiae for phosphatidylethanolamine andphosphatidylcholine biosynthesis which is used by the ethanolamine- and choline-requiring mutants defective in phosphatidylserine synthesis (2-4). These mutantssynthesize phosphatidylethanolamine and phosphatidylcholine by the CDPethanolamine- andCDP-choline-based pathways (5,6) by the * This work was supported by United States Public Health Service Grant GM-28140 from the National Institutes of Health, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund. This is New Jersey Agricultural Experiment Station Publication D10531-1-89.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence and reprint requests should be addressed. * The abbreviations used are: PA, phosphatidate; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-l-propanesulfonic acid.

reaction sequences PA -+ diacylglycerol + phosphatidylethanolamine and PA * diacylglycerol --z phosphatidylcholine. PA phosphatase catalyzes the formation of the diacylglycerol needed for the above reactions (5,7). The addition of inositol to thegrowth medium of wild-type S. cerevisiae cells results in an increase in PA phosphatase activity (8).PA phosphatase activity also increases when wildtype cells enter the stationary phase of growth (8, 9). PA phosphatase activity is associated with the membrane and cytosolic fractions of S. cerevisiae (8,9). The PA phosphatase activity associated with each of these cellular fractions is regulated by inositol (8) and the growth phase (8, 9) in a similar manner. The increase in PA phosphatase activity in response to inositol supplementation correlates with an increase in phospholipid content at the expense of triacylglycerol (8). On the other hand, the increase in PA phosphatase activity in the stationary phase of growth correlates with an increase in triacylglycerol content at theexpense of phospholipid (9, 10). The regulation of PA phosphatase is likely to influence the proportional synthesis of phospholipids and triacylglycerols as well as theprimary and auxiliary pathways for the synthesis of phosphatidylethanolamine andphosphatidylcholine in S. cereuisiae. A purified preparation of PA phosphatase is required for defined studies on the mechanism and regulation of this important enzyme of lipid metabolism in S. cereuisiae. In this communication, we report on the purification of the membrane-associated PA phosphatase 9833-fold to apparent homogeneity. This is the first report of the purification of any form of PA phosphatase (cytosolic or membrane-associated) from any organism. We also report on the enzymological properties of the pure enzyme. EXPERIMENTAL PROCEDURES AND RESULTS~

DISCUSSION

P A phosphatase is an important enzyme of lipid metabolism. The regulation of this enzyme is likely to influence phospholipid and triacylglycerol biosynthesis in S. cerevisiae as well as in higher eucaryotic organisms (27). Membrane and cytosolic forms of PA phosphatase exist in animals, plants, and bacteria (28,29). Unsuccessful attempts have been made to purify PA phosphatase from rat liver, rat lung, pig brain, adipose tissue, mung bean, and yeast (28-30). In this communication, we describe the purification and characterization of membrane-associated PA phosphatase from S. cerevisiae. This is the first report of the successful purification of PA Portions of this paper (including “Experimental Procedures,” “Results,” Tables I and 11, and Figs. 1-10) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

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phosphatase from any organism. The purification of PA phos- of purified PA phosphatase will permit further studies on the phatase required the solubilization of the enzyme from mem- mode of action and regulation of this important enzyme of branes with sodium cholate followed by several classical pro- lipid metabolism. tein purification techniques. The eight-step purification Acknowledgments-We thank Charles J. Belunis, Michael J. Kelscheme reported here resulted in a PA phosphatase preparation that was apparently homogeneous as evidenced by so- ley, Kelly R. Morlock, Myongsuk Bae-Lee, Paulette M. Gaynor, and Anthony J. Kinney for their many helpful suggestions during the dium dodecyl sulfate-polyacrylamide gel electrophoresis. The course of this work. enzyme was purified 9833-fold relative to the activity in the cell extract with a final specific activity of 30 Fmol/min/mg. REFERENCES The fold purification for PA phosphatase was in the same 1. Henry, S. A. (1982) in The Molecular Biology of the Yeast Sacrange of other phospholipid biosynthetic enzymes that have charomyces: Metabolism and Gene Expression (Strathern, J. N., Jones, E. W., and Broach, J. R., eds) pp. 101-158, Cold Spring been purified from S. cereuisiae (13,21,23,26). However, the Harbor Laboratory, Cold Spring Harbor, NY turnover number for PA phosphatase was about 5- to 20-fold 2. Atkinson, K., Fogel, S., and Henry, S. A. (1980) J. Biol. Chem. higher than other yeast phospholipid biosynthetic enzymes 255,6653-6661 (13, 21, 23, 26). Analysis of pure PA phosphatase by sodium 3. Kovac, L., Gbelska, I., Poliachova, V., Subik, J., and Kovacova, dodecyl sulfate-polyacrylamide gel electrophoresis indicated V. (1980) Eur. J. Biochem. 111,491-501 an apparent subunit molecular weight of 91,000. The native 4. Nikawa, J., and Yamashita, S. (1981) Bwchim.Biophys.Acta 665,420-426 molecular weight of the pure enzyme was estimated to be 5. Kennedy, E. P., and Weiss, S. B. (1956) J. Biol. Chem. 222,19393,000 by gel filtration chromatography with Superose 1 2 in 214 the presence of sodium cholate. Since, the micellar molecular 6. Christiansen, K. (1979) Biochim. Biophys. Acta5 7 4 , 448-460 weight of sodium cholate ranges from 900-1800 (31), the 7. Kates, M. (1955) Can. J. Biochem. 35,575-589 estimated molecular weight of PA phosphatase by gel filtra8. Morlock, K. R., Lin, Y.-P., andcarman, G. M. (1988) J. Bacteriol. tion chromatography was in close agreement with the molec170,3561-3566 9. Hosaka, K., and Yamashita, S. (1984) Biochim. Biophys.Acta ular weight estimated by sodium dodecyl sulfate-polyacryl796,110-117 amide gel electrophoresis. It appears that PA phosphatase 10. Taylor, F. R., and Parks, L. W. (1979) Biochim. Biophys. Acta exists as a monomer. 575,204-214 The cytosolic-associated PA phosphatase from S. cereuisiae 11. Greenberg, M.L., Klig, L. S., Letts, V. A.,Loewy,B. S., and has been partially purified 600-fold, and its basic properties Henry, S. A. (1983) J. Bacteriol. 1 5 3 , 791-799 have been studied (30). The molecular weight of the cytosolic 12. Klig, L. S., Homann, M. J., Carman, G. M., and Henry, S. A. (1985) J. Bacteriol. 1 6 2 , 1135-1141 form of the enzyme is 75,000 as determined by gel filtration chromatography with Sephadex G-100 (30). The basic enzy- 13. Fischl, A. S., and Carman, G. M. (1983) J. Bacteriol. 1 5 4 , 304311 mological properties (pH optimum, magnesium dependence, 14. Walsh, J. P., and Bell, R. M. (1986) J. Bwl. Chem. 261, 6239for PA) of the pure membrane-associated PA phosand K,,, 6247 phatase were similar to those of the partially pure cytosolic 15. Tillman, T. S., and Bell, R. M. (1986) J. Biol. Chem. 261,9144associated enzyme (30). 9149 P A phosphatase activity was inhibited by CDP-diacylglyc- 16. Steiner, S., and Lester, R. L. (1972) J. Bacteriol. 109,81-88 erol. CDP-diacylglycerol is the source of the phosphatidyl 17. Laemmli, U. K. (1970) Nature 227,680-685 Merril, C. R., Dunau, M.L., and Goldman, D. (1981) Anal. moiety in the primary route of synthesis of the major phos- 18. Biochem. 1 1 0 , 201-207 pholipids in yeast (1).It might be expected that thepartition- 19. Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254 ing of PA between CDP-diacylglycerol and diacylglycerol 20. Deems,R. A., Eaton, B.R., and Dennis, E. A. (1975) J. Biol. Chem. 250,9013-9020 would favor CDP-diacylglycerol.Therefore, the regulation of PA phosphatase by CDP-diacylglycerol might be expected. 21. Bae-Lee, M., and Carman, G. M. (1984) J. Biol. Chem. 2 5 9 , 10857-10862 The pure enzyme was also inhibited by diacylglycerol and 22. Carman, G.M., and Dowhan, W. (1979) J. Biol. Chem. 2 5 4 , triacylglycerol. The inhibition of PA phosphatase by these 8391-8397 lipids may be evidenceof regulation of triacylglycerol synthe- 23. Kelley,M. J., and Carman, G. M. (1987) J. Biol. Chem. 2 6 2 , sis by feedback inhibition. Future studiesfrom this laboratory 14563-14570 will be directed toward gaining a better understandingof the 24. Dowhan, W., Wickner, W. T., and Kennedy, E. P. (1974) J. Biol. Chem. 249,3079-3084 regulation of PA phosphatase and its relationship to overall 25. Dutt, A., and Dowhan, W. (1985) Biochemistry 24,1073-1079 lipid metabolism in S. cereuisiae. 26. Belunis, C. J., Bae-Lee, M., Kelley, M. J., and Carman, G. M. In animal cells, PA phosphatase is believed to play a major (1988) J. Biol. Chem. 263,18897-18903 role in the regulation of lipid synthesis (32). A number of 27. Kennedy, E. P. (1986) in Lipids and Membranes: Past, Present and Future (Op den Kamp, J. A. F., Roelofsen, B., and Wirtz, studies have shown that thecytosolic form of P A phosphatase K.W.A., eds) pp. 171-206, Elsevier Science Publishers B.V., represents an inactive reserve of enzyme which is activated Amsterdam upon its translocationto theendoplasmic reticulum (32). The 28. Brindley, D. N. (1988) in Phosphatidate Phosphohydrohe(Brintranslocation of the enzyme occurs in response to increases dley, D. N., ed) Vol. 1, pp. 2-19, CRC Press, Inc., Boca Raton, in the intraCellular concentrations of fatty acids and acylFL CoA ester6 (32). Thereis no evidence for the translocation of 29. Harwood, J. L., and Price-Jones, M. J. (1988) in Phosphatidate pp. 2-37, CRC PhosDhohvdrohe (Brindley, D. N., ed) Vol. 2, ~. PA phosphatase from the cytosol to membranes in yeast. The Press, In;, Boca Raton, FL availability of antibodies to boththe membrane and cytosolic K., and Yamashita, S. (1984) Biochim. Biophys.Acta forms of the enzymes would facilitate translocation studies in 30. Hosaka, 796,102-109 yeast. 31. Helenius., A.., and Simons,. K. (1975) . . Biochim. Bwphys. Acta4 1 5 , In summary, we have purified and characterized membrane29-79 associated PA phosphatase from s. cereuisiae. The availability 32. Brindley, D. N. (1985) Prog. Lipid Res. 2 3 , 115-133 "

Phosphatidate Phosphatase Supplemental naterial to Purification and Characterization of Phosphatidate Phosphatase from SaFCharOmVCeS cerevisiae by Y i - P m g L m and George n . Carman W E R I I E N T A L PROCEDURES Materials

from S. cerevisiae

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m %. GbrorPatmraDtir- R n Affi-Gel Blue c0112M (1.5 x 10 cm) vas equilibrated with 5 column VOlUmes of 50 mJ# Tris maleate buffer (pH 7.0) containing 10 mn HqCl2, l o mn 2-mercaptoethanol, 201 glycerol. and 0.1 H NaCl followed by equilibratlan with 1 column voluaa Of the (Iamt) buffer Containing 1% sodium cholate. The equilibration of the column with buffer containing 0.1 n NaC1 eliminated the need to desalt the enzyme preparation from the previous step. The DE-53 purified enzyme was applied to the column at a flow rate of 30 mVh. The column was washed with 3.5 column volumes Of 0.1 n NaC1 and 11 sodium cholate. PA equilibration buffer containing phoephatase was then eluted from the column with 9 column volluee Of D. linear NaCl gradlent ( 0 . 3 - 1.0 11) in the same buffer at a flaw rate of 30 ml/h. The peak Of PA phosphatase activity eluted from the column at a NaCl concentration of about 0.6 M (Fig. 2). The most active fractions were pooled and the enzyme preparation was desalted by dialysis against equilibration buffer.

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v-

A hydroxylapatite column (1.5 x 7 cm) vas equilibrated with 10 mn potas61um phosphate buffer (pH 7.0) containing 5 mn ngcl 10 mpI 2-marcaptoethanol. 20% glycerol and I t sodium cholate. Dialyzed hn;yme from the previous step was applied to the column at a flow rate of 15 ml/h. The column was washed with 2 column volumes Of equilibration buffer followed by elution of PA phosphatase with 8 column YolUmeB of Di linear 0.15 I ) in equilibration buffer. The potassium phosphate gradient (0.01 concentration of ngcl was increased to 10 M in the elution buffer. The peak of activity was elute$ from the column at a potassium phosphate concentration Of about 0.07 I4 (Fig. 3 ) . The most active fractions were pooled and Used for the next step in the purification.

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srt;p l+ 9 Q A anion exchange lono Q Column ( 0 . 5 X 5 equlllbrated wlth wlth 6 column volumes Of 50 IM Tris malaate buffer 10 mn 2-marcaptoethanol, 20a glycerol. 11 (pH 7.0) containing 10 mn ngcl sodium cholate. and 0.1 II N s 6 . The hydroxylapatite purified enzyme was applled to thecolumn at a flow rate Of 10 ml/h. The column ma8 washed with 8 column volumes Of equilibration buffer folloved by 2 column volumss of a linear NaC1 gradient (0.1 0.17 PI) in equilibration buffer. The encyme YaS then eluted from the column with 10 column volumes Of a linear NaCl gradient (0.17- 0.4 If) in equilibration buffer. The peak of enzyme activity eluted at a NaCl concentration of about 0.22 n (Fig. I ) . Fractions containing activity were pooled and used for the next step. Cm) was

Conditions- 5- cerevisiae strain ade5 MAT^ (11, 121 was used as a representative wild-type strain for enzyme purification. The organism was nalntained on YEPD (1% yeast extract, 2% peptone, 2% dextrose) media plate6 2% bacto-agar. Far enzyme purification, cells were grown in YEPD oontainlnq medium at 28 OC to late exwnential phase, harvested by centrifugation. and stored at -80 OC as described previous~iy (131. ion Qi substrates- [32P]PA was synthesized enzymatically frDm [32P)E;:"%?: diacylglycerol using Ei diacylglyceral kknase under the ssay conditions described previously ( 1 4 1 . 12- HIPA was prepared from [2qH)glycerol 3-phosphate and palmitoyl-COA using yeast nic=.090n.l-a5Soci.ted glycerol 3-phosphate acyltransferase under the assay conditions described previously (15). labeled PA vas purified by thin layer chromatography using the Solvent system chlaraforn/nethanol/vater (65:25:4).

wPA phosphataae activity w s routinely measur53 for 20 lnin by following the release of water-soluble 13'P]P. from 0.5 mld [ P]PA (1.000 (pH 7.0) containing s mn to 2,000 cpa/nrnol) ~n so mn TriS maleate buff& Triton X-100. 10 mM 2-mercaptoethanol 2 mM nqC1 and enzyme protein in a total volume of 0.1 ml at 30 C' ( 8 ) . 'The substrare PA was delivered to the assay as a uniform mixed micelle with Triton X - l o 0 at a molar ratio Of Triton X - 1 0 0 to PA of 1O:l ( 8 ) . Alternativyy. PA phosphatase activity was measured by following the fornation of (2- H]diacylglyoerol from (2-'H]PA (1.000 com/nmol> under the assaiy conditions described above. The chloroform-soluble l;didp;oduct of the riaction, diacylglycerol, vas analyzed with standard diacylglycerol by one-dimensional paper chromatography on EDTA-treated SG81 paper (16) using the solvent system hexane-d~sthylethe~-glaaialacetic acid (30:70:1). carrier standard diacylglycerol wae added to the chloroform phase prlor to separation and the position of the labeled diacylqlycerol on the chromatograms was determined after exposure to iodine vapor. The amount of labeled diacylglycerol was determined by lhquid scintillation counting. All assavs were linear with time and DrOteln concentration. One unit of enzymatic Of

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A?. A gel filtration Supsroae 12 column k 7.01 ( 1 x 30 cm) was aquilibrated with 50 m M Tris maleate buffer (pH 10 mu 2-mercaptoethanol. 201 glycerol, and 1% aodium containing 10 mn lqcl Cholate. The enzyme f& the previous nono p column was applied to the column at a flow rate Of 21 ml/h followed by elution o f the enzyme from the column With equilibration buffer. PA phosphatase activity and protein eluted from the column as a single peak (Fig. 5b. Fractions containing PA phoBphataBe activity were pooled and stored at - 80 C. A summary of the purification Of PA phosphatase is presented in Table 1. The overall purification Of PA phosphatase over the cell extract was 9811fold. with an activity yield of 4 % .

€ w A t Y Q f E a E k a m & m % & W o l e c u l a r l r S I g b f Purified PAphosphatase vas aubjected to palyscrglamide ?el eletrophorsais in the presence of sodium dodecyl sulfate &t 8 C. Fo1lowlng electrophoresis one lane from a slab gel wae stained with silver and analyzed slices and assayed COT by densitometry. A duplicate lane Was cut into 0.5" PA phoaphataae activity. This analysisshowed that theprotein preparation wan essentially homogeneous (Fig. 6A) with an apparent minimum subunit molecular weight Of 91,000 (Fig. 78). Furthermore. PA phosphatase activity was asso-eiated with the M 91,000 subunit [ F i g . 68). Gel filtration chromatography with Superose 12 wasrYsed to approximate the native molecular weight Of PA phoaphstase. PA phosphatase activity eluted from the Superose 12 column with a KaB, value corresponding to an apparent mo1eeule.r Weight Of 93,000 [Stokes ra lua of 38 A) (Flg. 7 A l .

protein.

ElectroDhoresis- Polyacrylamide gel electrophoresis in the presence of sodium dodecyi Sulfate (17) vas perfomed with 101 s l a b gels. Proteins on polyacrylamide gels Were visualzzed by the 611ver stain procedure ( 1 8 ) and analyzed with a scanning densitometer.

w n Deternination- Protein was determined by the method Of Bradford (19). urinq bovine serum albumin a s the Standard. Buffers which were identzcal to those contaming the protein samples were used IS blanks. Pratein was monitored during purification on columns by measuring the absorbance at 280nrn or 405 nm.

u

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L Cells (85 gl were disrupted with glass beads (diameter. 0.5 m) With a Biospec Products Bead-Beater in 50 mn Tris maleate buffer IDH 7.01 Eontainina 1 Iw Na. EDTA. 0.3 n SUCr08e. and 10

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extract. PA phoapha