Purification and Characterization of Phosphatidate Phosphatase from Saccharomyces cerevisiae*

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, Affi- Gel 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. phosphatase activity wag associated with the purified 91,000 subunit. The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7. The K,,, value for phosphatidate was 50 pM, and the V,, 30 pmol/min/mg. The turnover num- ber (molecular activity) for the enzyme was 2.7 X 10’ min“ at pH 7 and 30 “C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 “C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.

PA phosphatase catalyzes the formation of the diacylglycerol needed for the above reactions (5,7).
The addition of inositol to the growth 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 the expense of phospholipid (9, 10). The regulation of PA phosphatase is likely to influence the proportional synthesis of phospholipids and triacylglycerols as well as the primary 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.

DISCUSSION
PA 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)(29)(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. phosphatase from any organism. The purification of PA phosphatase required the solubilization of the enzyme from membranes with sodium cholate followed by several classical protein purification techniques. The eight-step purification scheme reported here resulted in a PA phosphatase preparation that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was purified 9833-fold relative to the activity in the cell extract with a final specific activity of 30 Fmol/min/mg. The fold purification for PA phosphatase was in the same range of other phospholipid biosynthetic enzymes that have been purified from S. cereuisiae (13,21,23,26). However, the turnover number for PA phosphatase was about 5-to 20-fold higher than other yeast phospholipid biosynthetic enzymes (13,21,23,26). Analysis of pure PA phosphatase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated an apparent subunit molecular weight of 91,000. The native molecular weight of the pure enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12 in the presence of sodium cholate. Since, the micellar molecular weight of sodium cholate ranges from 900-1800 (31), the estimated molecular weight of PA phosphatase by gel filtration chromatography was in close agreement with the molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It appears that PA phosphatase exists as a monomer.
The cytosolic-associated PA phosphatase from S. cereuisiae has been partially purified 600-fold, and its basic properties have been studied (30). The molecular weight of the cytosolic form of the enzyme is 75,000 as determined by gel filtration chromatography with Sephadex G-100 (30). The basic enzymological properties (pH optimum, magnesium dependence, and K,,, for PA) of the pure membrane-associated PA phosphatase were similar to those of the partially pure cytosolic associated enzyme (30).
PA phosphatase activity was inhibited by CDP-diacylglycerol. CDP-diacylglycerol is the source of the phosphatidyl moiety in the primary route of synthesis of the major phospholipids in yeast (1). It might be expected that the partitioning of PA between CDP-diacylglycerol and diacylglycerol would favor CDP-diacylglycerol. Therefore, the regulation of PA phosphatase by CDP-diacylglycerol might be expected. The pure enzyme was also inhibited by diacylglycerol and triacylglycerol. The inhibition of PA phosphatase by these lipids may be evidence of regulation of triacylglycerol synthesis by feedback inhibition. Future studies from this laboratory will be directed toward gaining a better understanding of the regulation of PA phosphatase and its relationship to overall lipid metabolism in S. cereuisiae.
In animal cells, PA phosphatase is believed to play a major role in the regulation of lipid synthesis (32). A number of studies have shown that the cytosolic form of PA phosphatase represents an inactive reserve of enzyme which is activated upon its translocation to the endoplasmic reticulum (32). The translocation of the enzyme occurs in response to increases in the intraCellular concentrations of fatty acids and acyl-CoA ester6 (32). There is no evidence for the translocation of PA phosphatase from the cytosol to membranes in yeast. The availability of antibodies to both the membrane and cytosolic forms of the enzymes would facilitate translocation studies in yeast.
In summary, we have purified and characterized membraneassociated PA phosphatase from s. cereuisiae. The availability of purified PA phosphatase will permit further studies on the mode of action and regulation of this important enzyme of lipid metabolism. 14.

32.
Henry com/nmol> under the assaiy conditions described above. The chloroform-soluble l;did p;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~-glaaial acetic 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  GbrorPatmraDtir-Rn Affi-Gel Blue c0112M (1.5 x 10 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 a flow rate of 30 mVh. The column was washed with 3.5 column volumes Of from the previous step. The DE-53 purified enzyme was applied to the column at equilibration buffer containing 0.1 n NaC1 and 11 sodium cholate. PA 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. cm) vas equilibrated with 10 mn potas61um phosphate buffer (pH 7.0) containing v-A hydroxylapatite column (1.5 x 7 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 potassium phosphate gradient (0.01 -0.15 I ) in equilibration buffer. The 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. 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 of 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.
The overall purification Of PA phosphatase over the cell extract was 9811-A summary of the purification Of PA phosphatase is presented in Table 1. fold. 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 eletrophorsais in the presence of sodium dodecyl sulfate &t 8 C. Fo1lowlng
Purified PA phosphatase v a s aubjected to palyscrglamide ?el electrophoresis one lane from a slab gel wae stained with silver and analyzed by densitometry. A duplicate lane Was cut into 0.5" slices and assayed COT PA phoaphataae activity. This analysis showed that the protein preparation wan weight Of 91,000 (Fig. 78) hand, the addition of 1 mu Kg + ions to the assay system resulted in complete 100% and 85% by 1 mY p-chlo='ole~curiphenyl~"lf~"i= acid and 1 N-inactivation of enzyme activity. PA phoaphataee activity vas a1110 inhibited ethylmaleimide, reepectively. The addition Of 2-msrcaptoethanol to the asmay system reversed the inhibition of PA phosphatase activity by these thioreactive compounds. Theae results imply that a sulfhydryl group is required for PA phosphatase activity. purification schema.