JOURNAL OF BACTERIOLOGY, Sept. 1992, p. 5702-5710 0021-9193/92/175702-09$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 174, No. 17

The Acyl Dihydroxyacetone Phosphate Pathway Enzymes for Glycerolipid Biosynthesis Are Present in the Yeast Saccharomyces cerevisiae PATRICIA V. RACENIS, JOYCE L. LAI, ARUN K. DAS, PRAKASH C. MULLICK, AMIYA K. HAJRA, AND MIRIAM L. GREENBERG* Department of Biological Chemistry and Neuroscience Laboratory, Mental Health Research Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109 Received 27 March 1992/Accepted 17 June 1992

The presence of the acyl dihydroxyacetone phosphate (acyl DHIAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide. Total membrane fraction of S. cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 1.1.1.101) activity, which has not been reported previously. The reductase has a Vm, of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHLAP (Km = 15 ,uM) by NADPH (Km = 20 ,uM). Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction. Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells. Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells. The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHIAP synthase) in S. cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.

Biosynthesis of phosphatidic acid, the precursor of glycerolipids in all organisms, proceeds via two different routes. In the glycerol phosphate pathway, phosphatidic acid is biosynthesized via the stepwise enzymatic acylation of snglycerol 3-phosphate (GP) by long-chain fatty acyl coenzyme A's (acyl-CoAs). In animals, phosphatidate is also biosynthesized by an alternate pathway, the acyl dihydroxyacetone phosphate (acyl DHAP) pathway. In this pathway, DHAP is acylated by a long-chain fatty acyl-CoA to form acyl DHAP. This compound is then reduced to 1-acyl GP, which is further acylated to yield phosphatidate (Fig. 1). The GP pathway is universal to all organisms, including yeasts (19, 27). In contrast, the acyl DHAP pathway is absent in bacteria and plants (11, 41). In animals, the acyl DHAP pathway is obligatory for ether lipid biosynthesis; the ether bond is synthesized by the substitution of a long-chain alcohol for the acyl group of acyl DHAP, with the release of free fatty acid to form alkyl DHAP (13). The enzymatic conversion of alkyl DHAP to glycerol ether lipids proceeds via reduction of alkyl DHAP to 1-alkyl GP followed by acylation (13). The importance of the acyl DHAP pathway for the biosynthesis of non-ether glycerolipids is controversial (6, 29, 30, 33, 34, 35). Except for some anaerobic bacteria which contain plasmalogens but not alkyl glycerol ethers, both aerobic bacteria and facultative anaerobes, as well as plants, lack ether lipids (9, 28). Consequently, it would seem that the key metabolic role of the acyl DHAP pathway is to provide the sole route for ether lipid biosynthesis. *

Ether lipids have not been detected in yeasts (28), yet Johnston and Paltauf (20) have reported the presence of an acyl DHAP pathway enzyme, DHAP acyltransferase (DHAPAT), in the yeast Saccharomyces carlsbergensis. This finding has been confirmed by Schlossman and Bell (36) for the yeast Saccharomyces cerevisiae. These authors have shown that the physical and biochemical properties of DHAPAT and GP acyltransferase (GPAT) are identical. However, they found no detectable acyl DHAP reductase activity in S. cerevisiae. On the basis of these findings, they ascribed the DHAPAT activity in yeasts to the nonspecificity of GPAT and concluded that the acyl DHAP pathway was absent in this organism. Nevertheless, we have shown that membranes prepared from commercial lyophilized S. cerevisiae contain acyl DHAP reductase (31). In the present report, we confirm the presence of this reductase in the membranes of freshly grown yeast cells and describe the properties of this enzyme in detail, along with those of DHAPAT and GPAT. Furthermore, we show that expression of these three enzymes is regulated to the same extent by oxygen. Our results indicate that the acyl DHAP pathway may be important for non-ether lipid biosynthesis in eukaryotes. MATERIALS AND METHODS Materials. Cytochrome c, succinate, DHAP, GP, NADH, NADPH, NADP+, palmitoyl-CoA, Tris base, EDTA, tergitol, and Tween 80 were obtained from Sigma. [1-'4C]palmitic acid was purchased from Du Pont-New England Nuclear and also from American Radiolabeled Products. Glass beads (0.45- to 0.50-mm diameter) for lysing yeast cells were from

Corresponding author. 5702

ACYL DHAP PATHWAY ENZYMES IN S. CEREVISME

VOL. 174, 1992 NAD

~

NADH

.DHAP

sn-GLYCEROL-3-P acyl CoA

CoASH

.4)

(GPAT)

NADP

1-ACYL-GLYCE,ROL 3-P acyl CoA CoASH

NADPH

(AcyllalkylDHAP reductase)

4)I

1,2-diacylglycerol-3-P (Phosphatidate)

(Alkyl DHAP

syndtase)

ROH CvR'COOH

ALKYL DHAP

(AcyllalkylDHAP

NADPH

reductase)

+

flNADP

ALKYL GP acyl CoA

CoASH

NON-ETHER LIPIDS

ETHER LIPIDS

FIG. 1. Pathways of phosphatidate biosynthesis.

B. Braun Biotech, Inc. Precoated Silica Gel 60 plates for thin-layer chromatography (TLC) were from E. Merck Co. Yeast extract was purchased from Difco. Vitamin-free yeast base was prepared as described in the Difco manual, omitting glucose, histidine, methionine, and tryptophan. Palmitoyl DHAP and hexadecyl DHAP were chemically synthesized as previously described (18). [32P]DHAP and [32P]GP were prepared by the enzymatic phosphorylation of dihydroxyacetone and glycerol, respectively, with [y-32P] ATP as described previously (16). B-[4-3H]NADPH was prepared and purified according to procedures described previously (3). Palmitoyl [32P]DHAP was prepared by the procedure of Hajra et al. (18). Growth conditions of the organism and preparation of subcellular membrane fractions. The wild-type S. cerevisiae strains used in this study were ade S (adeS AM Ta) and D273-1OB (met6 MATTa) (10). Strains were maintained in 15% glycerol at -80°C for long-term storage and on 1% yeast extract-2% peptone-2% glucose (YEPD) plates for shortterm storage. Synthetic minimal media consisted of vitaminfree yeast base (0.27%), vitamins (2), glucose (2%), and adenine (0.15 mM) or methionine (0.002%). In anaerobic growth experiments, media were supplemented with tergitol (10 g/liter), Tween 80 (2.5 ml/iter), and ergosterol (25 mg/liter). To prepare the ergosterol solution, 10 ml of 100% ethanol and 10 ml of melted tergitol were mixed; 0.25 g of ergosterol was added, and the solution was heated until the ergosterol was dissolved and the solution appeared uniform. Two milliliters of this stock was added per liter of medium. For the enzyme characterization studies, strain ade 5 cells grown on YEPD plates were inoculated into Erlenmeyer flasks containing synthetic minimal medium as described above and grown overnight at 30°C in a rotary shaker at 200 rpm. Erlenmeyer flasks containing 1 liter of synthetic minimal medium were inoculated from these overnight cultures and grown for approximately 18 to 24 h at 30°C in a rotary shaker at 200 rpm to the mid-log stage (A550 = 0.5). Cells were harvested by centrifugation at 5,000 rpm (3,000 x g) for 3 min at 4°C and washed once with buffer containing 50 mM Tris-HCl (pH 7.5)-l mM EDTA-0.3 M sucrose-10 mM

5703

P-mercaptoethanol (buffer 1), and pellets were immediately stored at -80°C. Frozen pellets were thawed and resuspended in 50 mM Tris-HCl (pH 7.5)-20% glycerol-10 mM ,-mercaptoethanol (buffer 2, 1 ml/g [wet weight]), and the cells were lysed by vortexing with approximately 0.5 volume of acid-washed glass beads for five 1-min intervals, with cooling of the cells on ice for 1 min between intervals (10). Mixtures were centrifuged in a Sorvall SS-34 rotor at 5,000 rpm (3,000 x g) for 5 min, and supernatants were saved. The pellets were resuspended in buffer 2, and the 5,000-rpm spin was repeated. The pellets were discarded, and supernatants were combined with the first supernatant. The pooled supernatants were centrifuged at 40,000 rpm (100,000 x g) in a Beckman L8-70 ultracentrifuge, using a Ti 70.1 rotor. After centrifugation, the supernatant was discarded and the pellet was resuspended and homogenized in ice-cold 0.25 M sucrose-10 mM Tris-HCl (pH 7.5)-i mM EDTA buffer and then centrifuged as before. The supernatant was again discarded, and the pellet was resuspended in sucrose-TrisHCI-EDTA buffer as described above. The membrane fractions isolated as described above were stored immediately at -20°C. Protein was determined by the method of Lowry et al. (26), using bovine serum albumin (BSA) as the standard. For studies of the effect of oxygen, strain D273-1OB cells were inoculated into Erlenmeyer flasks containing synthetic minimal medium as described above. The cells were then grown, harvested, and fractionated as described for the ade 5 cells. Anaerobic cells were grown under a continuous stream of deoxygenated nitrogen and chilled in ice water for 20 min prior to harvest. Nitrogen was deoxygenated by passage through alkaline dithionite (10 mg of sodium dithionite per ml in 0.1 M sodium phosphate buffer, pH 8.0). Membrane fractions were isolated as described above and stored immediately at -80°C. Enzyme assays. DHAPAT and GPAT were assayed by measuring the 32P-labeled lipids formed from [32PJDHAP and [32P]GP, respectively, and palmitoyl-CoA (17). The assay mixture contained 75 mM Tris-HCl (pH 7.5), 80 ,uM palmitoyl-CoA, 8.3 mM NaF, 8.3 mM MgCl2, 0.42 mM [32P]DHAP or [32P]GP (7,000 cpm/nmol), plus BSA (1.7 mg/ml for DHAPAT or 2.5 mg/ml for GPAT) in a total volume of 0.6 ml, including the subcellular membrane fraction. After a 20-min incubation with constant shaking at 30°C, lipids were extracted by an acidic Bligh and Dyer method and washed (1, 12), and aliquots of the CHCl3 extracts were transferred to scintillation minivials. Solvents were removed by blowing a gentle stream of air at 35°C. To the dried radioactive products was added 2 ml of scintillation solvent (Universol; ICN/Schwartz-Mann), and the radioactivity was determined by liquid scintillation counting. Acyl/alkyl DHAP reductase was assayed by measuring the formation of 3H-labeled lipid from either O-hexadecyl DHAP or palmitoyl DHAP and B-[4-3H]NADPH (23). Hexadecyl DHAP emulsions were prepared by combining 35 nmol of hexadecyl DHAP in chloroform-methanol (24:1) with 20 pug of Tween 20 detergent in CHCl3. The solvents were removed by evaporation with N2, and the residue was emulsified in 0.05 M Tris-HCl (pH 7.5) by sonication before addition to the incubation mixture. The incubation mixture contained 20 mM Tris-HCl (pH 7.5), 10 mM NaF, 0.7 mM EDTA, 60 FM hexadecyl DHAP (emulsified as described above), B-[4-3H]NADPH (70 ,uM, 5,000 cpm/nmol), the subcellular membrane fraction, and water in a total volume of 0.6 ml. The mixture was incubated at 30°C for 20 min with constant shaking, and the reaction was stopped by addition of 2.25 ml of chloroform-methanol (1:2). The product (1-0-

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J. BACTIE;RIOL.

TABLE 1. Requirements for incorporation of radioactivity into lipid from [32P]DHAP and [32P]GP by yeast membrane fractions' Radiolabeled lipid formed (nmol/min/mg of protein)

Conditions

Whole system Whole system Whole system Whole system Whole system

minus minus minus minus

BSA palmitoyl-CoA NaF MgCl2

DHAPAT

GPAT

0.46 0.20 0.00 0.38 0.16

3.60 1.11 0.00 3.08 2.49

_

0

_ E g _; _ E_

CL O

cX

_

a The whole system contained Tris-HCl buffer (75 mM, pH 7.5), NaF (8.3 mM), MgCl2 (8.3 mM), palmitoyl-CoA (80 ,uM), BSA (fatty acid poor, 1 mg), [32P]DHAP or [32P]GP (0.42 mM, 7,000 cpm/nmol), and membrane fraction from yeast cells in a total volume of 0.6 ml. The mixture was incubated at 30'C for 20 min, and radiolabeled lipids were determined as described in the text. The results are averages of data within the linear range obtained at two different protein concentrations (i.e., 25 and 50 ,ug) of yeast membrane fraction.

hexadecyl sn-[2-3H]glycerol 3-phosphate) was extracted under acidic conditions and washed, and its radioactivity was determined as described above. NADPH-cytochrome c reductase was assayed spectrophotometrically by measuring the rate of cyanide-insensitive NADPH-dependent reduction of cytochrome c catalyzed by the yeast subcellular membrane fraction (37). Cytochrome oxidase was assayed spectrophotometrically at 30°C by measuring the rate of cytochrome c oxidation (39). Product identification. The 32P-labeled products were identified by TLC (CHCl3-methanol-acetic acid-5% aqueous sodium metabisulfite, 100:40:12:4) followed by autoradiography as previously described (3). The 3H-labeled products were also identified by TLC (CHCl3-methanol-acetic acid-5% sodium metabisulfate, 100:40:12:4). After separation, the distribution of radioactivity from 3H-labeled products was directly determined by scraping out 1-cm TLC bands and extracting the powders twice with CHC13-methanol (1:1) containing HCl (0.1 M). The extracts were combined into scintillation minivials and dried by blowing a gentle stream of air. Two milliliters of scintillant (Universol; ICN/ Schwartz-Mann) was added to each vial, and the radioactivity was determined by liquid scintillation spectrometry (Beckman LS-133 spectrometer). Other materials and methods were the same as described previously (5, 16, 17, 21). RESULTS

Properties of the yeast total membrane fraction DHAPAT and GPAT activities. Yeast total membrane fraction catalyzes the acylation of both DHAP and GP, as other workers (20, 36) have previously reported. Table 1 shows the requirements for the formation of 32P-labeled lipid from [32P]DHAP and [32P]GP. The formation of labeled lipid is completely dependent on the addition of palmitoyl-CoA. As in mammalian systems, the reactions were facilitated by BSA, presumably by binding palmitoyl-CoA. This lipid substrate is inhibitory at high concentrations (23). Both NaF and Mg2+ stimulate the reaction, probably by inhibiting the phosphatase present in the total membrane fraction (38). Kinetic studies of the acyltransferases from yeast total membrane fraction at 80 ,uM palmitoyl-CoA disclose an apparent Km of 1.27 mM for DHAP with a Vm. of 5.9 nmol/min/mg of protein for DHAPAT (Fig. 2A and inset) and

0

1 [DHAP]

2 (mM)

12

10 1-OE

8

I-_

0 E ,-

7o 6

-

40

Pi

20 0

0

O0

-20

1-

0.0

1.0 [NEM] (mM)

2.0

FIG. 5. Inhibition of DHAPAT and GPAT activities by N-ethylmaleimide. Yeast total membrane fraction protein (25 and 50 p,g) was preincubated in the presence of 0 to 2 mM N-ethylmaleimide in 90 mM Tris-HCl (pH 7.5) in a total volume of 0.25 ml for 15 min on ice. After the preincubation step, the remainder of the assay incubation mixture was added to obtain a final volume of 0.6 ml, and the contents were assayed for DHAPAT and GPAT activities for 20 min at 30°C. The radioactivity in lipid was then determined as described in Materials and Methods.

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RACENIS ET AL.

J. BACTERIOL. 120

-

4 F.

e0

-2

0 :c

3

a

E

-

A

100

80

E -

60 40 20-

la E 2

0 -20

.

0

1

,

'

I

.

.

I

.

I

10 20 30 40 50 60 70 8(0

PREINCUBATION TEMPERATURE (DEG C)

-J

0 0

100 [NADPH] (uM)

200

F. 0 x-

2

$t

F

0 0

^

CD)



0

120

ANAERBKC

6000 0L

o80 4000

0

60

2000

20 0

0

8 7 6 5 CM FROM ORIGIN FIG. 8. Characterization of the 3H-labeled product formed by enzymatic reduction of hexadecyl DHAP with [3H]NADPH. Hexadecyl DHAP, [3H]NADPH, and yeast total membrane protein were incubated as described in Materials and Methods. The lipids were extracted, concentrated, and spotted on a silica gel TLC plate, 1

2

3

4

which was developed with chloroform-methanol-acetic acid-5% sodium bisulfite (100:40:12:4). The plate was divided into bands, and the bands were scraped and counted as described in Materials and Methods. The position of chemically synthesized 1-hexadecyl racglycerol 3-phosphate on the thin-layer plate is also shown.

derivatives. Figure 8 shows the distribution of radioactivity of 3H-labeled lipids formed from B-[4-3H]NADPH with hexadecyl DHAP as the substrate. The major radioactive component comigrated with synthetic 1-alkyl GP. The small amount of radioactivity present at the solvent front is probably due to the formation of the dephosphorylated product, hexadecyl glycerol, in the incubation mixture. Effect of oxygen. To determine the effect of oxygen on alkyl DHAP reductase activity, cells were grown aerobically and anaerobically in a synthetic medium containing glucose and supplements required for anaerobic growth (ergosterol and unsaturated fatty acids). Cytochrome oxidase, a mitochondrial marker enzyme repressed in the absence of oxygen (32), was assayed in both aerobically and anaerobically grown cells to confirm anaerobiosis. D273-1OB cells grown aerobically in glucose exhibit measurable cytochrome oxidase activity, in contrast to other strains tested (7). Therefore, cells of this strain were used for oxygen regulation studies, since they offer the most sensitive test for confirmation of stringent anaerobic conditions. As Fig. 9 shows, measurements of cytochrome oxidase activity confirmed anaerobiosis for cells grown in the absence of oxygen. In contrast, the activities of the endoplasmic reticulum marker enzyme (NADPH-cytochrome c reductase) were similar in both aerobic and anaerobic cells (Fig. 9). Wild-type cells grown in the absence of oxygen showed significantly lower alkyl DHAP reductase activity than did aerobically grown cells. Anaerobic cells exhibited a similar decrease in both DHAPAT and GPAT activities, showing approximately half the activity of aerobic cells. Formation of phosphatidate from DHAP in yeast cells and characterization of labeled lipid products. Figure 10 shows the formation of products from several 32P-labeled precursors. Palmitoyl [32P]DHAP was the only product formed from [32P]DHAP (lane A), whereas both labeled lysophosphatidate and phosphatidate were obtained from [3 P]GP (lane B). Upon addition of NADPH to an incubation mixture containing palmitoyl-CoA and [32P]DHAP, labeled products which comigrated with lysophosphatidate and phosphatidate

C D E B A FIG. 9. Effect of oxygen on various enzyme activities. Yeast membrane preparations from both aerobically and anaerobically grown cells were assayed for DHAPAT, GPAT, alkyl DHAP reductase, NADPH-cytochrome c reductase, and cytochrome c oxidase activities as described in Materials and Methods. Columns: A, alkyl DHAP reductase; B, DHAPAT; C, GPAT; D, NADPHcytochrome c reductase; E, cytochrome oxidase. Specific activities of membrane preparations from aerobically grown cells were 6.5, 0.44, 3.1, 10.9, and 114.5 nmol/min/mg of protein for alkyl DHAP reductase, DHAPAT, GPAT, NADPH-cytochrome c reductase, and cytochrome oxidase, respectively.

were formed along with palmitoyl [32P]DHAP (lane F). Incubation of palmitoyl [32P]DHAP with NADPH resulted in the formation of a single product which comigrated with lysophosphatidate. The other band corresponded to unreacted palmitoyl DHAP (lane C). Similar results were observed with use of control rat liver membrane fractions

PA

~*LPA

~~k

P 4 HAP

A

B

C

D

E

F

G

FIG. 10. Radioautogram of 32P-labeled lipid products formed from different labeled precursors in rat liver or yeast membrane fractions under various experimental conditions and after separation by TLC. Standard assay conditions were followed for each experiment, and the lipid products were isolated as detailed in Materials and Methods. The chromatogram was developed with CHC13methanol-acetic acid-5% aqueous sodium metabisulfite (100:40: 12:4) on a silica gel plate. Lanes: A, lipid formed by incubation of [32P]DHAP, palmitoyl-CoA, and yeast membrane fraction; B, lipid formed by incubation of [32P]GP, palmitoyl-CoA, and yeast membrane fraction; C, lipid formed by incubation of palmitoyl [32p] DHAP, NADPH, and yeast membrane fraction; D, lipid formed by incubation of palmitoyl [32P]DHAP, NADPH, and rat liver membrane fraction; E, zero-time control for lane C; F, lipid formed by incubation of [32P]DHAP, palmitoyl-CoA, NADPH, and yeast membrane fraction; G, lipid formed by incubation of [32P]DHAP, palmitoyl-CoA, NADPH, and rat liver membrane fraction. Abbreviations: PA, phosphatidate; LPA, lysophosphatidate; P-DHAP,

palmitoyl DHAP.

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RACENIS ET AL.

(lanes D and G). The small amount of phosphatidate formed from palmitoyl [32P]DHAP and NADPH (lane D) probably resulted from endogenous palmitoyl-CoA in the rat liver membrane protein preparation. The labeled acyl [32P]GP formed by incubating yeast membranes with palmitoyl [32P]DHAP and NADPH was further characterized by subjecting the mixture (similar to that in lane C) to alkaline methanolysis and high-voltage paper electrophoresis of the water-soluble 32P-labeled products (14). Of the two products obtained, one comigrated with Pi (the alkaline methanolysis product of palmitoyl [32P]DHAP) and the other comigrated with GP (the hydrolytic product of palmitoyl [32PlGP). The 3H- and 32P-labeled product formed from acyl [3 P]DHAP and B-14-3H]NADPH and purified by TLC had a molar 3H-to 3zP ratio of 1.05. DISCUSSION In higher eukaryotes, the acyl DHAP pathway is essential for ether lipid biosynthesis (15). In this pathway, acyl/alkyl DHAP reductase converts acyl DHAP into glycerolipids and alkyl DHAP, synthesized by alkyl DHAP synthase, into ether lipids. In the eukaryote S. cerevisiae, however, neither alkyl DHAP synthase nor ether lipids have been detected (33a). Nevertheless, we have shown in this report that S. cerevisiae contains both DHAPAT and acyl/alkyl DHAP reductase activities. Furthermore, we have shown that these enzymes, along with GPAT, are similarly regulated by oxygen. These data suggest that the acyl DHAP pathway may play a role in glycerolipid synthesis in this organism. This is the first demonstration of the acyl DHAP pathway in an organism lacking ether lipids. Our results demonstrate that S. cerevisiae is an ideal eukaryote in which to study the role of the acyl DHAP pathway in non-ether lipid synthesis. The first step in the acyl DHAP pathway is the DHAPATcatalyzed conversion of DHAP to acyl DHAP (Fig. 1). While Tillman and Bell (40) presented compelling genetic evidence that both GPAT and DHAPAT are dual functions of one enzyme encoded by a single nuclear gene, certain physicochemical properties of the two acyltransferases described in this report are not easily explained by a single enzyme. First, in our study, the two activities exhibited different kinetics of inactivation by N-ethylmaleimide (Fig. 5). In contrast, Schlossman and Bell (36) observed similar inactivation kinetics for the two enzymes, with both activities declining to approximately 20% of their initial activities following 10 min of exposure to N-ethylmaleimide. Second, we observed different pH optima for the two activities (Fig. 4), in contrast to the study by Schlossman and Bell (36), which showed no differences in pH activity profiles for GPAT and DHAPAT. Tillman and Bell (40), however, did show a significant difference in pH optima for the two enzymes in the wild-type strain. Apparently, pH activity profiles differ from strain to strain and under different assay conditions. Our results also suggest that the two activities are considerably more thermolabile than those observed by Schlossman and Bell (30% versus 40% activity following pretreatment of membrane fraction protein at 42°C for 10 min versus 48°C for 15 min). Finally, the kinetic parameters obtained from the two studies vary somewhat with respect to GPAT activity (Vm. of 12.6 nmol/min per mg of protein and Km of 0.28 mM in this study [Fig. 2B], compared with 3.4 nmol/min per mg of protein and 0.05 mM, respectively). The reasons for these differences are unclear, although strain differences may play some role in explaining these discrepancies. Interestingly, reductase expression was significantly re-

J. BACTERIOL.

duced in the absence of oxygen (Fig. 9). Furthermore, the extent of regulation by oxygen was almost identical to that observed for both GPAT and DHAPAT and clearly different from that for the marker enzymes tested. Regulation of these enzymes by oxygen might be related to their subcellular localization. Zinser and coworkers (42) have shown that most of the phospholipid biosynthetic enzymes, including GPAT, are present in both mitochondria and microsomes. Reduced enzyme expression may be due to reduced mitochondrial volume in anaerobically grown cells. Coordinate control of both GP and acyl DHAP pathway enzymes is consistent with the hypothesis that both pathways are utilized in phosphoglycerolipid synthesis. This is clearly seen in Fig. 10, which demonstrates that phosphatidate is formed from either DHAP or GP and necessary cofactors. Certain properties of the yeast reductase resemble those of the corresponding mammalian enzymes. The apparent Kms of the yeast enzyme for its two substrates are similar to those of the guinea pig liver enzyme (apparent Kms of 15 ,uM for NADPH and 20 pM for hexadecyl DHAP for yeast cells [Fig. 6]), compared with Kms of 20 ,uM for NADPH and 21 p,M for hexadecyl DHAP for guinea pig liver (4). Like the mammalian enzymes, NADPH, but not NADH, is the cofactor for the yeast enzyme (23; see above). The pH optima of both are about 6.5 (24; this study). However, the mammalian enzyme is more stable than its yeast counterpart. Peroxisomal and microsomal reductase activities from guinea pig liver were stable in the presence of high concentrations of N-ethylmaleimide (up to 10 mM). In addition, heating the mammalian enzymes for 15 min at 50°C failed to substantially diminish their activities (8). This report is the first demonstration of acyl/alkyl DHAP reductase in yeast cells. These results show not only that acyl/alkyl DHAP reductase activity is present in yeast cells but that it is present in substantial quantity. Although the activity of the enzyme (or enzymes) varied from one preparation to another, activities in the range of 3 to 6 nmol/ min/mg of protein were typical, and activities as high as 9 nmol/min/mg of protein were detected from some preparations. Other investigators did not detect this enzyme in membrane fractions of S. cerevisiae (36). At least three possibilities may account for this discrepancy. In the prior study (36), yeast cells were grown anaerobically and harvested in stationary phase. In this study, we showed that reductase activity was reduced in anaerobically grown yeast cells. Another possible explanation is that the assay system used by these workers was not sensitive enough to detect the lower levels of reductase activity present in membranes from anaerobically grown cells. In both direct and indirect assays for the enzyme, Schlossman and Bell (36) measured products by TLC. Furthermore, only a 5 ,uM concentration of the palmitoyl DHAP substrate was used, compared with 60 ,uM hexadecyl DHAP in our assay. Since the apparent Km of the yeast reductase is 20 ,uM (for hexadecyl DHAP; Fig. 6), the palmitoyl DHAP concentration used may have been too low to detect sufficient amounts of the radiolabeled lipid product. Additionally, the subsequent TLC extractions to quantify the product undoubtedly reduced the sensitivity even further. A third possibility is that the differences between these two studies may be accounted for by strain differences, as we have observed with other yeast enzymes (7). The question of the relative contributions of the GP and acyl DHAP pathways to phosphoglycerolipid and triacylglycerol biosynthesis in eukaryotes remains an important one (6, 29, 30, 33). In this study, we demonstrated that both pathways are present in the simple eukaryote S. cerevisiae,

ACYL DHAP PATHWAY ENZYMES IN S. CEREVISLAE

VOL. 174, 1992

and we characterized the properties of the acyl DHAP pathway enzymes in membrane fractions of this organism. The data support the hypothesis that the acyl DHAP pathway is utilized in glycerolipid synthesis in this yeast. Because S. cerevisiae is easily amenable to genetic and biochemical analysis, it serves as an excellent model in which to explore the relative contributions of the GP and acyl DHAP pathways to glycerolipid synthesis. ACKNOWLEDGMENTS This work was supported by NIH grants NS 08841 (A.K.H.) and GM 37723 (M.L.G.). REFERENCES 1. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. 37:911-917. 2. Culbertson, M. R., and S. A. Henry. 1975. Inositol-requiring mutants of Saccharomyces cerevisiae. Genetics 80:23-40. 3. Das, A. K., and A. K. Hajra. 1984. Estimation of acyl dihydroxyacetone phosphate and lysophosphatidate in animal tissues. Biochim. Biophys. Acta 796:178-189. 4. Datta, S. C., M. K. Ghosh, and A. K. Hajra. 1990. Purification and properties of acyl/alkyl dihydroxyacetone phosphate reductase from guinea pig liver peroxisomes. J. Biol. Chem. 265: 8268-8274. 5. Davis, P. A., and A. K. Hajra. 1981. Assay and properties of the enzyme catalyzing the biosynthesis of 1-0-alkyl dihydroxyacetone 3-phosphate. Arch. Biochem. Biophys. 211:20-29. 6. Declerq, P. E., H. P. Haagsman, P. Van Veldhoven, L. J. Debeer, L. M. G. Van Golde, and G. P. Mannaerts. 1984. Rat liver dihydroxyacetone phosphate acyltransferases and their contribution to glycerolipid synthesis. J. Biol. Chem. 259:9064-9075. 7. Gaynor, P. M., S. Hubbell, A. J. Schmidt, R. A. Lina, S. A. Minskoff, and M. L. Greenberg. 1991. Regulation of phosphatidylglycerolphosphate synthase in Saccharomyces cerevisiae by factors affecting mitochondrial development. J. Bacteriol. 173: 6124-6131. 8. Ghosh, M. K., and A. K. Hajra. 1986. Subcellular distribution and properties of acyl/alkyl dihydroxyacetone phosphate reductase in rodent livers. Arch. Biochem. Biophys. 245:523-530. 9. Goldfine, H., and P. 0. Hagen. 1972. Bacterial plasmalogens, p. 330-350. In F. Snyder (ed.), Ether lipids, chemistry and biology. Academic Press, Inc., New York. 10. Greenberg, M. L., S. Hubbell, and C. Lam. 1988. Inositol regulates phosphatidylglycerolphosphate synthase expression in Saccharomyces cerevisiae. Mol. Cell. Biol. 8:4773-4779. 11. Gurr, M. I. 1980. The biosynthesis of triacylglycerols, p. 205-248. In P. K. Stumpf (ed.), The biochemistry of plants, vol. 4. Lipids: structure and function. Academic Press, Inc., New York. 12. Hajra, A. K. 1974. On extraction of acyl and alkyl dihydroxyacetone phosphate from incubation mixtures. Lipids 9:502-505. 13. Hajra, A. K. 1983. Biosynthesis of O-alkylglycerol ether lipids, p. 85-106. In H. K. Mangold and F. Paltauf (ed.), Ether lipids: biochemical and biomedical aspects. Academic Press, Inc., New York. 14. Hajra, A. K., and B. W. Agranoff. 1968. Acyl dihydroxyacetone phosphate. Characterization of a 32P-labeled lipid from guinea pig liver mitochondria. J. Biol. Chem. 243:1617-1622. 15. Hajra, A. K., and J. E. Bishop. 1982. Glycerolipid biosynthesis in peroxisomes via the acyl dihydroxyacetone phosphate pathway. Ann. N.Y. Acad. Sci. 386:170-182. 16. Hajra, A. K., and C. L. Burke. 1978. Biosynthesis of phosphatidic acid in rat brain via acyl dihydroxyacetone phosphate. J. Neurochem. 31:125-134. 17. Hajra, A. K., C. L. Burke, and C. L. Jones. 1979. Subcellular localization of acyl coenzyme A: dihydroxyacetone phosphate acyltransferase in rat liver peroxisomes (microbodies). J. Biol. Chem. 254:10896-10900.

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The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae.

The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pa...
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