JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1368-13760021-9193/78/0133-1368$0.200/0Copyright 0) 1978 American Society for Microbiology

Vol. 133, No. 3

Printed in U.S.A.

Glycerolipid Biosynthesis in Saccharomyces cerevisiae: sn-Glycerol-3-Phosphate and Dihydroxyacetone Phosphate

Acyltransferase ActivitiesDAVID M. SCHLOSSMAN AND ROBERT M. BELL*

Departnent ofBiochemistry, Duke University Medical Center, Durham, North Carolina 27710

Received for publication 29 September 1977

Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAPacyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activityand that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a singlemembranous enzyme, (ii) estimate the relative contributions ofthe glycerol-P andDHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitabilityof yeast for future genetic investigations of the eucaryotic glycerol-P and DHAPacyltransferase activities. The membranous DHAP acyltransferase activityshowed an apparent Km of 0.79 mM for DHAP, with a V.. of 5.3 nmol/min permg, whereas the glycerol-P acyltwansferase activity showed an apparent Km of0.05 mM for glycerol-P, with a V,. of 3.4 nmol/min per mg. Glycerol-P was acompetitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, andDHAP was a competitive inhibitor (Ki, 0.91 mM) ofthe glycerol-P acyltransferaseactivity. The two acyltransferase activities exhibited marked similarities in theirpH dependence, acyl-coenzyme A chain length preference and substrate concen-tration dependencies, thermolability, and patterns of inactivation by N-ethyhnal-eimide, trypsin, and detergents. Thus, the data strongly suggest that yeastglycerol-P and DHAP acyltransferase activities represent dual catalytic functionsof a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxido-reductase activity could be detected in yeast membranes, the DHAP pathway forglycerolipid synthesis may not operate in yeast.

Glycerolipid biosynthesis in yeast (20, 33), asin mammals (22, 23, 36, 53), begins with thestepwise acylation of sn-glycerol 3-phosphate(glycerol-P) using long-chain fatty acyl-coen-zyme A's (acyl-CoA's). Recent studies on thebiosynthesis (12, 26, 49) and reduction (13, 16,27, 49) of acyl-dihydroxyacetone phosphate(acyl-DHAP) in liver particulate fractions haveled to the proposal of a separate, independentroute (the DHAP pathway) that allows DHAPto serve as a precursor for the backbone ofglycerolipid. Despite extensive work (1, 18, 35,37, 38-40, 43), the relative contributions of theglycerol-P and DHAP pathways to phospholipidand triacylglycerol synthesis remain to be estab-lished in eucaryotic systems (53, 55).The hepatic DHAP acyltransferase (12, 26,

49), acyl-DHAP oxidoreductase (25, 26, 49), andglycerol-P acyltransferase activities (46,55) havebeen localized in both the mitochondrialand microsomal subcellular compartments. Ourinvestigations have emphasized the microsomalactivities because only microsomes contain thediacylglycerol acyltransferase, diacylglycerol

choline-phosphotransferase, and diacylglycerolethanolaminephosphotransferase activities nec-essary for triacylglycerol, phosphatidylcholine,and phosphatidylethanolamine syntheses (46).Since these are the most abundant cellular glyc-erolipids, the endoplasmic reticulum is believedto be the principal site of glycerolipid synthesis(46).In previous studies of various mammalian tis-

sues (45, 46), we presented evidence challengingthe implicit assumption that the microsomalacylation of glycerol-P and DHAP occurs byseparate enzymes. This work strongly suggestedthat a single microsomal enzyme catalyzes theacylation of both glycerol-P and DHAP. Calcu-lations based on the determined kinetic param-eters (45, 46) and previously reported glycerol-Pand DHAP pools for fat cells (2) and liver (11)suggested that the glycerol-P pathway should bepredominant in these tissues.

Conclusive evidence that a single enzyme cat-alyzes two reactions could be obtained either bypurifying the enzyme to homogeneity or by ge-netic analysis of the structural gene in question.

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GLYCEROL-P/DIHYDROXYACETONE-P ACYLTRANSFERASE 1369

The microsomal glycerol-P acyltransferase hasthus far resisted all attempts to extensively pu-rify it (36, 52, 58, 59). Moreover, the only mu-tants of the glycerol-P acyltransferase activityare in the procaryote Escherichia coli (3, 47). E.coli does not contain significant DHAP acyl-transferase activity (54). In this paper, we reporta biochemical investigation designed to evaluatewhether Saccharomyces cerevisiae might besuitable for subsequent genetic investigation ofeucaryotic glycerol-P and DHAP acyltransfer-ase activities. This work extends our previousconclusion that a single enzyme catalyzes theacylation of both glycerol-P and DHAP in sev-eral mammalian tissues to a lower eucaryoticcell type.

MATERIALS AND METHODSMaterials. [2-3H]glycerol (200 mCi/mmol) was a

product ofNew England Nuclear Corp., Boston, Mass.Proteose peptone and yeast extract were from DifcoLaboratories, Detroit, Mich. GasPak 150 anaerobicculture systems were obtained from the BBL divisionof Becton, Dickinson, and Co., Cockeysville, Md. Allother chemicals and enzymes were the highest gradecommercially available and have been described pre-viously (45, 46).

Synthesis of radioactively labeled substrates.[2-3H]glycerol-P was synthesized by the glycerol ki-nase-dependent phosphorylation of [2-3H]glycerol ac-cording to the procedure of Chang and Kennedy (7),except that a step gradient from 0 to 2 N formic acidwas used to elute the product from the ion-exchangecolumn. Chromatography on Whatman no. 1 paper inmethyl cellosolve-methyl ethyl ketone-3 N NH4OH(7:2:3, vol/vol/vol) confirmed that the product con-tained no unreacted [3H]glycerol or other radioactiveimpurity. [3P]DHAP was prepared by the glycerolkinase-dependent phosphorylation of dihydroxyace-tone with [y-32PJATP as desribed previously (44),except that purification of the [3P]ATP from theGlynn-Chappell (10) reaction mixture was omittedwith some preparations. Chromatography on What-man no. 1 paper in t-butyl alcohol-water-picric acid(40 ml:10 ml:2 g) (15) confimed that the purified,lyophilized [3P]DHAP product contained less than5% 'Pi and no other radioactive impurity. The quan-tities of [2-3H]glycerol-P and (3P]DHAP synthesizedwere estimated spectrophotometrically by using glyc-erol-P dehydrogenase (41).

Palmitoyl-[32P]DIAP was synthesized enzymati-cally from [3PJDHAP and palmitoyl-CoA by usingmicrosomes from isolated fat cells under conditionsdescribed previously (45), scaled up 10- to 20-fold. Thecombined chloroform phase, containing lipid extractedfrom reaction mixtures, was evaporated to 1 ml andthen mixed with 2 ml of methanol and 0.8 ml of 1 MKCI containing 0.2 M tris(hydroxymethyl)amino-methane (Tris) base (48). The single phase of thisbasic Bligh and Dyer extraction (5) was immediatelybroken by the addition of 1 ml of chloroform and 1 mlof KCl-Tris solution, followed by brief centrifugation.Under these conditions, the monoacyl radioactive

product partitioned mainly into the upper phase, whilethe residual diacyl microsomal phospholipids re-mained in the lower phase (48). The upper aqueousmethanolic phase was brought to pH 1 with about 50pl of concentrated HCl and extracted with 3 ml ofchloroform to recover the purified radioactive product,which migrated simila to an authentic sample of acyl-DHAP on silica gel HR in chloroform-methanol-acetic acid-5% sodium metabisulfite in water(100:40:12:4, by volume) (26). The final chloroformphase was evaporated to dryness, and the palmitoyl-[32P]DHAP (20 nmol) was dispersed at 40C in 16 mMpotassium phosphate buffer (pH 7.4, 1 ml) by five 3-sbursts from a Branson Sonifier, with 30-s cooling pe-riods between bursts.Enzyme assays. Glycerol-P and DHAP acyltrans-

ferase activites were assessed by monitoring the incor-poration of [2-3H]glycerol-P and [3P]DHAP, respec-tively, into chloroform-extractable material under con-ditions similar to those described previously (45, 46).Assays were performed in screw-capped culture tubes(13 by 100 mm) at 23°C in a final volume of 100 ,il.The standard incubation mixture contained 75 mMTris-hydrochloride (pH 7.4), 4 mM MgCl2, 2 mg ofbovine serum albumin per ml, 8 mM NaF, 1 mMdithiothreitol, 80 pM palmitoyl-CoA, and either 0.3mM [2-3Hjglycerol-P (50 mCi/mmol) or 1.0 mM[32P]DHAP (10 to 100 mCi/mmol). Reactions wereinitiated by adding 0 to 25 pg of membrane proteinand were terminated after 10 min by the addition of 3ml of chloroform-methanol (1:2, vol/vol) and 0.7 ml of1% perchloric acid. The quantity of [3HJglycerol-P or[nP]DHAP converted to lipid was determined as de-scribed previously (45, 46), except that no carrier lipidswere added to the extraction and the chloroform phasewas only washed twice with 2-ml portions of 1% per-chloric acid. Both the glycerol-P and DHAP acyltrans-ferase activities were proportional to the amount ofadded protein up to at least 40 pg and to an incubationtime of up to 15 min. More than 95% of the labeledphospholipid product was extracted and recoveredunder the conditions used. Thin-layer chromatogra-phy on silica gel HR, using the sodium metabisulfitesystem (26), showed that 80% of the 3H-labeled ma-terial present in the final chloroform phase of glycerol-P acyltranderase assays migrated similar to phospha-tidic acid, while the remainder migrated simila tolysophosphatidic acid. On the same chromatogram,more than 95% of the 32P-labeled material in the finalchloroform phase of DHAP acyltransferase assays mi-grated similar to acyl-DHAP. In a previous study ofyeast membrane glycerol-P acyltransferase, Kuhn andLynen (24) reported that the ratio of glycerol-P-de-pendent CoA release to [32P]glycerol-P incorporationinto lipid was 1.5, indicating a mixture of monoacyland diacyl reaction products. However, their attemptto chromatograph the reaction products on KieselgelG yielded only a single spot tentatively identified aslysophosphatidic acid.Acyl-DHAP oxidoreductase activity was assessed

by two different methods. In the first, the reducednicotinamide adenine dinucleotide phosphate(NADPH) dependent conversion of ['P]DHAP to[32P]phosphatidic acid was followed (13, 25). Mem-branes were incubated in the standard DHAP acyl-

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1370 SCHLOSSMAN AND BELL

transferase assay mixtures, with and without 80,MNADPH, for 10 min at 23°C. The radioactive lipidproducts, extracted as described previously (45, 46),were concentrated, applied to a silica gel HR thin-layer plate, and developed in the sodium metabisulfitesystem (26). Spots migrating similarly to acyl-DHAP,lysophosphatidic acid, and phosphatidic acid stan-dards were localized by autoradiography, scraped intocounting vials, and counted in 8 ml of Aquasol 2 (NewEngland Nuclear Corp.). In the second method, theNADPH-dependent conversion of palmitoyl-[:cP]DHAP to [32P]phosphatidic acid was monitored underconditions similar to those used by LaBelle and Hajra(25, 27). The assay mixture (final volume, 0.1 ml)contained 16 mM potassium phosphate buffer (pH7.4), 10 mM NaF, 0.3 mM ethylenediaminetetraace-tate, 2.5 ,ug of Triton X-100, 0.5 nmol of palmitoyl-["2P]DHAP (5 x 104 cpm), 0 or 80 pLM NADPH, and0 or 50 puM oleoyl-CoA. After incubation at 23°C for10 min, lipids were extracted and quantitated as forthe first method.When samples from glycerol-P acyltransferase,

DHAP acyltransferase, and acyl-DHAP oxidoreduc-tase (method 1) assays were chromatographed onWhatman no. 1 paper in the picric acid system (15),no conversion of [3H]glycerol-P to [3H]DHAP or of[32P]DHAP to [nP]glycerol-P could be detected underany of the reaction conditions employed. Any varia-tions from the described reaction conditions for thevarious enzyme assays are noted in the appropriatefigure and table legends.Growth conditions of the organism and prep-

aration ofmembrane fractions. S. cerevisiae S288c(the kind gift of M. Vickers Hershfield, Duke Univer-sity Medical Center, Durham, N.C.) was grown, in 1-liter cultures at 26°C, anaerobically under a carbondioxide atmosphere in GasPak 150 anaerobic culturesystems. Methylene blue indicators were used to con-firm complete anaerobiosis. The culture medium con-tained proteose peptone (3 g/liter), yeast extract (3g/liter), and glucose (4 g/liter). Under these anaerobic,catabolite-repressing conditions, cells are devoid ofmature mitochondria. They contain only dedifferen-tiated promitochondrial structures, which differ dra-matically from normal mitochondria in several re-

spects including morphology, enzyme content, proteinand lipid contents, and potential for respiratory func-tion (29, 30, 44). Thus, the growth conditions werechosen to facilitate investigations on the microsomalglycerol-P and DHAP acyltransferase activities with-out extensive subcellular fractionation.

Cells were harvested in the stationary phase, 36 to40 h post-inoculation, after they had settled into a thinlayer on the bottom of the culture flasks. Subsequentoperations were carried out at 0 to 4°C. The culturemedium was decanted, and cells were suspended inmedium Ia (0.25 M sucrose; 10 mM Tris-hydrochlo-ride, pH 7.4; 1 mM ethylenediaminetetraacetate; 1

mM dithiothreitol), isolated by centrifugation at 1,000x g for 10 min, and resuspended in the same medium.The cells were then disrupted by three passagesthrough a French pressure cell at 20,000 lb/in2. Aftercentrifugation at 1,000 x g for 15 min to removeunbroken cells and cell debris, membranes were iso-lated by centrifugation at 160,000 x g for 1 h. Forsome experiments, membranes were isolated in me-

dium I (medium Ia with dithiothreitol omitted). Pro-tein was determined by the method of Lowry et al.(31) with bovine serum albumin as the standard. Mem-brane preparations diluted to a protein concentrationof 3 to 5 mg/ml in medium Ia were stored in 0.3-mlsamples at -15°C. Once thawed, the unused portionof a given membrane sample was discarded. Underthese conditions, the glycerol-P and DHAP acyltrans-ferase activities were stable for at least 2 months.

RESULTS

Preliminary experiments established thatyeast membranes contained DHAP acyltrans-ferase activity. The first systematic characteri-zation of this activity, along with studies on themembranous glycerol-P acyltransferase activityto determine whether the glycerol-P and DHAPacyltransferase activities are associated with asingle membrane-bound enzyme, is reported inthis paper. DHAP appeared to be a competitiveinhibitor of the glycerol-P acyltransferase (Fig.1A). At 80 AM palmitoyl-CoA, an apparent Kmof 0.05 mM for glycerol-P, with a V.. of 3.41nmol/min per mg of protein, was determined. ADHAP Ki of 0.91 mM was calculated. Likewise,glycerol-P was a competitive inhibitor of theDHAP acyltransferase activity (Fig. 1B). At 80,uM palmitoyl-CoA, an apparent Km of 0.79 mM

3

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7

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I

b 20 40 60 80 100I/ [GLYCEROL-P] (mM)'

I/[DHAP] (mM)'I

FIG. 1. (A) Dependence ofglycerol-P acyltransfer-ase activity on the concentration of P3H]glycerol-P inthe absence and presence of DHAP. Assays, as de-scribed in the text, employed 22.4 pg of protein. (B)Dependence ofDHAP acyltransferase activity on theconcentration of O'PIDHAP in the absence andpres-ence of glycerol-P. Assays, as described in the text,employed 22.4 pg ofprotein. AU data were plotted bythe method of Lineweaver and Burk (28) and sub-jected to computer-assisted least-squares analysis.

-Vmax 3.41 nmol/min/mg A

DHAP Ki - 0.9ImMGLYCEROL-P Km * 0.05mM

1 [OHAP) *0

[DHAP] *0.48mM

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3 - Vmax 5.29nmol/min/mg BDHAP Km -0.79mMi

GLYCEROLX-P K 0.07mM

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GLYCEROL-P/DIHYDROXYACETONE-P ACYLTRANSFERASE 1371

for DHAP, with a V,,. of 5.29 nmol/min per mgof protein, was determined. A glycerol-P Ki of0.07 mM was calculated. As expected, the ap-parent kinetic parameters determined for thetwo activities were strongly dependent on thelevel of acyl donor employed. However, at 50AM palmitoyl-CoA, glycerol-P and DHAP stillshowed a pattern of reciprocal competitive in-hibition (data not shown). At this level of pal-mitoyl-CoA, the glycerol-P acyltransferase ac-tivity had an apparent Km of .03 mM for glycerol-P, with a V.,. of 1.0 nmol/min per mg of proteinand a DHAP Ki of 0.54 mM. Data on the DHAPacyltransferase activity at 50,uM palnitoyl-CoAshowed an apparent Km for DHAP of 0.53 mM,with a Vmax of 2.0 nmol/min per mg of proteinand a glycerol-P Ki of 0.03 mM. The changes inkinetic parameters for the glycerol-P and DHAPacyltransferase activities caused by varying thepalmitoyl-CoA concentration were quite similar.Acyl-CoA dependencies. Saturated acyl-

CoA's containing four to eight carbon atomswere not substrates for either the yeast glycerol-P or DHAP acyltransferase activity. Figure 2Ashows the relative glycerol-P and DHAP acyl-transferase activities of various long-chain acyl-CoA's, which were employed at 80 ,M. The twoacyltransferase activities were quite similar intheir dependence on acyl-CoA chain length.Data for the various acyl-CoA's at 50 ,uM (notshown) confirmed this similarity. The glycerol-P and DHAP acyltransferase activities were alsoquite similar in their dependence on the concen-tration of palmitoyl-CoA added to the assay(Fig. 2B). Both activities increased to a maxi-mum at 80,uM and declined above this level.pH dependence. The pH dependencies of

the glycerol-P and DHAP acyltransferase activ-ities were very similar over the pH range inves-tigated (Fig. 3). Both activities were maximalabout pH 7 and declined about 50% above pH7.4. Control experiments demonstrated that the[32P]DHAP substrate was stable throughout thepH range employed.Thermolability of the glycerol-P and

DHAP acyltransferase activities. Since thekinetic observations presented above supportedthe hypothesis that the yeast glycerol-P andDHAP acyltransferase activites are associatedwith a single enzyme, a number of possible in-hibitory conditions were investigated to deter-mine whether both activities were affected sim-ilarly. Membrane preparations were heated at48°C, and samples were removed at varioustimes for assay of the*two acyltransferase activ-ities at 230C. The results (Fig. 4) demonstratethat both activities were inactivated with iden-tical kinetics and declined to 30% of their initialvalues within 30 min. The two activities were

O w so-

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ACYL-CoA CHAIN LENGTH

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0 40 80 120 160(PAL-CoA] (ALM)

FIG. 2. (A) Dependence of the glycerol-P andDHAP acyltransferase activities on acyl-CoA chainkngth. Assays containing 16.5 jg of protein wereperformed as described in the text, except that 80 uMacyl-CoA of the indicated chain length was substi-tuted for C16-CoA. (Because of the often confusingnomenclature identifying acyl-CoA's, we have electedto identify these chemicals by carbon atom number ofthe acyl chain and by the number of doubk bonds,where appropriate [451.) Maximum specific activitiesat Cv6-CoA were 2.8 and 2.4 nmol/min per mg for theglycerol-P and DHAP acyltransferase activities, re-

spectively. In this and all remaining figures, a filledsquare represents the superposition of the 100% con-trol values for the two activities. (B) Dependence ofglycerol-P and DHAP acyltransferase activities on

the concentration ofpalmitoyl-CoA (PAL-CoA). As-says, as described in the text, contained 23.5 pg ofprotein and the indicated level ofpalmitoyl-CoA. Thelevel ofI3H]glycerol-P in the glycerol-P acyltransfer-ase assays was reduced to 0.1 mM.

also similr in their time courses of inactivationat 430C (data not shown).

Inhibition by N-ethylmaleimide. Whenmembrane preparations were incubated with 0.4mM N-ethylmaleimide and samples were re-moved at various times for assay of the twoacyltransferase activities, the results (Fig. 5A)confirned Kuhn and Lynen's (24) report of thesensitivity of yeast membrane glycerol-P acyl-transferase to inactivation by this reagent. Thisactivity was inhibited 60% within 10 min. Inaddition, the time course for inactivation of theDHAP acyltransferase activity exactly paral-leled that ofthe glycerol-P acyltransferase activ-ity. When glycerol-P and DHAP acyltransferaseactivities were determined after 10 min of ex-

posure to various concentrations of N-ethylmal-eimide, both activities were again inactivatedquite similarly (Fig. 5B).

1.71 Za: _

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1372 SCHLOSSMAN AND BELL

6.6 7 7.4 7.8pH

FIG. 3. pH dependence ofDHAP acyltransferase activitjg ofprotein were performed a

except that the pH of the Tr!mixture was varied. The metmixtures was as indicated.

100

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ties. Assays using 13.25s aescrioea ILs-hydrochlorzsured pH o

5 10 15 20MINUTES OF HEATING

mf the text,ride bufferf reaction

g/ml. However, higher concentrations resultedparallel and severe losses of both activitiesig. 7A). When sodium deoxycholate was addedeach acyltransferase assay, the mild stimula-n of both activities persisted up to a detergentvel of 0.3 mg/ml. Above this concentration,

z 100I0z

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20

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0 5 I0 15 2C 0 .D 1.5 2MINUTES [N-ETHYLMALEIMIDE] (mM)

FIG. 5. Inhibition of the glycerol-P and DHAPacyltransferase activities by N-ethybnaleimide. (A)Time dependence. Membranes (1 mg/mi) prepared inthe absence of dithiothreitol were incubated with 0.4mM N-ethylmaleimide in medium I at 230C for var-ious times before determination of the activities. (B)Dose dependence. Membranes (1 mg/ml) prepared inthe absence of dithiothreitol were incubated withvarious levels of N-ethylmaleimide in medium I at23°C for 10 (glycerol-P acyltransferase) or 10.5(DHAP acyitransferase) min before determination ofthe activities. Assays containing 15pg ofprotein were

e performed as described in the text. The 1 mM dithi-othreitol contained in the assay mixtue was suffi-cient to quench any unreacted N-ethylmaleimide. The

I specific activities of untreated microsomes were 2.0

25 30 and 1.5 nmol/min per mg for the glycerol-P andDHAP acyltransferase activities, respectively.

FIG. 4. Thermolability of the glycerol-P andDHAP acyltransferase activities. Membranes (1

mg/ml) were incubated in medium Ia at 48C forvarious times before determination of the activities.Assays, as described in the text, contained 17 pg ofprotein. Specific activities of unheated microsomeswere 2.9 and 2.4 nmol/min per ng for the glycerol-PandDHAP acyltransferase activities, respectively.

Inhibition by trypsin and detergents. In-cubation of membranes with 60 ltg of trypsin perml for various times before assay showed thatboth glycerol-P and DHAP acyltransferase ac-tivities were more than 80% inhibited within 10min (Fig. 6). The kinetics of inactivation weresimilar for the two activities. When membraneswere incubated with varying concentrations oftrypsin for 5 min before assay, the glycerol-Pand DHAP acyltransferase activities were againinhibited imilarly (data not shown).The addition of Triton X-100 to each of the

acyltransferase assays showed that both glyc-erol-P andDHAP acyltransferase activities weremildly stimulated at a detergent level of 0.1

0,z 1001Z

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cFw 60

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FIG. 6. Inhibition of the glycerol-P and DHAPacyltransferase activities by trypsin. Membranes (1mg/ml) were incubated with 60 pug of typsin per mlin medium Ia at 23°C for various times before deter-minaton of the activities. Aways containing 15 pg ofprotein were carried out as described in the text.Specific activities of untreated microsomes were 2.2and 1.8 nmol/min per mg for the glycerol-P andDHAP acyltransferase activities, respectively.

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GLYCEROL-P/DIHYDROXYACETONE-P ACYLTRANSFERASE 1373

the two acyltransferase activities declined mark-edly (Fig. 7B). (The critical micellar concentra-tions of Triton X-100 and sodium deoxycholatehave been reported as 0.19 and 1.2 mg/ml, re-

spectively [32].)Yeast membranous acyl-DHAP oxidore-

ductase activity. Acyl-DHAP is an importantintermediate in the biosynthesis of ether lipids(6, 25, 27, 38, 40, 49, 57). Although we observeda significant DHAP acyltransferase activity inmembranes from S. cerevisiae, other workershave been unable to detect any trace of neutralor ionic alkoxylipids in this organism (34).Therefore, we investigated whether yeast mem-brane preparations contained detectable acyl-DHAP oxidoreductase, the enzyme activity re-

quired for further processing of acyl-DHAP into

_ X16C0 * A 6_B4.I- .2 .3 .4 .5 .1 .2.360.

120 20-

e'o 80

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CL.4~~~~~~~4

0 .1 .2 .3 .4 .5 0 .1 .2 .3 .4 .5

[TRITON X-l00] (mg/ml) [DEOXYCHOLATE] (mg/ml)

FIG. 7. Inhibition of the glycerol-P and DHAPacyltransferase activities by detergents. (A) Triton X-100. Assays, as described in the text, contained 10 pgofprotein and the indicated amount of Triton X-100.(B) Sodium deoxycholate. Assays, as described in thetext, contained 10 pg of protein and the indicatedamount ofsodium deoxycholate. Specific activities ofmicrosomes in the absence of detergent were 2.8 and2.2 nmol/min per mg for the glycerol-P and DHAPacyltransferase activities, respectively.

glycerol lipids (or of alkyl-DHAP into etherlipids).Acyl-DHAP constitutes the only radiolabeled

product formed by the palmitoyl-CoA-depend-ent acylation of[32P]DHAP by yeast membranes(see Materials and Methods) or liver microsomes(46) under the standard assay conditions. When80 uM NADPH was added to the standardDHAP acyltransferase assays, more than 98% ofthe 32P-labeled lipid product formed by yeastmembranes still consisted of acyl-DHAP (Table1, method 1). However, in the NADPH-supple-mented assays, more than 70% of the productformed by liver microsomes consisted of phos-phatidic acid, the remainder being acyl-DHAP.No lysophosphatidic acid was detected in eithercase. The total amount of lipid formed from[32P]DHAP by either liver microsomes or yeastmembranes was approximately the same in thepresence and absence of NADPH.

Similarly, whereas liver microsomes couldconvert up to 15% of a sample of acyl-[32P]-DHAP to [32P]phosphatidic acid within 10 min(Table 1, method 2), dependent upon NADPH,yeast membranes were unable to perform thisconversion. Again, no lysophosphatidic acid wasdetected in either case. That the conversion ofacyl-DHAP to phosphatidic acid by liver micro-

somes did not depend on added acyl-CoA maybe due to the ability of acyl-DHAP itself to serve

as an acyl donor (14).

DISCUSSION

Estimates of the relative quantities of glycer-olipid synthesized via the glycerol-P and DHAPpathways in various mammalian tissues remain

TABLE 1. Formation of ['2Plphosphatidic acid from ['2P]DHAP orpalmitoyl-[fP]DHAP by yeastmembranes and liver microsomesa

Yeast membranes Liver microsomes

Conditions DHAP LPA PA DAHAP LPA PADHApm (cpm) (cpm) (cp (cpm) (cpm)(cpm) (cpm)

(1) Conversion of ['2P]DHAP to [12P]phos-phatidic acidStandard DHAP acyltransferase assay 9,541 0 103 6,593 0 91Standard assay + 80 LM NADPH 8,145 0 137 2,661 0 6,501

(2) Conversion of pa1mitoyl-[:2F]DHAP to[:32P]phosphatidic acidStandard assay 8,733 0 114 7,712 0 133Standard assay + 80,IM NADPH 6,741 0 185 6,228 0 1,098Standard assay + 80,LM NADPH + 50,M 7,805 0 183 7,443 0 1,313oleoyl-CoA

a Assays to measure acyl-DHAP oxidoreductase activity, performed as described in Materials and Methods,contained 23.5 ug of yeast membranes. Rat liver microsomes (40,g), prepared as described previously (46), werealso tested as a positive control. Results are expressed as counts per minute of radioactive lipid productmigrating similar to acyl-DHAP, lysophosphatidic acid (LPA), and phosphatidic acid (PA).

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1374 SCHLOSSMAN AND BELL

controversial (9, 53, 5F, 57) despite the numerousinvestigations addressing this question (1, 18, 35,37, 38-40, 43). In previous studies ofseveral tissues that prominently synthesize lip-ids (45, 46), we presented evidence strongly sup-porting the hypothesis that the microsomal glyc-erol-P and DHAP acyltransferase activities ac-tually constitute dual catalytic functionm of asingle enzyme. This conclusion allowed a sim-plified view of metabolic regulation (45).Yeast membranes possess an easily measura-

ble DHAP acyltransferase activity. Several linesof evidence strongly support the hypothesis thatyeast membrane glycerol-P and DHAP acyl-transferase activities are catalyzed by a singleenzyme. Glycerol-P was a competitive inhibitorof the DHAP acyltransferase activity, and its Kiwas similar to its Km as a substrate for acylation.Likewise, DHAP was a competitive inhibitor ofthe glycerol-P acyltransferase activity, and itsK, was similar to its Km. This pattern of recip-rocal competitive inhibition observed with glyc-erol-P and DHAP is consistent with acylationoccurring at a single active site. In addition,shifts in kinetic parameters caused by varyingthe acyl-CoA concentration in the assay wereimilar for the two activities. Finally, the glyc-erol-P and DHAP acyltransferase actiyitiesshowed great similarities in their pH depend-ence, fatty acyl-CoA chain length and concen-tration dependencies, thennolability, and pat-terns of inactivation by N-ethylmaleimide, tryp-sin, and detergents. The present data are similarto those previously reported for the microsomalglycerol-P and DHAP acyltransferase activitiespresent in isolated fat cells (45), liver (46), andseveral other tissues (46). However, in a recentreport, Rock et al. observed that microsomesfrom rabbit harderian gland contained latentDHAP acyltransferase activity distinct fromglycerol-P acyltransferase activity by a numberof criteria (42). The growth conditions employedwere presumed essentially to inhibit the biogen-esis of mitochondria in yeast. The quantitativesimilarity of the glycerol-P and DHAP acyl-transferase activities in the membrane prepara-tion also suggests that under these conditionsmicrosomal enzyme activities are quantitativelypredominant or that any mitochondrial glycerol-P or DHAP acyltransferase activity present be-haves similar to the corresponding microsomalactivity.Although our assay procedures employed 20-

to 50-fold less protein and a lower temperature(23 instead of 300C) than previous investigations(8, 21, 24, 51, 56), the observed glycerol-P acyl-transferase reaction velocities are in good agree-ment with those of previous reports in which theincorporation of radioactive glycerol-P into lip-

ids was measured (8, 51, 56). Other investigators,who assessed glycerol-P acyltransferase activityby spectrophotometrically monitoring CoA re-leased from palmitoyl-CoA during the reaction(50), found values approximately fivefold higherthan we observed (21, 24). The single previousreport of DHAP acyltransferase activity in S.carlsbergensis agrees reasonably well with ourreaction velocities (21), even though an assaythat measured CoA release was employed. ThepH and acyl-CoA chain length dependenciesobserved for the glycerol-P acyltransferase arein substantial agreement with previous data (24,56). However, the optimal concentrations ofglycerol-P and palmitoyl-CoA required wereconsiderably lower than the values reported pre-viously (24, 56).The cellular contents of yeast metabolites are

dependent on the culture medium, state of oxy-genation, and other environmental factors (17).Glycerol-P and DHAP in S. cerevisiae growingin the presence of glucose have been estimatedto be in the ranges 1.2 to 2.0 and 0.8 to 1.0,umol/g of fresh anaerobically grown yeast, re-spectively (19). Assuming that 71% of a yeast isaqueous (19) and that there is no compartmen-talization of substrates, intracellular glycerol-Pand DHAP concentration ranges of 1.7 to 2.8and 1.1 to 1.4 mM, respectively, can be calcu-lated. Using these estimated cellular glycerol-Pand DHAP concentrations and the kinetic pa-rameters (K., Vm,a Ki) determined (Fig. 1) forthe yeast membranous glycerol-P and DHAPacyltransferase activities, the ratio of glycerol-Pto DHAP acylation would be greater than 12:1.Thus, the glycerol-P pathway for phospholipidsynthesis would be expected to be predominantin yeast cells in vivo in comparison to the DHAPpathway.The failure to detect acyl-DHAP oxidoreduc-

tase activity in our membrane preparationsmight reflect repression of this activity underthe growth conditions employed. However, thelack of this enzyme activity coupled with thepreviously reported absence of alkyl-linked lip-ids in yeast (34) suggests that the DHAP path-way may not operate in S. cerevisiae. If theDHAP acyltransferase activity detected in vitrofunctions in vivo, the acyl-DHAP formed mightserve as an acyl donor (14) in biosynthetic re-actions for the formation of complex lipids inthe cell.The data suggest that S. cerevisiae can pro-

vide a system for genetic analysis of the eucar-yotic glycerol-P and DHAP acyltransferase ac-tivities. In addition, experiments employing hy-brid E. coli plasmids bearing yeast DNA tocorrect the E. coli pisB glycerol-P acyltransfer-ase lesion (3, 4) might provide a system with

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GLYCEROL-P/DIHYDROXYACETONE-P ACYLTRANSFERASE 1375

which to investigate the expression of a eucar-

yotic membrane enzyme in E. coli. Since thisorganism does not contain significant DHAPacyltransferase activity (54), expression of theyeast enzyme should be relatively easy to dem-onstrate.

ACKNOWLEDGMENTS

D.M.S. was supported by Public Health Service traininggrant GM 01678 from the National Institute of General Med-ical Sciences. This work was done during the tenure of an

Established Investigatorship of the American Heart Associa-tion. It was supported by a grant-in-aid from the AmericanHeart Association with funds contributed by the North Car-olina Heart Association, by a grant-in-aid from the NorthCarolina Heart Association (1976-77-A-18), and by PublicHealth Service grant AM 20205 from the National Institute ofArthritis, Metabolism, and Digestive r-qeases.

LlTERATURE CrrED

1. Agranoff, B. W., and A. K. Hajra. 1971. The acyldihydroxyacetone phosphate pathway for glycerolipidbiosynthesis in mouse liver and Ehrlich ascites tumorcells. Proc. Natl. Acad. Sci. U.S.A. 68:411-415.

2. Ballard, F. J. 1972. Effects of fasting and refeeding on

the concentrations of glycolytic intermediates and theregulation of lipogenesis in rat adipose tissue in vivo.Biochim. Biophys. Acta 273:110-118.

3. Bell, R. M. 1974. Mutants of Escherichia coli defective inmembrane phospholipid synthesis: macromolecularsynthesis in an sn-glycerol 3-phosphate acyltransferase,K. mutant. J. Bacteriol. 117:1065-1076.

4. Bell, R. M. 1975. Mutants of Escherichia coli defective inmembrane phospholipid synthesis. Properties of wildtype and Km defective sn-glycerol 3-phosphate acyl-transferase activites. J. Biol. Chem. 250:7147-7152.

5. Bligh, E. G., and W. J. Dyer. 1959. A rapid method oftotal lipid extraction and purification. Can. J. Biochem.Physiol. 37:911-917.

6. Chae, K., C. Piantadosi, and F. Snyder. 1973. Reduc-tase, phosphatase, and kinase activities in the metabo-lism of alkyldihydroxyacetone phosphate and alkyldi-hydroxyacetone. J. Biol. Chem. 248:6718-6723.

7. Chang, Y., and E. P. Kennedy. 1967. Biosynthesis ofphosphatidylglycerate in Escherichia coli. J. Lipid Res.8:447-455.

8. Cobon, G. S., P. D. Crowfoot, and A. W. Linnane.1974. Biogenesis of mitochondria. Phospholipid synthe-sis in vitro by yeast mitochondrial and microsomalfractions. Biochem. J. 144:265-275.

9. Curstedt, T. 1974. Biosynthesis of molecular species ofphosphatidylcholines in bile, liver, and plasma of ratsgiven [1,1'H2]ethanol. Biochim. Biophys. Acta369:196-208.

10. Glynn, I. M., and J. B. Chappell. 1964. A simple methodfor the preparation of 32P-labeled adenosine triphos-phate of high specific activity. Biochem. J. 90:147-149.

11. Greenbaum, A. L., K. A. Gumaa, and P. McClean.1971. The distribution of hepatic metabolites and thecontrol of the pathways of carbohydrate metabolism inanimals of different dietary and hormonal status. Arch.Biochem. Biophys. 143:617-663.

12. Hajra, A. K. 1968. Biosynthesis of acyl dihydroxyacetonephosphate in guinea pig liver mitochondria. J. Biol.Chem. 243:3458-3465.

13. Hajra, A. K. 1968. Biosynthesis of phosphatidic acid fromdihydrozyacetone phosphate. Biochem. Biophys. Res.Commun. 33:929-935.

14. Hajra, A. K. 1974. Enzymatic transfer of the acyl groupfrom acyl dihydroxyacetone phosphate to different sub-strates. Biochem. Biophys. Res. Commun. 57: 668-674.

15. Hajra, A. K., and B. W. Agranoff. 1968. Acyl dihydrox-yacetone phosphate. Characterization of a 32P-labeledlipid from guinea pig liver mitochondria. J. Biol. Chem.243:1617-1622.

16. Hajra, A. K., and B. W. Agranoff. 1968. Reduction ofpalmitoyl dihydroxyacetone phosphate by mitochon-dria. J. Biol. Chem. 243:3542-3543.

17. Hess, B., A. Boiteux, and J. Kriiger. 1968. Cooperationof glycolytic enzymes. Adv. Enzyme Regul. 7:149-167.

18. Hill, E. E., and W. E. M. Lands. 1970. Formation of acyland alkenyl glycerol derivatives in Clostridium butyri-cum. Biochim. Biophys. Acta 202:209-211.

19. Holger, H., W. Bernhardt, and S. Schneider. 1963. ZurGlycerinbildung in Backerhefe. Biochem. Z. 336:495-509.

20. Hunter, K., and A. H. Rose. 1971. Yeast lipids andmembranes, p. 211-270. In A. H. Rose and J. S. Harrison(ed.), The yeasts. Academic Press Inc., New York.

21. Johnston, J. M., and F. Paltauf. 1970. Lipid metabolismin inositol deficient yeast, Saccharomyces carlsbergen-sis. II. Incorporation of labeled precursors into lipids bywhole cells and activities of some enzymes involved inlipid formation. Biochim. Biophys. Acta 218:431-440.

22. Kennedy, E. P. 1953. Synthesis of phosphatides in iso-lated mitochondria. J. Biol. Chem. 201:399-412.

23. Kornberg, A., and W. E. Pricer, Jr. 1953. Enzymaticesterification of a-glycerophosphate by long chain fattyacids. J. Biol. Chem. 204:345-357.

24. Kuhn, N. J., and F. Lynen. 1965. Phosphatidic acidsynthesis in yeast. Biochem. J. 94:240-246.

25. LaBelle, E. F., Jr., and A. K. Hajra. 1972. Enzymaticreduction of alkyl and acyl derivatives of dihydroxyac-etone phosphate by reduced pyridine nucleotides. J.Biol. Chem. 247:5825-5834.

26. LaBelle, E. F., Jr., and A. K. Hajra. 1972. Biosynthesisof acyl dihydrozyacetone phosphate in subcellular frac-tions of rat liver. J. Biol. Chem. 247:5835-5841.

27. LaBelle, E. F., Jr., and A. K. Hajra. 1974. Purificationand kinetic properties of acyl and alkyl dihydroxyace-tone phosphate oxidoreductase. J. Biol. Chem.249:6936-6944.

28. Lineweaver, H., and D. Burk. 1934. The determinationof enzyme dissociation constants. J. Am. Chem. Soc.56:658-666.

29. Linnane, A. W., and J. M. Haslam. 1970. The biogenesisof yeast mitochondria. Curr. Top. Cell. Regul.2:102-172.

30. Linnane, A. W., and H. B. Lukins. 1975. Isolation ofmitochondria and techniques for studying mitochon-drial biogenesis in yeasts, p. 285-309. In D. M. Prescott(ed.), Methods in cell biology, vol. 12, Yeast cells. Aca-demic Press Inc., New York.

31. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

32. Makino, S., J. A. Reynolds, and C. Tanford. 1973. Thebinding of deoxycholate and Triton X-100 to proteins.J. Biol. Chem. 248:4926-4932.

33. Mangnall, D. G., and G. Getz. 1973. Phospholipids, p.146-196. In J. A. Erwin (ed.), Lipids and membranes ofeukaryotic microorganisms. Academic Press Inc., NewYork.

34. Mangold, H. K. 1972. The search for alkoxylipids inplants, p. 399-405. In F. Snyder (ed.), Ether lipids,chemistry and biology. Academic Press Inc., New York.

35. Manning, R., and D. N. Brindley. 1972. Tritium isotopeeffects in the measurement of the glycerol phosphateand dihydroxyacetone phosphate pathways of glycero-lipid biosynthesis in rat liver. Biochem. J.130:1003-1012.

36. Numa, S., and S. Yamashita. 1974. Regulation of lipo-genesis in animal tissues. Curr. Top. Cell Regul.8:197-246.

VOL. 133, 1978

on August 5, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

1376 SCHLOSSMAN AND BELL

37. Okuyama, H., and W. E. M. Lands. 1970. A test for thedihydroxyacetone phosphate pathway. Biochim. Bio-phys. Acta 218:376-377.

38. Pollock, R. J., A. K. Hajra, and B. W. Agranoff. 1975.The relative utilization of the acyl dihydrozyacetonephosphate and glycerol phosphate pathways for synthe-sis of glycerolipids in various tumors and normal tissues.Biochim. Biophys. Acta 380:421-435.

39. Poliock, R. J., A. K. Hajra, and B. W. Agranoff. 1976.Incorporation of D-[3-3H, U-'4C]glucose into glyceroli-pid via acyl dihydroxyacetone phosphate in untrans-formed and viral-transformed BHK-21-C13 fibroblasts.J. Biol. Chem. 251:5149-5154.

40. Pollock, R. J., A. K. Hajra, W. R. Folk, and B. W.Agranoff. 1975. Use of [1 or 3-3H, U-'4C]glucose toestimate the synthesis of glycerolipids via acyl dihy-droxyacetone phosphate. Biochem. Biophys. Res. Com-mun. 65:658-664.

41. Racker, E. 1947. Spectrophotometric measurement ofhexokinase and phosphohexokinase activity. J. Biol.Chem. 167:843-854.

42. Rock, C. O., V. Fitzgerald, and F. Snyder. 1977. Prop-erties of dihydroxyacetone phosphate acyltransferase inthe harderian gland. J. Biol. Chem. 252:6363-6366.

43. Rognstad, R., D. G. Clark, and J. Katz. 1974. Pathwaysof glyceride glycerol synthesis. Biochem. J.140:249-251.

44. Schatz, G. 1970. Biogenesis of mitochondria, p. 251-314.In E. Racker (ed.), Membranes of mitochondria andchloroplasts. Van Nostrand Reinhold Co., New York.

45. Schlossman, D. M., and R. M. Bell. 1976. Triacylgly-cerol synthesis in isolated fat cells. Evidence that thesn-glycerol 3-phosphate and dihydrozyacetone phos-phate acyltransferase activities are dual catalytic func-tions of a single microsomal enzyme. J. Biol. Chem.251:5738-5744.

46. Schlossman, D. A., and R. M. Bell. 1977. Microsomalsn-glycerol 3-phosphate and dihydrozyacetone phos-phate acyltransferase activities from liver and othertissues. Evidence for a single enzyme catlyzing bothreactions. Arch. Biochem. Biophys. 182:732-742.

47. Silbert, D. F. 1975. Genetic modification of membranelipid. Annu. Rev. Biochem. 44:315-339.

48. Snider, M. S., and E. P. Kennedy. 1977. Partial purifi-

cation of glycerophosphate acyltransferase from Esch-erichia coli. J. Bacteriol. 130:1072-1083.

49. Snyder, F. 1972. The enzymic pathways of ether-linkedlipids and their precursors, p. 121-156. In F. Synder(ed.), Ether lipids, chemistry and biology. AcademicPress Inc., New York.

50. Stansly, P. G. 1955. Estimation of the enzymic conden-sation of a-glycerophosphate and palmityl coenzyme A.Biochim Biophys. Acta 18:411-415.

51. Steiner, M. R., and R. LX Lester. 1972. In vitro studiesof phospholipid biosynthesis in Saccharomyces cerevi-siae. Biochim. Biophys. Acta 260:222-243.

52. Tamai, Y., and W. E. M. Lands. 1974. Positional speci-ficity of sn-glycerol 3-phosphate acylation during phos-phatidate formation by rat liver microsomes. J. Bio-chem. 76:847-60.

53. Van den Bosch, H. 1974. Phosphoglyceride metabolism.Annu. Rev. Biochem. 43:243-273.

54. Van den Bosch, H., and P. R. Vagelos. 1970. Fattyacyl-CoA and fatty acyl-acyl carrier protein as acyldonors in the synthesis of lysophosphatidate and phos-phatidate in Escherichia coli. Biochim. Biophys. Acta218:233-248.

55. Van Golde, L. M. G., and S. G. Van den Bergh. 1977.Liver, p. 35-150. In F. Snyder (ed.), Lipid metabolismin mammals, vol. 1. Plenum Press, New York.

56. White, G. L, and J. N. Hawthorne. 1970. Phosphatidicacid and phosphatidylinisitol metabolism in Schizosac-charomycespombe. Biochem. J. 117:203-213.

57. Wykle, R. L, C. Piantadosi, and F. Synder. 1972, Therole of acyldihydroxyacetone phosphate, reduced nico-tinamide adenine dinucleotide, and reduced nicotina-mide adenine dinucleotide phosphate in the biosyn-thesis of O-alkyl glycerolipids by microsomal enzymesof Ehrlich ascites tumor. J. Biol. Chem. 247:2944-2948,

58. Yamashita, S., K. Hosaka, and S. Numa 1972. Reso-lution and reconstitution of the phosphatidate synthe-sizing system of rat liver microsomes. Proc. Natl. Acad.Sci. U.S.A. 69:3490-3492.

59. Yamashita, S., and S. Numa. 1972. Partial purificationand properties of glycerophosphate acyltransferasefrom rat liver. Formation of 1-acylglycerol 3-phosphatefrom sn-glycerol 3-phosphate and palmityl coenzyme A.Eur. J. Biochem. 31:565-573.

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