JOURNAL OF BACTERIOLOGY, Nov. 1994, p. 6861-6868 Vol. 176, No. 220021-9193/94/$04.00+0Copyright C 1994, American Society for Microbiology

Functional Redundancy of CDP-Ethanolamine and CDP-CholinePathway Enzymes in Phospholipid Biosynthesis: Ethanolamine-Dependent Effects on Steady-State Membrane Phospholipid

Composition in Saccharomyces cerevisiaeTODD P. McGEE,t HENRY B. SKINNER, AND VYTAS A. BANKAITIS*

Department of Cell Biology, University ofAlabama at Birmingham, Birmingham, Alabama 35294-0005

Received 7 July 1994/Accepted 7 September 1994

It has been established that yeast membrane phospholipid content is responsive to the inositol and cholinecontent of the growth medium. Alterations in the levels of transcription of phospholipid biosynthetic enzymescontribute significantly to this response. We now describe conditions under which ethanolamine can exertsignificant influence on yeast membrane phospholipid composition. We demonstrate that mutations whichblock a defined subset of the reactions required for the biosynthesis of phosphatidylcholine (PC) via theCDP-choline pathway cause ethanolamine-dependent ellects on the steady-state levels of bulk PC in yeastmembranes. Such an ethanolamine-dependent reduction in bulk membrane PC content was observed for bothcholine kinase (cki) and choline phosphotransferase (cptl) mutants, but it was not observed for mutantsdefective in cholinephosphate cytidylyltransferase, the enzyme that catalyzes the penultimate reaction of theCDP-choline pathway for PC biosynthesis. Moreover, the ethanolamine effect observed for cki and cptl mutantswas independent of the choline content of the growth medium. Finally, we found that haploid yeast strainsdefective in the activity of both the choline and ethanolamine phosphotransferases experienced an ethanol-amine-insensitive reduction in steady-state PC content, an effect which was not observed in strains defective ineither one of these activities alone. The collective data indicate that specific enzymes of the CDP-ethanolaminepathway for phosphatidylethanolamine biosynthesis, while able to contribute to PC synthesis when yeast cellsare grown under conditions of ethanolamine deprivation, do not do so when yeast cells are presented with thisphospholipid headgroup precursor.

Phosphatidylcholine (PC) is a major phospholipid (PL) ofmost eukaryotic cells and is an important structural moleculeof both cellular membranes and lung surfactant (19). PC issynthesized either by the incorporation of choline into PC viaa cytidine-based process (i.e., the CDP-choline pathway) or viathe methylation of phosphatidylethanolamine (PE) by theterminal enzymes of the PE methylation pathway (reviewed inreference 5). The CDP-choline pathway involves three distinctreactions: (i) the phosphorylation of choline by a cholinekinase (CKIase), (ii) the production of CDP-choline fromphosphorylcholine by the action of the cholinephosphate cyti-dylyltransferase (CCTase), and (iii) the choline phosphotrans-ferase (CPTase)-catalyzed condensation of CDP-choline withdiacylglycerol to form PC (5, 13). In addition to its role in thegeneration of net PC from exogenously supplied choline, theCDP-choline pathway is also involved in the salvage of choline-containing metabolites resulting from PC turnover. A directlyanalogous pathway for the synthesis of PE from free ethanol-amine exists (i.e., the CDP-ethanolamine pathway). Both theCDP-choline and CDP-ethanolamine pathways are operativein the yeast Saccharomyces cerevisiae, and strains defective inCKIase (cki), CCTase (cct), CPTase (cptl), and ethanolaminephosphotransferase (EPTase) (eptl) activity have been isolated(13). An entirely unanticipated cellular involvement of the

* Corresponding author. Mailing address: Department of Cell Biol-ogy, 668 Basic Health Sciences Building, University of Alabama atBirmingham, Birmingham, AL 35294-0005. Phone: (205) 934-6151.Fax: (205) 934-0950.

t Present address: Department of Biological Sciences, StanfordUniversity, Stanford, CA 94305-5020.

CDP-choline pathway has also recently been discovered. Ge-netic analyses of the yeast Golgi secretory process havedemonstrated the activity of the CDP-choline pathway to beintimately related to the essential requirement for phosphati-dylinositol/phosphatidylcholine transfer protein function in themaintenance of the secretory competence of the yeast Golgicomplex, even under conditions in which the CDP-cholinepathway does not contribute to the generation of net cellularPC (4, 12). These findings have raised a number of questionsconcerning the role of PL metabolism in eukaryotic secretorypathway function, organelle membrane composition, and or-ganelle biogenesis.There exists ample evidence to indicate that the transcrip-

tion of at least several structural genes for yeast membranephospholipid biosynthetic enzymes is regulated in response tothe inositol and choline content of the growth medium (re-viewed in reference 13). In this report, we describe conditionsunder which ethanolamine exerts clear effects on bulk mem-brane PL composition. We demonstrate that mutations whichblock defined reactions in the biosynthesis of PC via theCDP-choline pathway exert variable effects on the steady-statelevels of bulk PC in yeast membranes. We show that thedifferential effects on steady-state PC content resulting fromthe various CDP-choline pathway defects correlate with theactivity of biochemically redundant enzymes whose PC biosyn-thetic capabilities are inhibited by the presence of ethanol-amine in the growth medium. Moreover, we have obtainedboth biochemical and genetic data to indicate that thesebiochemical homologs represent structural enzymes of theCDP-ethanolamine pathway. The collective data identify asubset of enzymes of the CDP-ethanolamine pathway that,

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while able to effect a significant contribution to the steady-statePC content of yeast cells grown under conditions of ethanol-amine deprivation, fail to contribute to bulk membrane PCcontent when yeast cells are cultured in the presence ofexogenously supplied ethanolamine.

MATERIALS AND METHODS

Yeast strains, media, and reagents. YPD and syntheticdefined complete media were prepared as previously describedwith the exception that the agar concentration was held at2.5% (2, 14). myo-Inositol, choline chloride, and ethanolaminehydrochloride were purchased from Sigma (St. Louis, Mo.)and were added, when appropriate, to a final concentration of1 mM each. 32Pi was purchased from American RadiolabeledChemicals (St. Louis, Mo.). Butylated hydroxytoluene (BHT)and Rhodamine 6G were purchased from Sigma. All otherchemicals and materials were of reagent grade.

Yeast strains used for this study were CTY182 (ALATaura3-52 Ahis3-200 lys2-801) (10), CTY105 (AL4Ta ura3-52Ahis3-200 lys2-801 sec14-1ts bsr2-5) (5), CTY392 (ALTaura3-52 Ahis3-200 lys2-801 sec14-1's cki-284:HIS3) (5), CTY434 (AL4Ta ura3-52 his5-519 ade2-101 leu2-3,112 sec14-1tscptl::LEU2) (6, 12), C`TY436 (MATa ura3-52 Ahis3-200 lys2-801 sec14-1's eptl::URA3) (this study), CTY479 (MATa ade2-101 ura3-52 Ahis3-200 bsd2-1) (this study), CTY618 (MATaura3-52 his5-519 ade2-101 leu2-3,112 sec14-1s cptl::LEU2eptl::URA3) (this study), and CTY394 (A ATa ura3-52 Ahis3-200 lys2-801 secl4-88::URA3 cki-284::HIS3) (this study). Yeaststrains CTY436 and CTY618 were created by direct transfor-mation with the eptl::URA3 disruption cassette (pRH511)described and constructed by Hjelmstad and Bell (7).

Steady-state PL analysis. To determine the steady-state PLcomposition of cells grown in synthetic defined completemedia (9), cells were grown for five to six generations in themedium of interest supplemented with 32Pi to a final concen-tration of 10 ,uCi/ml. Following labeling, cells were harvestedby centrifugation at 500 x g and the incorporation of label wasterminated by resuspension of the cell pellet in 5% ice-coldtrichloroacetic acid. PLs were extracted from the fixed cellpellet by incubation in polar extraction solvent (16) at 65°C for20 min. Lipid extractable material was separated from theaqueous environment by the addition of CHCl3-CH30H-BHT(2:1:0.0005), and the medium was vortexed and centrifuged toseparate the aqueous and organic phases. The organic phasewas removed and brought to dryness under a stream ofnitrogen gas. Extracted lipid material was resuspended in 60 ,ulof CHCl3-CH30H-BHT (2:1:0.0005) for separation and iden-tification. PLs were subsequently resolved by two-dimensionalpaper chromatography with Whatman SG81 chromatographypaper which had been treated with 2% EDTA and heated to100°C for at least 20 min prior to sample application. First-dimension resolution was achieved by using the solvent systemCHCl3-CH3OH-NH4OH-H20 (22:9:1:0.26), while the second-dimension solvent consisted of CHC13-CH30H-CH3COOH-H20(8:1:1.25:0.25). Resolved PLs were located by autoradiogra-phy, and the identified PL spots were excised and individuallyquantitated by scintillation counting.The PL composition of cells grown in YPD was determined

by similar methods, with the exception that individual PLspecies were quantitated by a nonisotopic method. Aftergrowth to mid-logarithmic growth phase in YPD (14), cellswere harvested, ice-cold trichloroacetic acid (5% final concen-tration) was added, and the PLs were extracted from the cellpellet as described above. Following resolution by two-dimen-sional paper chromatography, extracted PLs were located by

staining the chromatograph with a 0.0012% solution of Rho-damine 6G and inspecting the chromatograph under UVillumination (3). The regions of the chromatograph containingthe major PLs were excised, and the PLs were eluted from thechromatography paper by the addition of 2.0 ml of CHCl3-CH30H-BHT (2:1:0.0005%). The isolated PL species weresubsequently dried under gentle vacuum, and the relativeamount of each PL was determined by assay for total phos-phorous as described by Ames (1).

[14C]ethanolamine labeling of yeast lipids. The appropriateyeast strains were grown to mid-logarithmic growth phase indefined medium lacking choline and were presented with[14C]ethanolamine hydrochloride (1 ,uCi/ml) for 20 min at25°C with shaking. Incorporation of label was measured byremoving 1/10 of the culture, immobilizing the culture aliquoton 0.5-,um-pore glass fiber filters, washing the filters four timeswith 2.5 volumes of a 10 mM ethanolamine solution per wash,and resuspending the dried cell pellets (including filters) inscintillation fluid for counting. ['C]ethanolamine incorpora-tion values were employed to normalize the loading of lipidsamples derived from each of these cultures for furtheranalysis. PLs were extracted from the remainder of eachculture, as described above, and the incorporation of radiola-bel into normalized amounts of lipid sample from each culturewas quantitated by scintillation counting.

Assessment of opi phenotype. The inositol excretion capa-bilities of yeast strains were tested on plates lacking inositol,choline, and ethanolamine (I-C-E- plates) that had a re-duced agar concentration (1.2%) relative to our standard solidmedia. The yeast strain to be tested was patched onto theI-C-E- tester plate and permitted to grow for 72 h at 25°C.The inositol auxotroph reporter strain was then dilutionstreaked away from the patch, and crossfeeding was scoredafter an additional 72-h incubation at 25°C. The inositolauxotroph used in this inositol excretion bioassay was haploidstrain CTY479 (bsd2-1 SEC14). The bsd2-1 allele manifestsitself in a dominant inositol auxotrophy (20), one that is eventighter than the Ino- phenotype of strains defective in inositolbiosynthesis (e.g., strains carrying inol-13).

RESULTS AND DISCUSSION

Bulk membrane PL composition of CDP-choline pathwaymutants as a function of growth medium. We have observedthat mutations which block specific reactions of the CDP-choline pathway for PC biosynthesis elicit differential effects onthe steady-state membrane PL composition when the corre-sponding mutants are grown in defined medium supplementedwith inositol and choline (I+C+). An example of such an effectis provided by a comparison of the bulk membrane PLcompositions of mutant strains bearing individual cki-284::HIS3, bsr2-5, and cptl::LEU2 alleles, i.e., loss-of-function mu-tations in the CKIase, CCTase, and CPTase structural genes,respectively (Table 1). The cki-284::HIS3 mutant, when grownin I+C+ medium, displayed a PL composition of 25% phos-phatidylinositol (PI), 42% PC, 7% phosphatidylserine (PS),and 12% PE, a profile which was essentially indistinguishablefrom those of wild-type and cptl::LEU2 yeast strains. Thebsr2-5 mutation represents a loss-of-function mutation in theCCTase structural gene as evidenced by its very tight linkage tothe CCTase structural gene (28 parental ditype asci: 0 nonpa-rental ditype asci: 0 tetratype asci) and the absence of CCTaseactivity in cell extracts prepared from bsr2-5 yeast strains (notshown), and it results in a CDP-choline pathway PC biosyn-thetic defect of a magnitude similar to that of the defectelicited by either cki-284::HIS3 or cptl::LEU2 (12). However,

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TABLE 1. Steady-state PL composition of yeast strains grown in I+C+E- and I+C+E+ defined mediaa

Mole fraction of the following PL species (% of total extractable PL):Type of medium Strain Relevant genotype

PC PI PS PE

I+C+E- C1Y182 WTV 44 ± 1.2 25 ± 0.9 9 ± 1.4 15 ± 0.6C11Y105 bsr2-5 30 ± 0.9 40 ± 1.1 8 ± 0.2 7 ± 1.4CTY392 cki-284:JHIS3 42 ± 1.5 25 ± 1.2 7 ± 0.7 12 ± 0.4CTY434 cptl::LEU2 41 ± 1.3 22 ± 0.8 9 ± 1.1 13 ± 1.0CTY436 eptl::URh43 40 ± 2.0 23 ± 1.0 8 ± 0.3 19 ± 1.2C1Y618 cptl::LEU2 eptl::UR,43 30 ± 1.8 32 ± 2.3 9 ± 0.4 19 ± 0.8

I+C+E+ CTY182 WT 42 ± 1.5 24 ± 2.1 8 ± 0.4 17 ± 0.8CTY105 bsr2-5 29 ± 1.0 28 ± 1.9 8 ± 0.6 23 ± 0.9C1fY392 cki-284::HIS3 27 ± 1.7 27 ± 0.9 9 ± 0.3 25 ± 0.5CTY434 cptl::LEU2 34 ± 1.3 26 ± 1.1 9 ± 0.2 21 ± 1.0C1TY436 eptl::URA3 40 ± 2.0 21 ± 1.4 8 ± 0.4 14 ± 0.4CTY618 cptl::LEU2 eptl::URA3 30 ± 1.6 26 ± 1.0 8 ± 0.5 20 ± 0.8

a The indicated strains were grown for five to six generations in synthetic defined medium containing inositol and choline only (I+C+E-) or inositol, choline, andethanolamine (I+C+E+) and supplemented with 32Pi to 10 ,uCiml. The PL headgroup precursors were added to growth medium to a final concentration of 1 mM each.Glycerophospholipids were subsequently extracted, resolved, and quantitated as described in Materials and Methods. The mole fraction of each of the major PLs ofyeast cells is shown. Radiolabeled material that failed to migrate from the origin was also counted and included in the total PL value used in the mole percentagecalculations for the individual PL species. For this reason, the sum of the PI, PC, PS, and PE mole percentages does not equal 100%. Only trace amounts of phosphatidicacid and monomethylated and dimethylated forms of PC were detected (a composite total of <5% of the total PL content), and these trace values were not includedin the mole percentage calculations. The values shown represent the averages of those from three independent trials ± standard deviations. The complete genotypeof each yeast strain employed in these experiments is given in Materials and Methods.

b WT, wild type.

this CCTase defect reduced the steady-state bulk membranePC level to only 30% of the total PL and increased the bulkmembrane PI level to 40% of the total PL (Table 1). Thus,although the bsr2-5, cptl::LEU2, and cki-284::HIS3 mutantsare all impaired for the activity of the CDP-choline pathway,only bsr2-5 mutants experienced steady-state reductions inbulk membrane PC when cells were grown in I+C+ medium.The significant elevation in the level of bulk membrane PI wasuniquely observed for several bsr2 alleles, including a disrup-tion allele, but it was a function of the growth medium thatexhibited no discernible pattern that might provide some cluesas to the mechanism of elevation of bulk PI levels (see"Conclusions" below).

In contrast to the case which occurred when the cki-284::HIS3 and cptl: LEU2 mutants were grown in I+C+ medium,the PL composition exhibited by these same mutants (whenthey were cultured in complex [YPD] medium) revealed auniformly reduced bulk membrane PC content relative to thebulk membrane PC content recorded for the isogenic wild-typestrain. Whereas bulk membranes prepared from wild-type cellsconsisted of 21% PI, 50% PC, 4% PS, and 20% PE, thecki-284:HIS3 and cptl: LEU2 mutants displayed abnormal PLcompositions of 33% PI, 40% PC, 7% PS, and 20% PE and29% PI, 42% PC, 6% PS, and 22% PE, respectively (data notshown). The CCTase-deficient bsr2-5 strain again exhibited anabnormal bulk membrane PL composition when grown inYPD medium (36% PI, 30% PC, 10% PS, and 26% PE; datanot shown). We have previously shown that, under the growthconditions employed in this study, these various strains exhibitvery similar PL mass per unit cell equivalent (12). Thus,quantitation of each individual PL species as mole percent oftotal PL allows direct comparison of PL values (obtainedunder a given set of growth conditions) between these variousstrains. As a result, these data indicate that the cki-284::HIS3and cptl::LEU2 mutants exhibited growth medium-dependentabnormalities in bulk PL composition, whereas the bsr2-5strain exhibited constitutive abnormalities. A significant com-

ponent of these compositional abnormalities was a markedreduction in bulk membrane PC content.

Bulk membrane PL composition of CDP-choline pathwaymutants is sensitive to ethanolamine. The CKIase, whilerepresenting the major CKJase activity of the cell, is not theonly source of this activity in yeast cells. Evidence to this effectcomes from the demonstration that Acki strains of yeast stillexhibit CKlase activity in vivo (8, 12). We therefore consideredthe possibility that this residual CKIase activity contributed tothe normal PL composition of cki mutants in I+C+ mediumand that this activity was inhibited by some compound presentin YPD medium but not contained in the defined medium.One plausible scenario was that the residual CKIase activitypresent in cki mutants represented a yeast ethanolaminekinase (EKIase) that, while primarily dedicated to PE synthesisvia the CDP-ethanolamine pathway, could also utilize cholineas a substrate. Thus, when cki yeast cells are grown inethanolamine-free defined medium supplemented with cho-line, the EKIase would utilize choline as a substrate andcontribute to the essentially wild-type steady-state PC compo-sition exhibited by cki strains in minimal defined I+C+ me-dium. However, the availability of ethanolamine in rich growthmedium would diminish, by substrate-level competition, thecontribution of the EKIase to PC biosynthesis and result in thereduced bulk membrane PC content of cki-284::HIS3 strains inYPD medium. By direct analogy, as the EPTase of theCDP-ethanolamine pathway utilizes both ethanolamine andcholine derivatives as substrates in vitro (6, 7), we consideredit likely that the action of EPTase was responsible for themedium effect recorded for the cptl::LEU2 strain.To provide experimental support for the hypothesis that

enzymes of the CDP-ethanolamine pathway provide an eth-anolamine-sensitive contribution to the steady-state bulk PClevels of CDP-choline pathway mutants, it was necessary toestablish (i) that ethanolamine was the medium componentresponsible for the observed medium effects and (ii) that theobserved medium effects were eliminated by inactivation of theappropriate enzyme of the CDP-ethanolamine pathway. Totest the former point, yeast strains unable to execute definedreactions of the CDP-choline pathway were labeled to steadystate in I+C+ synthetic medium containing (E+) or lacking

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(E-) ethanolamine. Following this steady-state labeling, thePL composition of each strain was determined as a function ofthe growth medium (see Materials and Methods). In I+C+E+medium, the bulk PL profiles of the wild-type strain closelyresembled those displayed by this same strain when grown inI+C+E- medium (Table 1). This result demonstrated thatbulk PC of wild-type yeast was insensitive to the mediumeffects related to ethanolamine supplementation. In markedcontrast, strains bearing either the cki-284::HIS3 or the cptl::LEU2 allele experienced a decrease in steady-state bulk PCcontent when grown in E+ medium, relative to the bulkmembrane PC content recorded for these same strains grown

in E- medium. Bulk membranes prepared from the cki-284::HIS3 and cptl::LEU2 strains grown in I+C+E+ medium were

composed of only 28 and 34% PC, respectively (Table 1),compared with the steady-state PC content of approximately42% of the total PL content measured for these same strainsgrown in I+C+E- medium (Table 1). These data clearlydemonstrated that ethanolamine was the medium componentresponsible for effecting the reduction in bulk membrane PCcontent measured for the cki-284::HIS3 and cptl::LEU2 mu-

tant strains. Moreover, the ethanolamine sensitivity of bulk PCin cki-284::HIS3 yeast strains was consistent with the idea thatthe correction of bulk membrane PC content, at steady state,was mediated by an ethanolamine-sensitive CKIase.

Ethanolamine elect reflects the differential contributions ofCDP-ethanolamine pathway enzymes to PC biosynthesis. Wewished to test the hypothesis that the differential intercessionof CDP-ethanolamine enzymes in PC biosynthesis accountedfor this ethanolamine effect. Unfortunately, a direct test of theprediction that yeast EKIase is responsible for the steady-statecorrection of CKIase deficiency (at the level of bulk membranePC) is not yet possible, as the structural gene for the yeastEKIase has not been identified. Thus, to test the question ofwhether inactivation of the appropriate CDP-ethanolaminepathway enzyme would eliminate the observed ethanolamineeffects, we focused our attention on the final reactions of theCDP-choline and CDP-ethanolamine pathways, which are

catalyzed by the CPT1 and EPTI gene products, respectively(6, 7). The CPTI and EPTI genes encode aminoalcohol phos-photransferases which are capable of utilizing either choline or

ethanolamine as a substrate in vitro, in direct analogy to thescenario considered above for the CKIase and the EKIase.Thus, the intercession hypothesis predicted that cptl eptldouble mutants would display a reduced, and ethanolamine-insensitive, bulk membrane PC content at steady state. Thedata are shown in Table 1.As expected, the eptl::URA3 mutant, which retains normal

CPT1 gene product function, displayed a wild-type PC content(40% of the total PL content) when grown in either E+ or E-medium (Table 1). In agreement with the test prediction, thecptl::LEU2 eptl::URA3 double mutant, which should be com-

pletely deficient in PL synthesis by both the CDP-choline andCDP-ethanolamine pathways, displayed reduced bulk mem-

brane PC content (only 30% of the total PL content) regard-less of the ethanolamine content of the growth medium (Table1). We interpret these data to indicate that the correction ofPC biosynthetic defects at the steady-state level in the cptlmutant was due to the ethanolamine-sensitive contribution toPC synthesis by EPTase. As the eptl::URA3 mutation does notexert a significant effect on PC synthesis through the CDP-choline pathway, as measured by pulse-radiolabeling experi-ments following the incorporation of either [14C]choline or 32pinto PL (12), the demonstration that EPTase can contributesignificantly to steady-state bulk membrane PC levels indicated

TABLE 2. Steady-state PL composition of strains grownin I-C-E- mediuma

Mole fraction of the following PL species

Strain Relevant (% of total PL content):genotype

PC PI PS PE

CTY182 WV 42±2.4 12± 1.7 10± 1.6 19± 1.7CTY105 bsr2-5 36 ± 2.2 21 ± 1.4 9 ± 0.9 20 ± 2.5CTY434 cptl::LEU2 43 ± 2.1 14 ± 0.8 10 ± 1.4 19 ± 1.2CIY436 eptl::URA3 43 ± 1.7 13 ± 0.9 10 ± 0.7 18 ± 1.5CfY618 cptl::LEU2 37 ± 1.4 12 ± 1.1 10 ± 1.2 19 ± 1.6

eptl::URA3a The indicated strains were grown for five to six generations in I-C-E-

medium supplemented with 32Pi to 10 ,uCi/ml. Glycerophospholipids wereextracted, resolved, and quantitated as described in Materials and Methods.Details regarding the determination of the mole percentage values are presentedin footnote a to Table 1. Data are averages of those from three independent trials± standard deviations. Complete genotypes for the strains are provided inMaterials and Methods.

b WT, wild type.

that in vivo catalysis of PC biosynthesis by this enzyme isinefficient at best.Another noteworthy feature of the data described above was

that the CCTase-deficient bsr2-5 mutant consistently displayedreduction in bulk membrane PC content and that this reduc-tion was entirely insensitive to the ethanolamine content of thegrowth medium. This observation was in complete agreementwith biochemical data demonstrating that the correspondingenzyme of the CDP-ethanolamine pathway, the ethanolamine-phosphate cytidylyltransferase (ECTase), is incapable of utiliz-ing choline phosphate as a substrate (12, 17, 18).CDP-ethanolamine pathway enzymes contribute to choline

salvage. When yeast cells are grown in choline-free media, theactivity of the CDP-choline pathway is relegated to a salvagepathway that does not contribute to net PC biosynthesis butserves to recycle soluble choline-containing compounds pro-duced by PC turnover back into PC. Thus, we wished todetermine the effect of ethanolamine on bulk membranecomposition in CDP-choline pathway mutants grown in theabsence of exogenously supplied choline. As shown in Table 2,PC represented 42% of the total PL recovered from bulkmembranes prepared from wild-type cells cultured in I-C-E-medium. Under these same growth conditions, CCIase-defi-cient bsr2-5 mutants experienced a reduction in bulk PCcontent to only approximately 36% of the total PL content.While neither the cptl:.LEU2 mutation nor the eptl::URA3mutation alone exerted any significant effect on cellular PClevels, the combination of both cptI::LEU2 and eptl:URA3effected a decrease in the steady-state bulk membrane PCcontent to only 37% of the total PL content (Table 2).Moreover, incubation of the cptl::LEU2 and cki-284:.HIS3mutants in I-C-E+ medium was sufficient to reduce the PCcontent of each of these mutants to the levels measured forbsr2-5 mutants and cptl::LEU2 eptl::URA3 double mutants(data not shown).We consider the observed reductions in steady-state bulk

membrane PC content described above to be significant fortwo reasons. First, these reductions were highly reproducible,and they were unusual in that the contribution of PC to bulkmembrane PL content at steady state was very consistent underany given set of experimental conditions. Second, the reduc-tion in bulk membrane PC content manifested itself in a clearoverproduction of the inositol (opi) phenotype. The opi phe-notype has been correlated with unbalanced PL biosynthesis inS. cerevisiae (11). This opi effect is revealed by the ability of

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wildtype cptl::LEU2

eptl::URA3 cptl,::LEU2, eptl::URA3

bsr2-5FIG. 1. A subset of CDP-choline pathway mutants excrete inositol. A bioassay for inositol excretion is described in Materials and Methods.

Briefly, an inositol-requiring strain, CTY479 (bsd2-1 SEC14), was dilution streaked away from a patch of the strain whose inositol excretion (i.e.,opi) phenotype was to be tested. Thus, each streak (from left to right and top to bottom in the figure) of CTY479 exhibited progressively fewercells. The relevant genotype of each strain tested is shown below the corresponding panel.

inositol-overproducing strains to excrete inositol and crossfeedan appropriate inositol auxotroph reporter strain. As demon-strated in Fig. 1, neither wild-type nor cptl::LEU2 nor eptl::URA3 strains exhibited crossfeeding capability. All of thesestrains had similar wild-type bulk membrane PL profiles (seeabove). By contrast, both the bsr2-5 strain and the cptl::LEU2eptl::URA3 double-mutant strain not only shared the propertyof constitutively reduced bulk membrane PC content, but theyalso had unambiguous opi phenotypes. We wish to emphasizethat these opi phenotypes, while clearly scorable, were none-theless qualitatively weaker than those elicited by classical opimutations, e.g., opil mutations that result in transcriptionalderepression of INO1, the structural gene for the key inositol

biosynthetic enzyme inositol-1-phosphate synthase (10). Curi-ously, addition of ethanolamine to the medium failed to elicitan opi phenotype in cptl::LEU2 and cki-284::HIS3 mutants(data not shown), even though ethanolamine supplementationeffected reductions in bulk membrane PC content in thesestrains. We interpret this result to indicate that ethanolaminesupplementation of the medium does not lead to a quantita-tively equivalent reduction in the involvement of EKIase andEPTase in PC biosynthesis to that elicited by genetic disruptionof at least the EPTase structural gene. Thus, the opi phenotypeappears to be a less sensitive (or less direct) indicator of PCbiosynthetic defects than is reduced bulk membrane PC con-tent itself. These collective data indicate that the salvage

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120 -

100-

80-

60-

40-

20

0-

100% 102%

92.1%

14.6%

-.7.7%~~~~~~~~~.

~ o1ss1

FIG. 2. Incorporation of ethanolamine into PL in CDP-cholinepathway mutant strains. The indicated strains were grown to mid-logarithmic growth phase in 2.0 ml of minimal defined I+C-E- growthmedium and pulse-radiolabeled with ['4C]ethanolamine (1 RCi/ml) for20 min. Aliquots were removed from the labeled culture and evaluatedfor total uptake of radiolabel into cells, and radiolabeled PLs wereextracted from the remainder of the culture. Values shown representthe percentages of total incorporated radiolabel recovered in the PLfraction (determined as described in Materials and Methods).

activity of the CDP-choline pathway was required for themaintenance of normal bulk membrane PC content when yeastcells were grown in the absence of exogenously suppliedcholine and that intercession of specific CDP-ethanolaminepathway enzymes was sufficient to correct PC salvage pathwaydefects at the level of either CKIase or CPTase when thecorresponding mutants were grown in ethanolamine-free me-dium.PE synthesis in CDP-choline pathway mutants. To further

assess the extent of cross-pathway intercession by enzymes ofthe CDP-choline and CDP-ethanolamine pathways, we wishedto determine the extent to which CDP-choline pathway en-zymes could contribute to the apparent rate of bulk membranePE synthesis. Thus, the ability of the CDP-choline pathwaymutants to incorporate radiolabeled ethanolamine into lipidwas examined. Strains bearing individual cki-284:HIS3, bsr2-5,cptl::LEU2, or eptl::URA3 mutations were grown to mid-logarithmic growth phase in I+C-E- medium and pulse-radiolabeled with [14C]ethanolamine for 20 min. Radiolabelingwas terminated by the addition of trichloroacetic acid, and thelipids were extracted from each culture and analyzed asdescribed in Materials and Methods. Representative data arepresented in Fig. 2. In agreement with previous reports that theCKJ gene product contributes to PE synthesis in vivo (18), thecki-284::IS3 disruption reduced the efficiency of incorpora-tion of [14C]ethanolamine into lipid by 55% relative to that ofthe isogenic wild-type strain. The bsr2-5 allele, however, failedto exert a discernible effect on the apparent rate of PE

TABLE 3. Steady-state PL composition of strains grownin I+C-E+ mediuma

Mole fraction of the following PL speciesStrain Relevant (% of total PL content):genotype

PE PI PS PC

CTY182 WT 24 ± 1.2 26 ± 1.2 7 ± 0.2 27 ± 2.0CIsY105 bsr2-5 24 ± 0.5 25 ± 1.8 6 ± 0.3 27 ± 1.8CTY392 cki-284::HIS3 21 ± 0.7 29 ± 0.8 7 ± 0.4 26 ± 1.5CT'Y434 cptl::LEU2 23 ± 0.6 27 ± 1.1 7 ± 0.4 28 ± 1.6CIY436 eptl::URA3 18 ± 1.4 26 ± 1.1 8 ± 0.5 31 ± 2.1CIY618 cptl::LEU2 18 ± 1.3 28 ± 1.5 9 ± 0.8 27 ± 1.8

eptl::URA3

aThe indicated yeast strains were cultured for five to six generations incholine-free synthetic defined medium supplemented with inositol and ethanol-amine (each at a final concentration of 1 mM) and 32p; (10 ,uCi/ml). Glycero-phospholipids were extracted and resolved by paper chromatography, and eachindividual PL species was quantitated as described in Materials and Methods.Details regarding determination of the mole percentage values are presented infootnote a to Table 1. Values are averages of those from three independent trials± standard deviations.

b WT, wild type.

synthesis via the CDP-ethanolamine pathway, as evidenced bythe wild-type level of incorporation of [14C]ethanolamine intolipid displayed by the bsr2-5 strain. In contrast, disruption ofthe EPTI gene resulted in an 85% decrease in the incorpora-tion of [14C]ethanolamine into lipid, while disruption of CPT1effected only an approximately 8% reduction in the activity ofthe CDP-ethanolamine pathway (Fig. 2). As expected, thecptl::LEU2 eptl::URA3 double mutant was very defective inthe incorporation of radiolabeled ethanolamine tracer into PL(92% reduction relative to the wild-type level).

Steady-state PE levels in bulk membranes of CDP-cholinepathway mutants. Intercession of CDP-ethanolamine enzymesin the CDP-choline pathway is apparent only at the steady-state level in mutant strains that are defective for the corre-sponding CDP-choline pathway enzyme (see above). Thus, wewished to assess the influence of CDP-choline pathway enzymeactivity on bulk membrane PE levels at steady state in a mutantdefective for CDP-ethanolamine pathway function. To inves-tigate the extent to which CDP-choline pathway enzymes couldcontribute to bulk membrane PE composition at the steady-state level, we determined the bulk membrane PL compositionof CDP-choline pathway mutants grown in I+C-E+ medium.These experiments involved the steady-state labeling of theappropriate yeast strains with 32Pi followed by extraction,resolution, and quantitation of bulk membrane PLs (seeMaterials and Methods). By analogy to the ethanolamineeffects characterized above, the I+C-E+ medium cannot serveas a source for the PL headgroup precursor (i.e., choline) thatmight interfere with the involvement of CDP-choline pathwayenzymes in the CDP-ethanolamine pathway. As a result, theI+C-E+ growth medium was expected to represent the mostpermissive condition for detecting potential CDP-cholinepathway enzyme-mediated correction of reduced bulk mem-brane PE content resulting from CDP-ethanolamine pathwaydysfunction. The most satisfactory system to analyze thisquestion again involved CPTase and EPTase, as these repre-sent the only corresponding pair of CDP-choline and CDP-ethanolamine enzymes for which loss-of-function mutants af-fecting both enzymes presently exist.The representative data presented in Table 3 show that PE

accounted for approximately 24% of the total PL in wild-typeyeast membranes at steady state under these growth condi-tions. As expected, while bsr2-5 and cptl::LEU2 mutants also

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exhibited wild-type bulk membrane PE profiles, disruption ofCDP-ethanolamine pathway function by the eptl::URA3 mu-tation elicited a reproducible reduction in bulk membrane PEcontent, to approximately 18% of the total PL content. IfCPTase contributed to bulk membrane PE synthesis in amanner analogous to the contribution of EPTase to bulkmembrane PC synthesis (Table 1), the cptl::LEU2 eptl::URA3double mutant would exhibit an even greater reduction in bulkmembrane PE content than that measured for the eptl::URA3single mutant. The data show that the cptl::LEU2 eptl::URA3double mutant exhibited a bulk membrane PE content that wasindistinguishable from that recorded for the eptl::URA3 singlemutant (Table 3). These results indicate that CPTase did notcontribute to bulk membrane PE synthesis at steady state wheneptl::URA3 yeast cells were grown in choline-free medium.Two possible explanations for this result are offered. First, asCDP-ethanolamine is a poor substrate for CPTase in vitro (6),it is possible that the lack of contribution by CPTase to PEsynthesis simply reflects this aspect of CPTase substrate spec-ificity. Alternatively, the flux of choline through the CDP-choline pathway via salvage may be sufficiently high, even whencells are grown in choline-free medium, to effectively precludeparticipation of CPTase in CDP-ethanolamine pathway func-tion by virtue of substrate-level competition. Consistent withthe latter possibility, we have demonstrated that the activity ofthe CDP-choline pathway in PC synthesis via choline salvagecontributes significantly to the apparent rate of PC synthesis inyeast cells cultured in choline-free medium (12).As CKIase significantly contributes to bulk cellular EKIase

activity in yeast cells (Fig. 2) (8, 12), we expected this enzymeto contribute to bulk membrane PE synthesis at steady state.Analysis of bulk membrane PE in cki-284::HIS3 mutantsrevealed only a very modest, but reproducible, reduction inbulk membrane PE content (21% of the total PL) relative tothat measured for wild-type cells (24% of the total PL; Table3). This marginal reduction in bulk membrane PE content inthe cki-284::HIS3 mutant likely reflected not only the loss ofCKIase-associated EKIase activity, but also the continuedinvolvement of the dedicated EKIase.

Cellular requirement for SEC14p is not related to theethanolamine effect. We had previously demonstrated thatinactivation of the CDP-choline pathway is sufficient to com-

pletely abrogate the normally essential cellular requirementfor SEC14p function (4). These data were interpreted as

suggesting that Golgi membranes required a particular PLcomposition in order to execute Golgi secretory functions andthat it was the purview of SEC14p to maintain such an

appropriate Golgi membrane PL composition. An extendedinterpretation of those data that could be considered is thatinactivation of the CDP-choline pathway might result in alter-ations in bulk membrane PL composition that are compatiblewith a membrane PL composition potentially required foryeast Golgi secretory function. The imposition of such alteredbulk membrane composition on Golgi membranes (by bulkmembrane flow) might then obviate the requirement forSEC14p. Our demonstration that cki and Cpt mutants showedreductions in bulk membrane PC content when grown in thepresence of ethanolamine, and that cCt mutants exhibitedconstitutively reduced bulk membrane PC content, was at leastsuperficially consistent with such an idea. Therefore, we testedwhether the "bypass SEC14p" phenotype associated with ckimutations required the presence of ethanolamine in thegrowth medium. These bypass SEC14p mutants were initiallyisolated on YPD, an ethanolamine-containing medium (4).The isogenic strains CTY182 (wild type), CTY1-1A (sec14-1J),and CTY394 (secl4-88::URA3 cki-284:WIIS3) were streaked

out for isolation on I+C+E+ and I+C+E- plates. The abilitiesof these strains to form isolated colonies on E+ and E- mediaat 30°C, a permissive temperature for sec14-1ts strains, wereassessed. The secl4-88::URA3 allele is normally a haploidlethal mutation under all growth conditions, but in strainCTY394, viability is conferred by the cki-284::HIS3 allele. Asexpected, both the wild-type and sec14-1's strains grew underboth the E+ and E- conditions. Growth of the secl4-88::URA3cki-284::HIS3 double mutant was similarly insensitive to theethanolamine content of the medium (data not shown). Thus,the presence of ethanolamine in the medium was not obliga-torily required for cki-mediated suppression of the normallylethal secl4-88::URA3 allele.

Conclusions. Collectively, the data presented in this reportdemonstrate an interrelationship between the CDP-cholineand CDP-ethanolamine PL biosynthetic pathways in yeast cellsand demonstrate that, in the face of a dysfunctional CDP-choline pathway, biochemically redundant enzymes of theCDP-ethanolamine pathway can significantly contribute tobulk membrane PC biosynthesis-as long as ethanolamine isabsent from the growth medium. Although this ethanolamine-sensitive contribution to PC biosynthesis is relatively ineffi-cient, as indicated by the lack of effect of the eptl::URA3 alleleon apparent rates of PC synthesis via the CDP-choline pathwayin in vivo pulse-radiolabeling experiments (12), it is significantthat it is sufficient to correct, at steady state, the PC biosyn-thetic defects of cki-284::HIS3 and cptl::LEU2 mutant strains.By contrast, mutants defective in CCTase activity (i.e., bsr2-5mutants) experienced an ethanolamine-insensitive reductionin bulk membrane PC content. This finding is readily explainedby the demonstration that the phosphoaminoalcohol cytidylyl-transferases display tight lipid headgroup precursor specifici-ties. CCTase does not utilize ethanolaminephosphate as asubstrate in vitro (17) or in vivo (Fig. 2), while the ECTase wasnot capable of correcting steady-state PC defects resultingfrom bsr2-5 defects. On the basis of the collective datareported here, we make the following two predictions. First, wepredict that yeast strains defective for both CKIase activity andEKIase activity will exhibit an ethanolamine-insensitive reduc-tion in bulk membrane PC content of the order exhibited bythe cptl::LEU2 eptl::URA3 double mutant. Second, we predictthat ECTase defects will have no effect on the bulk membranePC of otherwise wild-type strains, regardless of the ethanol-amine content of the medium in which such strains are grown,and that inactivation of ECTase will not further exacerbate theethanolamine-insensitive reduction in bulk membrane PC con-tent exhibited by CCTase-deficient strains. One puzzling fea-ture of the data was our demonstration that bsr2-5 mutantsuniquely exhibited elevated PI levels, relative to those of theother strains employed in these studies, when grown inI+C+E- and I-C-E- media (Tables 1 and 2) but not whengrown in I+C+E+ or I+C-E+ medium (Tables 1 and 3). Thebasis for this phenomenon remains unclear.

Finally, the data reported here provide additional insightinto the relationship between CDP-choline pathway functionand the normally essential involvement of a phosphatidylino-sitol/phosphatidylcholine transfer protein (SEC14p) in thestimulation of Golgi secretory function in S. cerevisiae (2, 4).We proposed that SEC14p functions to control the PI and/orPC content of yeast Golgi membranes so that Golgi secretoryfunction can be maintained. Two possible mechanisms bywhich inactivation of the CDP-choline pathway could bypassSEC14p function were entertained: (i) that SEC14p exertedsome local control of PL composition in the Golgi membranesthat was in principle antagonistic to CDP-choline pathwayfunction, a local control that was in effect reimposed in

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6868 McGEE ET AL.

SEC14p-deficient yeast cells by specific inactivation of theCDP-choline pathway, or (ii) that inactivation of the CDP-choline pathway effected a bulk membrane PL compositionthat was compatible with Golgi secretory function and, uponimposition of this bulk PL composition on Golgi membranesby bulk membrane flow, that rendered SEC14p-mediatedcontrol of Golgi membrane PL composition unnecessary (4).Consideration of the latter possibility was prompted by ourfinding that CDP-choline pathway mutants exhibited reducedbulk membrane PC content when grown in YPD medium. Ourdemonstration that this reduction in bulk membrane PCcontent is a simple function of the ethanolamine content of thegrowth medium, when coupled with our observation thatsuppression of SEC14p defects by loss of CDP-choline path-way activity does not obligatorily require ethanolamine in thegrowth medium (see above), provides a strong argumentagainst the latter proposal for how CDP-choline pathwaydysfunction effects bypass of the SEC14p requirement forGolgi function and cell viability. Rather, the data are mostconsistent with a local relationship between SEC14p andCDP-choline pathway function in Golgi membranes. Thisconclusion is further supported by (i) quantitative analyses ofGolgi membrane PL composition, as a function of SEC14pactivity, in yeast strains in which Golgi secretory function isuncoupled from its usual SEC14p requirement (12) and (ii) invivo and in vitro data suggesting that SEC14p repressesCDP-choline pathway activity in a PL-ligand-modulated fash-ion by downregulating CCTase, the rate-determining enzymeof this pathway (15).

ACKNOWLEDGMENTThis work was supported by grant GM44530 from the National

Institutes of Health to V.A.B.

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