Good Fat, Essential Cellular Requirements for TriacylglycerolSynthesis to Maintain Membrane Homeostasis in Yeast*□S

Received for publication, May 25, 2009, and in revised form, July 16, 2009 Published, JBC Papers in Press, July 16, 2009, DOI 10.1074/jbc.M109.024752

Julia Petschnigg‡1, Heimo Wolinski‡, Dagmar Kolb‡, Gunther Zellnig§, Christoph F. Kurat‡, Klaus Natter‡,and Sepp D. Kohlwein‡2

From the ‡Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/II, A8010 Graz and §Institute of Plant Sciences,University of Graz, A8010 Graz, Austria

Storage triacylglycerols (TAG) and membrane phospholipidsshare commonprecursors, i.e. phosphatidic acid and diacylglyc-erol, in the endoplasmic reticulum. In addition to providing abiophysically rather inert storage pool for fatty acids, TAG syn-thesis plays an important role to buffer excess fatty acids (FA).The inability to incorporate exogenous oleic acid into TAG in ayeast mutant lacking the acyltransferases Lro1p, Dga1p, Are1p,and Are2p contributing to TAG synthesis results in dysregula-tion of lipid synthesis, massive proliferation of intracellularmembranes, and ultimately cell death. Carboxypeptidase Y traf-ficking from the endoplasmic reticulum to the vacuole isseverely impaired, but the unfolded protein response is onlymoderately up-regulated, and dispensable formembrane prolif-eration, upon exposure to oleic acid. FA-induced toxicity is spe-cific to oleic acid and much less pronounced with palmitoleicacid and is not detectable with the saturated fatty acids, palmiticand stearic acid. Palmitic acid supplementation partially sup-presses oleic acid-induced lipotoxicity and restores carboxypep-tidase Y trafficking to the vacuole. These data show the follow-ing: (i) FA uptake is not regulated by the cellular lipidrequirements; (ii) TAG synthesis functions as a crucial intracel-lular buffer for detoxifying excess unsaturated fatty acids; (iii)membrane lipid synthesis and proliferation are responsive toand controlled by a balanced fatty acid composition.

In the aqueous cellular environment, fatty acyl chains ester-ified in glycerophospholipids constitute the hydrophobic bar-rier of biological membranes. Thus, fatty acid (FA)3 composi-tion is a crucial determinant of cellular membrane function.Establishment of the specific FA profiles in lipid species of var-ious organelle membranes (1) relies on an intricate balancebetween endogenous FA synthesis, recycling of FA from lipidbreakdown, and perhaps uptake from the exterior. Glycero-

phospholipids and triacylglycerols (TAG), which serve as themajor storage form of FA, share the similar precursors phos-phatidic acid (PA) and diacylglycerol (DAG), both generated inthe endoplasmic reticulum (ER)membrane. TAG are packagedinto lipid droplets and are thus sequestered away from the ERmembrane by a mechanism not yet understood. In addition,membranes and lipid storage pools (2, 3) undergo significantturnover and intracellular flux, e.g. during secretion or endocy-tosis and cellular growth, which must be accounted for bymechanisms that establish and maintain lipid homeostasis inthese dynamicmembrane systems (4).We have recently shownthat TAG degradation provides metabolites that are critical forefficient cell cycle progression at the G1/S transition (3). Netsupplywith FA in growing cells, however, is procured by endog-enous synthesis (5); nevertheless, yeast also has a high capacityfor FAuptake (6), whichmay become essential in the absence ofendogenous synthesis, e.g. in fatty-acid synthase mutants or inthe presence of the fatty-acid synthase inhibitor cerulenin. Thebroad substrate specificity of up to six acyl-CoA synthetasesmay explain the ability of yeast to take up various “non-natural”FA (6), which may be present in their environment. FA derivedfrom exogenous sources, endogenous de novo synthesis, orfrom lipid turnover react, as coenzyme derivatives, with glyc-erol 3-phosphate to form PA, which is the central precursorboth for membrane phospholipids and for storage TAG. Ester-ification of the phosphate residue gives rise to cellular phospho-lipids, whereas PA dephosphorylation and one additional acy-lation step yield TAG, which are stored in lipid droplets. Themechanisms that direct and regulate the flux of activated FAeither into membrane lipid or storage lipid (i.e. TAG) synthesisare unknown.However, upon FAoverload, cells produce excessTAG, which results in proliferation and accumulation of lipiddroplets.In mammals, adipocytes are the preferred sites of excess

TAG storage, whereas cells of nonadipose tissues have a ratherlimited capacity for neutral fat deposition; if this storage capac-ity is exceeded, FA and lipid overloadmay lead to abnormal cellfunction and ultimately cell death, also referred to as lipotoxic-ity (7). Thus, TAG synthesis appears to play an important rolein buffering excess FA to prevent their incorporation into met-abolically critical molecules, such as diacylglycerols (8) andsphingolipids (7, 9, 10), whichmay serve essential signaling andstructural functions. Ultimately, after prolonged exposure tolipotoxic FA, cells may undergo apoptosis or necrosis (8–11).In this studywe have established and further explored a yeast

model system for FA-induced lipotoxicity to address themolec-

* This work was supported in part by grants from the Federal Ministry forScience and Research (Project GOLD, Genomics of Lipid-associated Disor-ders, in the framework of the GEN-AU Program) and the Austrian ScienceFund Project SFB Lipotox F3005 (to S. D. K.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1 and 2.

1 Recipient of a DOC-fFORTE stipend of the Austrian Academy of Sciences.2 To whom correspondence should be addressed. Tel.: 43-316-380-5487; Fax:

43-316-380-9854; E-mail: sepp.kohlwein@uni-graz.at.3 The abbreviations used are: FA, fatty acid; DAG, diacylglycerol; TAG, triacyl-

glycerol; PA, phosphatidic acid; ER, endoplasmic reticulum; ERAC, ER-asso-ciated compartment; UPR, unfolded protein response; LD, lipid droplet;GFP, green fluorescent protein; SE, steryl ester; CPY, carboxypeptidase Y.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 45, pp. 30981–30993, November 6, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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ular mechanisms that control FA fluxes and to understand thephysiological consequences of a lipotoxic insult. This yeaststrain lacks all four acyltransferases involved in TAG synthesis,namely the diacylglycerol acyltransferase ortholog Dga1p (12),lecithin-cholesterol acyltransferase-related open reading frameLro1p (lecithin-cholesterol acyltransferase ortholog (13)), andboth acyl-CoA:cholesterol O-acyltransferase-related enzymes,Are1p and Are2p (acyl-CoA:cholesterol O-acyltransferaseorthologs (14, 15)). Quite astonishingly, this mutant is viabledespite the complete absence of TAG and steryl esters and,accordingly, also the lack of lipid droplets, which are the storagecompartment for both types of neutral lipids (16, 17). However,the are1� are2� dga1� lro1� quadruple mutant YJP1078becomes highly sensitive to the presence of unsaturated FA,such as oleic acid and palmitoleic acid. These fatty acids, whichare preferentially incorporated into TAG in wild-type cells,promote cell death in a time- and concentration-dependentmanner, which becomes apparent only after 3–6 h of incuba-tion with the lipotoxic FA. Short term incubation, however,leads to massive alterations of cellular phospholipid profilesand the accumulation of intracellular membranes; the deliveryof carboxypeptidase Y to the vacuole is blocked; however, theunfolded protein response (UPR) is only moderately up-regu-lated during membrane proliferation upon unsaturated FAchallenge. Notably, UPR is not required for the proliferation ofmembranes, as a pentuple mutant additionally deleted for theUPR sensor kinase, Ire1p, displays a similar time course ofmembrane proliferation upon exposure to oleic acid. Thesedata demonstrate the following: (i) FA uptake is not limited bythe cellular requirements; (ii) in the absence of TAG synthesisexcess FA are channeled into membrane phospholipid synthe-sis; (iii) UPR is not involved in excessmembrane proliferation inmutants defective in TAG synthesis; and (iv) FA-induced celldeath is not an immediate response to oleic acid exposure, butrather the consequence of induced defects of cellular mem-brane trafficking.

MATERIALS AND METHODS

Media and Culture Conditions—Standard YPD media con-tained 1% yeast extract (Difco), 2% glucose (Merck), 2% Bacto-peptone (Difco); YND minimal media with 2% glucose, 2%galactose (Sigma), or 2% raffinose (Acros Organics) as the car-bon sources contained 0.17% Yeast Nitrogen Base withoutamino acids and ammonium sulfate (Difco), 0.5% ammoniumsulfate, supplemented with the respective amino acids andbases (adjusted to pH 6). Yeast transformants carrying expres-sion plasmids were grown in uracil-free minimal medium.Sporulation medium contained 0.25% yeast extract, 1% potas-sium acetate, and 0.1% glucose. Ampicillin-resistant Esche-richia coli transformants were selected in LBA media (1%Bacto-tryptone (Difco), 0.5% yeast extract (Difco), 0.5% NaCl,100 �g/ml ampicillin (Amresco)).

Geneticin resistance was determined on YPD plates contain-ing 200 mg/liter geneticin (G418, Calbiochem). Expression ofGFP fusions under control of aCUP1 promoter was induced bythe addition of 0.5 mM CuSO4 to the medium. Expression ofGFP fusions under the control of the MET25 promoter wasinduced in the absence of methionine in the media. Expression

of GFP fusions or glutathione S-transferase fusions under thecontrol of the GAL1/10 promoter was induced in media con-taining 2% galactose instead of glucose.For fatty acid treatment, palmitic acid, palmitoleic acid, ste-

aric acid, and oleic acid (Sigma) were dissolved in pre-warmedBrij58 (Sigma) and added to the culture media or agar plates atthe indicated concentrations (final concentration of Brij58 was1%). For liquid cultures, cells were grown overnight, shifted tofresh medium, grown to log phase, and exposed to the respec-tive fatty acids for the indicated periods of time. Control mediacontained 1%Brij58, which, however, did not affect growth. Forgrowth tests, cells were cultivated overnight in liquid media,adjusted toA600� 0.5, and serially diluted in 1:10 steps, and 2�lof the respective dilutions were spotted onto media plates con-taining 2% agar.Yeast Strains and Plasmids—Yeast strains used in this study

were wild-type BY4742 (MATa his3�1 leu2�0 lys2�0 ura3�0),are1� mutant (MAT� his3�1 leu2�0 lys2�0 ura3�0yor048w�::KanMX4), are2� mutant (MAT� his3�1 leu2�0lys2�0 ura3�0 ynr019w�::KanMX), are1� are2� doublemutant, YJP1076 (MAT� his3�1 leu2�0 lys2�0 ura3�0ycr048w�::KanMX4 ynr019w�::KanMX), dga1� lro1� doublemutant, YJP1075 (MAT� his3�1 leu2�0 lys2�0 ura3�0yor245c�::KanMX4 ynr008w�::KanMX4), an are1� are2�dga1� lro1� quadruple deletion strain, YJP1078 (MAT�his3�1 leu2�0 lys2�0 ura3�0 ycr048w�::KanMX4 ynr019w�::KanMX4 yor245c�::KanMX4 ynr008w�::KanMX4), and anire1� are1� are2� dga1� lro1� pentuple mutant (MAT�his3�1 leu2�0 lys2�0 ura3�0 ycr048w�::KanMX4ynr019w�::KanMX4 yor245c�::KanMX4 ynr008w�::KanMX4yhr079c�::URA3). Deletion strains were obtained from Euro-scarf. Double mutants and the quadruple mutant were con-structed by standard genetic crosses and tetrad dissection; dou-blemutations (i.e. dga1� lro1� and are1� are2�) were typicallyidentified in nonparental di-type tetrads (based on geneticinresistance) and verified by colony PCR, using gene deletion-specific primers. These double mutants were used for furthercrosses to select triple and quadruple mutants, which wereidentified by colony PCR.The construction of the ire1� are1� are2� dga1� lro1� pen-

tuple mutant was performed as follows. The IRE1 open readingframe was replaced by the URA3 gene by homologous recom-bination in the are1� are2� dga1� lro1� background using theprimers 5�-CATTAAAAAAACAGCATATCTGAGGAATT-AATATTTTAGCACTTTGAAAAGCTTTTCAATTCAAT-TCATC-3� and 5�-TAACATTAATGCAATAATCAACCAA-GAAGAAGCAGAGGGGCATGAACATGCAGGGTAATAA-CTGATATAA-3�.URA3was amplified by PCR, using the plas-mid pCGCU as the template. PCR products were transformedinto the are1� are2� dga1� lro1� background, and positivetransformants were selected on SC plates lacking uracil. Pen-tuple mutants were verified by colony PCR, using gene dele-tion-specific primers.E. coli TOP10F� ([proAB, laqIq, lacZDM15, Tn10(Tetr)],

mcrA�(mrr-hsdRMS-mcrBC), �80�lacZM15, �lacX74, deoR,recA1, araD139�(ara, leu), 7697galU, galK, �-rps(streptomy-cinr), endA1, nupG) was used for plasmid amplification and

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purification. Transformation of yeast and E. coli cells was donefollowing standard procedures (18).Cloning Procedures—For localization studies, reading

frame ELO3 was amplified by PCR and cloned into thepUG35 vector (19), harboring a MET25 promoter and theGFP reading frame 3� to the multiple cloning site. Yeast wild-type BY4742 genomic DNA was used as the template, andprimers 5�-ACGTATCTAGAATGAACACTACCACATCT-ACTGTTATAGCA-3� and 5�-ACGTAGTCGACAGCTTTC-CTGGAAGAGACCTTGG-3� were used for ELO3 amplifica-tion. The fragments were cleaved with XbaI and SalI andinserted into the multiple cloning site of pUG35, to yield aC-terminal GFP fusion. Plasmid pCGAU (GFP-skl) expressingthe peroxisomal marker under control of the CUP1 promoterand harboring aURA3 selectionmarker was kindly provided byDr. Uros Petrovic, Ljubljana, Slovenia.ARE1, ARE2, LRO1, andDGA1were cloned into pCGCUand

pCGGU plasmids, containing monomeric GFP, a URA3 selec-tion marker, and the CUP1 or GAL10 promoter, respectively.Primers used were as follows: 5�-ACGTAGAATTCATGACG-GAGACTAAGGATTTGTTG-3� and 5�-ACGTAGCATGCT-AAGGTCAGGTACAACGTCATAATGA-3� for ARE1 ampli-fication; 5�-ACGTAGAATTCATGGACAAGAAGAAGGAT-CTACTGG-3� and 5�-ACGTAGCATGCGAATGTCAAGTA-CAACGTACACATGAC-3� for ARE2 amplification; 5�-ACG-TAGAATTCATGGGCACACTGTTTCGAAGA-3� and 5�-ACGTAGCATGCCATTGGGAAGGGCATCTGAG-3� forLRO1 amplification; and 5�-ACGTAGAATTCATGTCAGGA-ACATTCAATGATATAAGAAG-3� and 5�-ACGTAGCATG-CCCCAACTATCTTCAATTCTGCATC-3� for DGA1 ampli-fication. Fragments were cut with EcoRI and SphI and ligatedinto the multiple cloning site of pCGCU or pCGGU.LRO1 andDGA1were also cloned into pYES263 (Euroscarf),

harboring aGAL1/10 promoter, glutathione S-transferase cod-ing sequence, and the URA3 marker. Primers used were asfollows: 5�-ACGTAGGATCCATGTCAGGAACATTCAAT-GATATAAGAAG-3� and 5�-ACGTAGCGGCCGCTTACAT-TGGGAAGGGCATCTGA-3� for LRO1 amplification, and 5�-ACGTAGGATCCATGGGCACACTGTTTCGAAGA-3� and5�-ACGTAGCGGCCGCTTACCCAACTATCTTCAATTC-TGCAT-3� for DGA1 amplification. Fragments were cleavedwith BamHI and NotI and inserted into the multiple cloningsite of pYES263.

�-Galactosidase Assay—Induction of the UPRwasmeasuredusing a reporter construct with aCYC1minimal promoter con-taining 4� UPRE fused to the lacZ gene (pCJ104 plasmid,kindly provided by Stephen Jesch, Ithaca, NY). Cells weregrown overnight in YND medium lacking uracil, transferredinto fresh medium to an A600 � 0.1, and grown for additional4 h. 0.02% oleic acid was added for the indicated times. �-Ga-lactosidase activity was determined as described (18). As a pos-itive control, full induction of UPR in wild-type cells wasachieved by addition of 2 �g/ml tunicamycin for 2 h (20).Cell Viability Measurements—For survival assays, cells were

grown overnight, diluted to 105 cells/ml, and grown for 4 h infresh minimal medium. Fatty acids were added at concentra-tions ranging from 0.0001 to 0.1% for 0–12 h. Aliquots werewithdrawn and the cell number determined using CASY1�

technology (Scharfe SystemGmbH). 300 cells were plated ontoYPDplates and incubated for 2 days at 30 °C. Strains expressingDga1-GFP and Lro1-GFP constructs were grown overnight inthe presence of 0.5 mM CuSO4 in minimal medium lackinguracil, shifted to fresh medium for 4 h, and viable cell countsanalyzed as described above.Carboxypeptidase YProcessing—Cellswere grownovernight,

shifted to freshmedium, and incubated for 4 h. Fatty acids wereadded at a concentration of 0.1%; cells were harvested at indi-cated time points, and proteins were precipitated with 5% tri-chloroacetic acid (final concentration) for 10min on ice. Pelletswere spun down at 12,000 rpm for 5 min, resuspended in 1�SDS loading buffer, and incubated for 5 min at 80 °C. Proteinswere separated on 8% SDS-polyacrylamide gels and electro-blotted to nitrocellulose membranes (Bio-Rad); after blockingwith 5% dry milk powder in TBST buffer (10 mM Tris-HCl, 150mM NaCl, 0.05% Tween 20, pH 8.0), blots were probed withanti-CPY antibody (Rockland Inc.).Lipid Analysis—For total lipid analyses, yeast cells were

homogenized with glass beads in a Merckenschlager homoge-nizer (B. Braun Biotech International) under CO2 cooling, andlipids were extracted with chloroform/methanol, 2:1 (v/v) (21,22). Neutral lipid separation and analysis was performed byTLC on silica gel plates (Merck), essentially as described (23–25), using light petroleum/diethyl ether/acetic acid (32:8:0.4,per volume) as the solvent. Lipidswere visualized onTLCplatesby carbonization after dipping plates into 3.2%H2SO4 and 0.5%MnCl2, followed by heating at 120 °C for 30 min. Lipids werequantified by densitometric scanning at 450 nm (Camag TLCscanner 3), using triolein as the standard (24, 25). Whole celllipid extracts were converted to fatty acid methyl esters using14% boron trifluoride/methanol (26). Gas chromatography/mass spectrometry analysis was performed on a Trace-GCUltra-DSQ-MS system (ThermoElectron, Waltham, MA).Electron Microscopy—Cells were prepared for transmission

electron microscopy essentially as described (27). Yeast cellswere fixed with 1.5% KMnO4 in distilled water for 15 min atroom temperature, dehydrated in a graded series of ethanol(50–100%), embedded in Epon resin, and specimens polymer-ized for 48 h at 60 °C. Ultrathin sections were stained with 2%lead citrate for 5 min and 2% uranyl acetate for 15 min. Ultra-thin sections were observed with a Philips CM 10 transmissionelectron microscope.Fluorescence Microscopy—Vital staining was typically per-

formed with 1 ml of cell suspension. Fluorescence dyes, NileRed, MitoTracker� Red CM-H2XRos (Invitrogen), and theyeast vacuole membrane marker, FM4-64 (Invitrogen), wereadded from stock solutions inDMSOat a final concentration of1 �g/ml. Fluorescence microscopy was performed after 10–20min of incubation with the vital dye, without subsequent wash-ing of cells. Nile Red fluorescence was excited at 543 nm andemission detected simultaneously between 550 and 570 nm forlipid droplets, and between 600 and 650 nm for lipid dropletsplus intracellular membranes, as described previously (28).MitoTracker� Red CM-H2XRos was excited at 543 nm andemission detected between 580 and 650 nm. The yeast vacuolemembranemarker FM4-64was excited at 488 nmand emissiondetected between 580 and 660 nm. SytoxGreenTM (Invitrogen)

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was used as a viability stain, and fluorescence was excited at 488nm and emission detected between 500 and 550 nm; the samesettings were used for GFP detection. Microscopy was per-formed on Leica TCS4d and Leica SP2 confocal microscopes,the latter equippedwith acousto optical beam splitter and spec-tral detection, using 100� oil immersion (NA 1.4) and 40� oilimmersions objectives (NA 1.25). Transmission images wereacquired using differential interference contrast optics. Images

were adjusted for contrast and colorand assembled using Adobe Photo-shopTM CS (Adobe Inc.).

RESULTS

Mutant Lacking Neutral LipidsDisplays Delayed Growth and Mor-phological Defects—The are1�are2� dga1� lro1� quadruplemutant lacks all four enzymesresponsible for the synthesis ofTAG and steryl esters (SE) (16, 29).Accordingly, as shown by stainingwith the lipophilic dyeNile Red (Fig.1A), this mutant is devoid of lipiddroplets (LD). Notably, in thismutant, the Nile Red fluorescenceemission spectrum appears differ-ent from that in the wild type (28);fluorescence of the endomembranesystem between 600 and 650 nm islow inwild-type cells, and inmarkedcontrast, the quadruple mutant dis-plays intense membrane fluores-cence in this emission range,consistent with a changed hydro-phobicity of the environment of dye(30). The fluorescence characteris-tics of Nile Red make it particularlysuited to differentiate the morehydrophobic lipid droplets (emis-sion detection between 550 and 570nm) and the less hydrophobic ERmembrane (emission detectionbetween 600 and 650 nm) in themutants (Fig. 1A) (28); however, italso requires defined spectral detec-tion to avoid mis-interpretation ofmembrane-associated fluorescencesignals as “lipid droplets.” To assessin greater detail the impact of lack-ing TAG and SE synthesis on cellu-lar organelle and membrane mor-phology, we analyzedmitochondria,vacuoles, endoplasmic reticulum,and peroxisomes, by fluorescencemicroscopy, using vital dyes andorganelle-specific GFP fusion con-structs. The ER marker Elo3-GFP(31, 32) was expressed in wild type

and the YJP1078 quadruple mutant. No gross alterations of ERmorphology were observed between the wild-type and themutant at the level of light microscopy (supplemental Fig. 1).Mitochondria stained with MitotrackerTM also displayed nosignificant morphological alterations in the mutant, consistentwith normal respiratory function that was also assessed by test-ing growth on nonfermentable carbon sources (data notshown). Interestingly, and despite recent observations that per-

FIGURE 1. are1� are2� dga1� lro1� quadruple mutant lacks lipid droplets and exhibits growth defects.A, Nile Red staining of wild-type and the are1� are2� dga1� lro1� mutant YJP1078, discrimination of LD andmembranous structures based on different fluorescence emission characteristics of Nile Red. Left panels, �ex/�em excitation/emission at 543/550 –570 nm detects preferentially LD; these are completely absent in themutant (lower row). Right panels, �ex/�em 543/600 – 650 nm fluorescence emission range detects, in addition toLD in wild type, labeled membranous structures both in wild type and in the mutant. Scale bar, 5 �m. B, growth(cell number/ml) of wild type, dga1� lro1� double and are1� are2� dga1� lro1� quadruple mutants in com-plete media. �, wild type; f, dga1� lro1; Œ, are1� are2� dga1� lro1�. Growth of dga1� lro1� and the quad-ruple mutants is severely delayed during the first 7 h of cultivation (inset). All cell types reach similar celldensities in stationary phase.

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oxisomes and lipid droplets closely interact (33), peroxisomeformation in the quadruple mutant upon exhaustion of glucoseappeared very similar to wild type as well. Peroxisome numberand overall shape, as determined by microscopic inspection ofthe peroxisomalmarker GFP-skl (34, 35), was indistinguishablefrom wild type (supplemental Fig. 1A). As a major morpholog-ical phenotype, we observed a highly fragmented vacuole in themutant (supplemental Fig. 1A, panel i); similar fragmentationphenotypes suggestive of impaired membrane fusion are fre-quently observed inmutants defective in lipidmetabolism, suchas fatty acid elongation mutants (31) or phospholipase Cmutants (36).Next, we tested the cellular TAG and SE requirements for

growth. Growth of the YJP1078 mutant strain was severelyimpaired in comparison with the wild type during the first 6–8h of cultivation (Fig. 1B), similarly to the lipase-deficient tgl3�tgl4� mutants, which are unable to degrade TAG (2, 3). Thisobservation is consistent with specific requirements for TAG,presumably to provide lipid precursors for membrane prolifer-ation, during the initial phase of growth (2, 3, 37, 38). In station-ary phase, mutant cells reached about 80–90% of the wild-typecell density. The dga1� lro�1 double mutant displayed a lesssevere growth defect, as it still has residual capacity to synthe-size TAG; in contrast, the are1� are2� doublemutant grew likewild type (not shown), demonstrating that not the formation ofSE but rather TAG is limiting for growth of the mutants.The contribution of the individual acyltransferases to cellular

growth and lipid droplet formation was assessed by expressingARE1, ARE2, DGA1, and LRO1 individually in the quadruplemutant (39). As shown in supplemental Fig. 1, B and C, expres-sion of theDGA1 gene encoding the acyl-CoA-dependentDAGacyltransferase restored growth and induced LD formation to alevel comparable with wild type. Expression of the LRO1 gene(phospholipid-dependent DAG acyltransferase), on the otherhand, was less effective, and the acyl-CoA:cholesterol O-acyl-transferase-related sterol acyltransferase Are2p restoredgrowth and LD formation only to aminor extent.ARE1 expres-sion was not sufficient to drive LD formation, although it waspreviously shown to display minor TAG synthesizing capacity(16, 29). The capacity to synthesize TAG and LD, based onmicroscopic analysis, parallels the growth characteristics of thequadruple mutant transformed with the individual acyltrans-ferases (supplemental Fig. 1, B and C). These data underscorethe cellular requirements for TAG for rapid initiation of growthand demonstrate the specific requirement for TAG rather thansteryl ester synthesis, for lipid droplet formation (39).Lack of TAG Synthesis Renders Mutant Cells Highly Sensitive

to Unsaturated Fatty Acids—The metabolic regulators thatcontrol the flux of FA into membrane or storage lipids are cur-rently unknown. Because TAG are a preferred storage form forunsaturated FA, we next tested the response ofmutants lackingenzymes involved in TAG and SE synthesis to treatment withvarious FA. In a first approach, saturated (palmitic acid, C16:0,and stearic acid, C18:0) and unsaturated FA (palmitoleic acid,C16:1, and oleic acid, C18:1) were applied over a wide range ofconcentrations (0.0001–0.1%) to wild-type and single, double,and quadruple mutants, lacking diacylglycerol and sterol acyl-transferases; these FA represent the most abundant species in

yeast (40). As shown in Fig. 2A, C16:0 and C18:0 saturated FAdo not affect growth of either wild-type or of any of the mutantstrains. Addition of unsaturated FA, on the other hand, stronglyinhibited growth of the dga1� lro1� double mutant and theare1� are2� dga1� lro1� quadruple mutant. Growth of thequadruple mutant was completely abolished at a concentrationof 0.01% oleic acid, whereas the dga1� lro1� double mutantwas more tolerant and required 0.1% oleic acid for completegrowth inhibition. Similarly, supplementation with palmitoleicacid also severely inhibited growth of the double and quadruplemutants, albeit at a 2-fold higher concentration than oleic acid.This unsaturated FA-induced toxicity was indeed due to thelack of TAG synthesis because ectopic expression of DAG acyl-transferases in the mutants fully (Dga1p) or partially (Lro1p)reversed the growth deficiencies on oleic and palmitoleic acid-containing media (Fig. 2A); expression of the sterol acyltrans-ferases, Are1p or Are2p, in the quadruple mutant did notrestore growth in the presence of unsaturated FA (data notshown). These data demonstrate that TAG synthesis, catalyzedby DAG acyltransferases Dga1p and Lro1p, is indispensable forexcess unsaturated FA detoxification. Notably, palmitic acidsupplementation partially restored growth of the quadruplemutant in the presence of oleic acid (see below).Specificity and time dependence of FA toxicity was also

assessed by survival analyses, which showed a clear time andconcentration dependence, and oleate concentrations as low as0.0005% (�15 �M, which corresponds to the critical micellarconcentration) already significantly impaired viability (supple-mental Fig. 2,A and B); incubation with 0.005% palmitoleate oroleate for 3 h rendered more than 70 or 90%, respectively, ofmutant cells nonviable. By 12 h of incubation, less than 10% ofmutant cells survived palmitoleate treatment, and virtuallynone of the cells survived oleate treatment (supplemental Fig. 2,C andD). However, in time course experiments we noticed thatshort term incubation up to 30 min with potentially toxic con-centrations did not result in a marked change in survival rates,demonstrating that yeast cells apparently have a significant butobviously limited capacity to buffer excess FA, and that FA tox-icity is not an acute response to the inability to synthesize TAG.In addition to plating assays, we also assessed viability in liquidculture by incubating cells with 0.001% of the respective FA, asindicated, for up to 12 h prior to SytoxGreenTM staining andfluorescence microscopy (Fig. 2B). Whereas wild type was fullyresistant to oleic acid treatment, a significant fraction of cells ofthe quadruple mutant became SytoxGreenTM-positive afterpalmitoleic or oleic acid supplementation, indicating loss ofviability (41). Unsaturated fatty acid toxicity was significantlysuppressed by ectopic expression ofDGA1 and LRO1 genes andalso by additional supplementation with palmitic acid.Taken together, these data show the following: (i) uptake of

FA is not regulated by intracellular fatty acid requirements inyeast; (ii) saturated FA supplementation is not toxic, even in theabsence of TAG synthesis; (iii) TAG synthesis is the criticaldeterminant of unsaturated FA detoxification.Phospholipid Profiles Are Altered in the Quadruple Mutant—

We next analyzed TAG and phospholipid content and compo-sition of wild-type andmutant cells, with and without FA treat-ment (Tables 1 and 2). Cells were grown to early log phase, and

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FA was added at a concentration of0.001% for 30 min and up to 12 h. Inthe quadruple mutant, as expected,TAGwere below the detection limitthroughout the duration of theexperiment, whereas TAG levelsincreased by 20–25% in the wildtype supplemented with palmitateor oleate and doubled as cellsreached stationary phase. The totalphospholipid to ergosterol ratioremained largely unchanged inwild-type cells under untreated aswell as FA supplemented condi-tions. In marked contrast, the quad-ruple mutant displayed 20% ele-vated phospholipid to ergosterolratio already in the absence of FA,which increased even further in thepresence of palmitate by 1.5-foldand, more pronounced, oleic acid(3-fold) after 12 h of fatty acid incu-bation. It should be noted, however,that the quadruple mutant alsolacks steryl ester synthesis, whichincreases free ergosterol levels by20% (29). Notably, the relativechanges in FA profiles in phospho-lipids both in wild type and thequadruple mutant clearly indicatedsubstantialmodification of incorpo-rated palmitic acid by elongationand desaturation, maintaining amore balanced ratio of C16 to C18FA, and degree of desaturation inthe membrane lipids. In wild type,oleic acid is preferentially incorpo-rated into TAG, whereas the oleicacid content in its phospholipids isonly moderately elevated (from 25to 32%, after 30 min of incubation).In striking contrast, in the quadru-ple mutant treated with oleic acid,the ratio of saturated to unsaturatedFA in the phospholipids dropped by25–30%, and the ratio of C16 to C18FA decreased by 2–4-fold, after 30min and 12 h of incubation, respec-tively, compared with wild type.Mutants harbored about 70% oleateand only about 8% palmitoleate intheir phospholipids, whereas wildtype contained equal amounts ofthese unsaturated FA in its phos-pholipids, after incubation with0.001% oleic acid for 12 h (Table 2).Thus, TAG synthesis provides anessential regulating valve to dispose

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of excess unsaturated FA. Because palmitic acid supplementa-tion is not toxic to the mutants defective in TAG synthesis anddoes not significantly change the ratios of saturated versusunsaturated and 16 versus 18 carbon atoms containing acylchains, we suggest that oleic acid-mediated toxicity is becauseof massively altered acyl chain distribution in membrane phos-pholipids. In support of this notion is the observation that co-supplementation with oleic acid and palmitic acid restored FAdistribution in phospholipids to levels more closely resemblingwild-type distribution. In particular, ratios of saturated tounsaturated FA are wild-type-like under these conditions(Table 2).ER Membranes Proliferate upon Unsaturated Fatty Acid

Supplementation—The observed severe changes in phospho-lipid acyl chain distribution suggested amajor impact onmem-branemorphology and function. Indeed, in YJP1078 quadruplemutant cells treated with unsaturated FA, highly light-diffract-ing structures appeared in transmission microscopy (Fig. 3A).Fluorescencemicroscopy, using the lipophilic dye Nile Red andrecording in the 600–650 nm “membrane detection channel”(see also Fig. 1A), showed highly aberrant structures thatformed in the presence of unsaturated FA but not in the pres-ence of palmitate. These structures extended from the endo-plasmic reticulum and co-localized with the fluorescent ERmarker protein Elo3-GFP (Fig. 3B). Thus, in the absence ofTAG synthesis, excess unsaturated FA appear to be channeledinto phospholipids and induce major membrane proliferationsof the ER. This view is supported by transmission electronmicroscopy, which unveils a remarkable proliferation of highlyordered, parallel sheets ofmembranes extending from the ER inmutants treated with unsaturated fatty acid (Fig. 3C). Thesemembrane proliferations are much more pronounced in thepresence of oleate compared with palmitoleate; palmitate had

no apparent effect on ERmorphology; however, mitochondrialmorphology in the mutant appeared somewhat dilated onpalmitate treatment, which is currently under investigation.Wild-type cells showed massive lipid droplets in the vicinity ofER membranes, as expected, upon treatment with saturated orunsaturated FA (Fig. 3C).Induction ofMembrane Proliferations Is a Very Rapid Process

and Generates ER Stress—The potential correlation betweenERmembrane proliferations and FA-induced loss of viability ofthe quadruple mutant prompted us to compare the timecourses for both events. SytoxGreenTM andNile Red staining ofthe quadruple mutant and control mutant cells transformedwith the DGA1-expressing plasmid were performed in parallelupon supplementation with 0.001% oleic acid. Rearrangementof the ERmembrane, as indicated by Nile Red staining, becameevident within 15–30 min after addition of oleic acid (Fig. 4A),consistent with a rapid incorporation of the FA intomembranelipids. In contrast, significant loss of viability of the mutant wasdetectable only after about 2–3 h of treatment, and viabilitydecreased steadily thereafter. Thus, the appearance of mem-brane proliferations did not correlate with loss of viability,which suggests that the mutant cells are able to tolerate sub-stantial membrane rearrangements and ER proliferations thatneed to exceed a certain threshold level for the induction of celldeath. The massive ER proliferation also suggested an impacton the UPR, which is a major signaling pathway involved in ERquality control (42, 43). UPR in wild-type and the quadruplemutant was determined using a UPRE-lacZ reporter construct,which expresses �-galactosidase under control of the UPR pro-moter element (44). Not unexpectedly, the quadruple mutantshowed about 2-fold higher basal levels of UPR-controlledreporter gene expression, compared with wild type (Fig. 4B).UPR was not affected in wild-type cells treated with oleic acid,whereas the quadruple mutant showed up to 9-fold increasedUPR-dependent �-galactosidase expression after 12 h of oleatesupplementation. As a positive control, UPR induction in wild-type cells by tunicamycin occurred 60–150-fold comparedwith untreated cells (data not shown and see Refs. 43, 44)and demonstrates that UPR induction in mutant cells chal-lenged with oleic acid represents a rather attenuated anddelayed response.We next investigated whether UPR was required for the

induction of membrane proliferations by deleting the IRE1gene in the quadruple mutant. Ire1p is the major mem-brane kinase in the nuclear membrane involved in transmittingthe unfolded protein signal to the level of a transcriptionalresponse to activateUPR target genes (42). UPR is tightly linkedto the lipid metabolic pathways (45), and changes in FA com-

FIGURE 2. are1� are2� dga1� lro1� quadruple mutant is highly sensitive to exogenously supplied oleic and palmitoleic acid. A, growth tests of yeaststrains on agar plates containing different FA. Wild-type, are1� are2�, dga1� lro1�, are1� are2� dga1� lro1�, DGA1[are1� are2� dga1� lro1�] (quadruplemutant transformed with a plasmid expressing DGA1 wild-type gene), and LRO1[are1� are2� dga1� lro1�] (quadruple mutant transformed with a plasmidexpressing the LRO1 gene) were grown to stationary phase and diluted to A600 � 0.5, and serial dilutions of 1:10 were spotted onto agar plates containingdifferent FA dissolved in 1% Brij58. Palmitic acid (C16:0), stearic acid (C18:0), palmitoleic acid (C16:1), and oleic acid (C18:1), control is in the presence of 1% Brij58only. w/o, without FA supplementation. B, determination of cell vitality of yeast strains on FA addition using SytoxGreenTM staining. Wild-type (top panel), are1�are2� dga1� lro1� (1st middle panel), and DGA1[are1� are2� dga1� lro1�] and LRO1[are1� are2� dga1� lro1�] (2nd middle panel) were grown overnight,shifted to fresh YND medium, and grown for 4 h. Cells were incubated in the presence of indicated FA (0.001%) for 12 h prior to SytoxGreenTM staining. FAtoxicity is most pronounced for oleate � palmitoleate and not detectable on supplementation with saturated FA. Dga1p and, to a lesser extent, Lro1pexpression in the quadruple mutant rescues unsaturated FA toxicity. Addition of palmitic acid (bottom panel) restored viability to the quadruple mutant, in thepresence of oleic acid. Scale bar, 10 �m; DIC, differential interference contrast.

TABLE 1Triacylglycerol and phospholipid content and composition of wildtype and the are1� are2� dga1� lro1� quadruple mutant in theabsence or presence of FA supplementationCells were grown to early log phase, and palmitate or oleatewas added at a sub-lethalconcentration of 0.001%. Lipids were extracted at the indicated time points. Theupper panel shows the relative triacylglycerol content normalized to ergosterol; thelower panel shows the relative phospholipid content normalized to ergosterol. Lipidanalyses were performed in duplicate; the S.E. was below �10%. ND means notdetectable.

Without � Palmitate � Oleate0.5 h 12 h 0.5 h 12 h 0.5 h 12 h

TG/ERG ratioWild type 0.90 1.98 1.15 2.20 1.11 2.51are1� are2� dga1� lro1� ND ND ND ND ND ND

PL/ERG ratioWild type 1.43 1.71 1.40 1.51 1.64 1.20are1� are2� dga1� lro1� 1.90 2.00 2.14 2.56 2.30 3.10

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position in the ER may lead to ER stress and subsequent UPRinduction and cell death (46). Interestingly, this pentuplemutant lacking Ire1p in addition to the four acyltransferasesrequired for TAG synthesis is viable in the absence of unsatur-ated FA, or if supplemented with palmitic acid, and did notdisplay any increased sensitivity to oleic acid, compared withthe quadruple mutant lacking the acyltransferases only (Fig.4C). On oleic acid supplementation, membrane proliferationsappeared after 30–60 min also in the pentuple mutant lackingIre1p (Fig. 4,D and E). Notably, the morphology of these mem-brane proliferations resembles tubuloreticular structures (Fig.4E) rather than membrane sheets, as observed in mutants withfunctional Ire1p (Fig. 3C). These tubuloreticular membranesare highly reminiscent of ERAC structures, an ER-associatedcompartment that is induced on overexpression of severalmembrane proteins (73). UPR is dispensable for ERAC induc-tion, consistent with the observation that thesemembrane pro-liferations appear quite prominent in oleic acid-challengedpentuple mutants lacking Ire1p (Fig. 4E).CPY Trafficking Is Defective in the Quadruple Mutant upon

Addition of Oleic Acid—CPY is a post-translationally modifiedglycoprotein, which allows monitoring of protein traffickingbetween the ER, the Golgi, and the vacuole (47). CPY is synthe-sized as a 67-kDa pre-protein in the ER, processed to a 69-kDaform in the Golgi, and finally cleaved in the vacuole to a 61-kDaprotein. To investigate defects in membrane trafficking as aconsequence of ER proliferations in the quadruple mutant sup-plemented with oleic acid, we monitored CPY processing afteraddition of saturated as well as unsaturated FA. As a control, asec13-1 mutant was analyzed, which is defective in a compo-nent of the COPII complex involved in ER to Golgi secretoryvesicle formation. After a shift to the restrictive temperature of37 °C, Sec13p is rapidly inactivated, and a prominent 67-kDaER form of CPY accumulates already 20min after the shift (Fig.5). In the absence of exogenous FA or on supplementation withpalmitic acid, CPY processing in the YJP1078 quadruplemutant was like wild type, and it led to the complete conversionto the vacuolar 61-kDa form of the protein. In contrast, growthin the presence of oleic acid resulted in a block in CPY process-

ing from the 67-kDa ER to the 69-kDa Golgi form within 20min, comparablewith the block in the sec13-1mutant after shiftto the restrictive temperature, demonstrating that the ER exit isseverely impaired in the mutant under these conditions. Mostnotably, CPY processing was reverted to wild-type levels if botholeic acid and palmitic acid were supplemented to the quadru-ple mutant. Also, the formation of membrane proliferationswas reverted if both FAwere present, demonstrating the criticalrequirement of a balanced FA composition for maintainingmembrane homeostasis, in the absence of TAG synthesis.

DISCUSSION

What limits the amount of membrane in a cell? Establishingand maintaining a balanced FA composition during cellulargrowth are of utmost importance for biological membranestructure and function (48). The molecular mechanisms con-trolling composition of themolecular species of individual sub-cellular membranes (1), however, are largely unknown. Inrecent years, a number of acyltransferases have been identified,which catalyze the incorporation of activated FA that arederived either from de novo synthesis or lipid turnover (49, 50).In addition, acyl transfer may also be governed by directexchange from a phospholipid to diacylglycerol (13), adding tothe complexity of cellular lipid remodeling andmembrane lipidhomeostasis. Membrane-forming glycerophospholipids andTAG, which primarily serve as biophysically rather inert FAstorage compounds, are synthesized from common precursors,phosphatidic acid and diacylglycerol, in the endoplasmic retic-ulum and are diverted either way dependent on the physiolog-ical requirements. Recent evidence suggests a cell cycle-regu-lated switch that controls the flux of FA into and out ofTAG (3),underscoring an important buffering function for TAG synthe-sis to provide membrane lipid precursors, presumably FA, in atimely coordinated manner.Yeast cells typically donot feed onFA; thus, the net supply for

membrane and storage lipid synthesis in growing cells mostlyrelies on endogenous FA synthesis (5), which in wild-type cellsis well controlled by several levels of regulation (5, 51). In par-ticular, the level of unsaturated FA is exquisitely controlled by a

TABLE 2Triacylglycerol and phospholipid content and composition of wild type and the are1� are2� dga1� lro1� quadruple mutant in the absenceor presence of FA supplementationCells were grown to early log phase, and palmitate or oleate was added at a sub-lethal concentration of 0.001%. Lipids were extracted at the indicated time points, and FAwas analyzed by gas chromatography/mass spectrometry after conversion to FA methyl esters. Mean values are of two independent measurements; S.E. was typicallybelow � 5%. Sat means saturated FA and unsat means unsaturated FA.

Strain Time Fatty acid

Fatty acid distribution

Triacylglycerols PhospholipidsSat/unsat 16:18

16:0 16:1 18:0 18:1 16:0 16:1 18:0 18:1

h % %Wild type 0.5 30.6 35.5 10.3 22.4 21.2 39.8 10.0 25.2 0.48 1.73

0.5 Palmitate 36.2 31.0 11.4 21.0 23.3 40.2 8.1 26.0 0.47 1.860.5 Oleate 28.5 25.5 5.3 40.3 20.4 37.2 7.6 32.6 0.40 1.4312 28.2 35.8 5.6 26.5 15.6 43.5 11.2 28.0 0.47 1.5212 Palmitate 37.5 31.6 8.6 20.3 21.6 41.3 7.2 28.4 0.41 1.7812 Oleate 25.4 19.5 5.0 48.0 16.7 36.0 6.9 37.6 0.32 1.18

are1� are2� dga1� lro1� 0.5 17.4 43.5 8.1 28.2 0.36 1.680.5 Palmitate 19.3 47.4 7.7 25.2 0.37 2.030.5 Oleate 13.5 23.0 7.9 51.8 0.29 0.6112 16.5 40.3 9.1 32.7 0.35 1.3612 Palmitate 22.6 43.5 12.2 21.4 0.54 1.9712 Oleate 12.0 9.7 6.4 69.5 0.23 0.29

are1� are2� dga1� lro1� 12 Palmitate � oleate 18.1 27.5 8.2 46.3 0.35 0.84

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regulatory circuit that involves the membrane-bound tran-scription factorsMga2p and Spt23p that are processed depend-ent on the membrane environment, and regulate expression ofthe only yeast desaturase OLE1 (52, 53). In the absence ofendogenous synthesis, i.e. in mutants defective in FA synthaseor in Ole1p desaturase-deficient cells, cellular growth dependson the presence of exogenous fatty acids, which are readilytaken up by the cell and incorporated into all cellular lipids (5).Indeed, yeast has evolved efficient mechanisms to take up FAfrom the environment, a process that appears to be directlycoupled to the activation step, which provides the substrates foracyltransferases or �-oxidation enzymes (6). In a recent study,

Lockshon et al. (41) identified numerous yeast mutants that arehighly sensitive to unsaturated FA treatment. Initially designedto identify novel factors involved in peroxisome biogenesis, itbecame evident from that study that unsaturated FA are actu-ally growth-inhibitory to some mutants. It was concluded thatlack of peroxisomal �-oxidation of excess unsaturated FAmight have a negative impact on membrane fluidity, in partic-ular of the plasma membrane (41). FA degradation, which inyeast takes place exclusively in peroxisomes, may thus berequired for establishing a balanced FA composition of subcel-lular membranes. This view is also supported by a recent study,which uncovered a close association of peroxisomes with lipid

FIGURE 3. are1� are2� dga1� lro1� quadruple mutant displays massive membrane proliferations on unsaturated fatty acid treatment. A, transmis-sion image (differential interference contrast (DIC)) of are1� are2� dga1� lro1� quadruple mutants treated with 0.001% oleic acid for 3 h. Note the appearanceof highly light diffractive structures (arrowheads) corresponding to membrane proliferations. B, visualization of membrane proliferations in the are1� are2�dga1� lro1� mutant treated with fatty acids. The quadruple mutant was grown for 4 h prior to treatment (3 h) with palmitic, oleic, and palmitoleic acid (0.001%each); wild-type treated with 0.001% oleic acid for 3 h served as a control. Upper row, Nile Red staining in the mutant shows the appearance of massivemembrane proliferations detectable in the 600 – 650 nm emission range, on unsaturated FA supplementation. Lower row, expression of Elo3-GFP confirms therearrangement and proliferation of the ER membrane in the mutant exposed to unsaturated FA. Scale bar, 5 �m. C, electron micrographs of wild-type andmutant cells. Cells were grown in minimal medium in the absence or presence of 0.001% FA. Lipid droplets are readily detectable in the wild type grown in thepresence of oleic acid but are absent from the are1� are2� dga1� lro1� quadruple mutant under all conditions analyzed. Addition of oleic acid inducedmassive membrane proliferations in the quadruple mutant (arrowheads). M, mitochondrion; N, nucleus; ER, endoplasmic reticulum; LD, lipid droplet; V, vacuole;w/o, without FA supplementation. Scale bar, 1 �m.

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droplets, storage sites of TAG, in oleate-induced cells (33). Adifferent mechanism of unsaturated FA-induced toxicity, how-ever, must exist in the are1� are2� dga1� lro1� quadruplemutant lacking TAG synthesis and lipid droplets altogether;oleic acid was toxic in the presence of glucose as the carbonsource, which efficiently suppresses peroxisome induction (54).Furthermore, repression of peroxisome formation uponexhaustion of glucosewas not affected in the quadruplemutant,which suggests that peroxisome biogenesis is independent ofTAG synthesis and lipid droplet formation.Because TAG and glycerophospholipids share similar pre-

cursors in the endoplasmic reticulum, namely PA and DAG, itappears likely that an excess of FA interfereswith balanced lipidformation. Indeed, DAG and PA levels are increased in thequadruple mutant lacking TAG synthesis,4 which leads to adefective transcriptional regulation of phospholipid synthesis(55). The distribution of the DAG backbone, and thus of cellu-lar FA toward phospholipids or TAG, is mainly controlled bythe activity of Mg2�-dependent phosphatidate phosphatasePah1p (56–58), which is the yeast ortholog of humanLipin (59),and mutants lacking active Pah1p enzyme are characterized bya nuclear membrane expansion phenotype and reduced levelsof TAG (57, 58, 60). However, pah1-deficient mutants are notsensitive to (unsaturated) FA at concentrations employed inthis study because of sufficient residual cellular phosphatidatephosphatase activity and a limited capacity to synthesize TAG.Oleic acid becomes toxic to pah1 mutants at 10 times higherconcentrations (0.1%),4 further corroborating the requirementfor TAG synthesis as a means to detoxify excess unsaturatedFA.The inability ofmutant cells lackingTAGsynthesis to sustain

unsaturated FA supplementation implies a number of conclu-sions. First, uptake of (unsaturated) FA in yeast is not regulatedby the cellular requirements and is largely dependent on theavailability of FA in the culturemedium (61). The rapid appear-ance of oleic acid in phospholipids of the quadruple mutantdemonstrates the absence of an efficient feedback mechanismto regulate lipid acylation in the presence of excess unsaturatedacyl-CoA. Indeed, this is a very artificial situation because of thefollowing: first, any excess of unsaturated FA is typicallyshunted into TAG, and second, endogenous desaturation isexquisitely regulated at the level of the only FA desaturase in

4 J. Petschnigg and S. D. Kohlwein, unpublished observations.

FIGURE 4. Proliferation of membranes in the are1� are2� dga1� lro1� quadruple mutant is a fast response to oleic acid addition and independent of theunfolded protein response and does not directly correlate with a loss of viability. A, quadruplemutantcellsand DGA1[are1�are2�dga1� lro1�]cellsweregrownovernight, transferred to fresh medium for 4 h, and treated with 0.001% oleic acid for the indicated time points. Staining with Nile Red and detection in the 600–650nm fluorescence emission range demonstrate that appearance of membrane proliferations precedes loss of viability (indicated by SytoxGreenTM staining) by about2 h. Scale bar, 5 �m. DIC, differential interference contrast. B, wild-type and mutant cells harboring a UPRE-lacZ construct were cultivated overnight and grown for 4 hin fresh SD media prior to 0.001% oleic acid addition. At indicated time points, aliquots were withdrawn and subjected to �-galactosidase assay. Activity (fold increase;black bars in the presence of oleate) is given relative to untreated wild-type cells (white bars) at time point 0. Assays were performed in triplicate. C, growth tests of yeaststrains on agar plates containing different FA. Wild-type, are1� are2� dga1� lro1� quadruple mutants, and ire1� are1� are2� dga1� lro1� pentuple mutants weregrown to stationary phase and diluted to A600 � 0.5, and serial dilutions of 1:10 were spotted onto agar plates containing oleic acid dissolved in 1% Brij58 at indicatedconcentrations. Additional deletion of the IRE1 gene in the quadruple mutant background does not affect fatty acid sensitivity of the strain. D, membrane proliferationsalso occur in the ire1� are1� are2� dga1� lro1� pentuple mutant treated with oleic acid. The pentuple mutant was grown for 4 h prior to treatment with 0.001% oleicacid. Nile Red staining and fluorescence detection in the 600–650 nm range shows massive membrane proliferations in the mutant on oleic acid supplementation,within 30–60 min. Scale bar, 5 �m. E, electron micrographs of the pentuple mutant treated with oleic acid. Cells were grown in minimal medium for 4 h prior to oleicacid addition (0.001%) for 3 h. Induction of membrane proliferations (arrowheads) in the ire1�are1�are2�dga1� lro1�pentuple mutant on oleic acid is very rapid andindependent of Ire1p. Note the altered morphology of these membrane proliferations, compared with the quadruple mutant (Fig. 3C), which show striking similarityto ERAC structures (73). V, vacuole; ER, endoplasmic reticulum; N, nucleus; w/o, without FA supplementation. Scale bar, 1 �m.

FIGURE 5. ER-to-Golgi trafficking is blocked in the quadruple mutant on addi-tion of oleic acid. Cells were grown overnight, shifted to fresh medium, and incu-batedfor4h.FAwereaddedatconcentrationsof0.001%,andcellswereharvestedatindicated time points. Proteins were extracted and separated by 8% SDS-PAGE. Car-boxypeptidaseYprocessingwasmonitoredusingaCPY-specificantibody(RocklandInc.). proCPY, 67-kDa ER form; mCPY, mature 61-kDa vacuolar form; w/o, without FAsupplementation. Anti-Tcm (ribosomal protein) was used as a loading control.

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yeast, Ole1p (52, 53, 62); accordingly, overexpression of OLE1under control of the strong GAL1/10 promoter neither stimu-lated desaturation, nor did it enhance cell death of the quadru-plemutant (data not shown), consistent with the notion ofmul-tiple levels of control of desaturase activity in yeast (53).Second, saturated FA supplementation to mutants unable tosynthesize TAG had no apparent effect on viability, in yeast.Although suchFAare also takenupby the cell and incorporatedinto lipids (5, 6), their uptake rate may be limited by their solu-bility (critical micellar concentration for palmitic acid is about10-fold lower, compared with oleic acid). Also, and mostimportantly, saturated FA are subject to elongation and/ordesaturation (5, 51), resulting in a FA distribution more closelyresembling the “natural” spectrum typically present in yeastmembrane lipids. In the fission yeast, Schizosaccharomycespombe, TAG synthesis is essential for long term survival and tosustain saturated FA treatment (8). Caspase-dependent (apo-ptotic) and independent (necrotic) lipotoxic pathways exist,triggered by enhanced synthesis of DAG, a potent signalingmolecule, and may involve mitochondria, production of ROS,and cell-death regulators such as Pca1p (11). Chinese hamsterovary cells drastically expand their ER on palmitate supplemen-tation, which activates the unfolded protein response (42, 63)and eventually induces apoptosis (64, 65). In fibroblasts, thecytotoxic effect of palmitic acid is presumably because of its roleas a precursor for ceramides (7, 9, 10) rather than through amechanism that involves TAG synthesis (66), and it can be sup-pressed by overexpression of stearoyl-CoA desaturase, SCD1.Indeed, palmitic acid also becomes toxic in yeast mutants thatare compromised in desaturase activity (46).4 The cumulativeeffects of altered chain length distribution (C16:C18 ratio) andthe degree of unsaturation are likely to have profound effects onmembrane structure and function, in particular of the endo-plasmic reticulum. A general effect on membrane fluidity as acause of death (41) by an excess of oleic acid containing lipids ofthe quadruple mutant (up to 70% after 12 h of incubation)appears unlikely, however, because maintaining cells at lowertemperatures of 16 or 25 °C, which is expected to require higherlevels of unsaturated FA, did not improve oleate tolerance. Inline with a crucial balance between saturated and unsaturatedFA in membrane lipids is the observation that oleic acid-in-duced toxicity can be partially suppressed by the addition ofpalmitic acid. A simple competitionmechanism for uptake andactivation is unlikely; this observation rather suggests that theactivity of acyltransferases discriminates and limits incorpora-tion of activated FA into lipids, and that this activity may bedependent on the lipid environment.Third, in the absence of TAG synthesis, excess unsaturated

FA are channeled into phospholipid synthesis, which inducesmembrane proliferations and a block of secretion. This is a veryrapid process, taking place within a fewminutes after challeng-ing mutant cells with FA. Loss of viability of the mutants anddownstreamexecution of either apoptotic or necrotic cell deathpathways lack significantly behind and argue against an instanttoxic insult by unsaturated FA. ER stress response and lipidmetabolism are tightly linked; for example, one of the mosthighly regulated genes involved in lipid synthesis, INO1, is atarget of the UPR-triggered transcription factor, Hac1p (42,

67–69). Opi1p-mediated repression of UASINO-regulatedphospholipid biosynthetic genes is suspended upon Hac1pexpression, which stimulates phospholipid synthesis (45, 68).It came as a surprise to discover that the unfolded protein

response was only moderately up-regulated upon oleic acid-induced membrane proliferation in the absence of TAG syn-thesis, and that membranes continued to proliferate in theabsence of the central UPR signaling kinase, Ire1p. This obser-vation of UPR-independent membrane growth is not unprece-dented, as shown for the induction of karmellae (70) or similarmembrane proliferations (71, 72). However, the morphology ofthe membrane proliferations changed in the absence of Ire1p,giving rise to tubuloreticular structures, rather than parallelsheets of membrane, strikingly resembling ERACs (73), whichare implicated in ER quality control. ERAC formation was alsoshown to be independent of UPR (73). It remains to be deter-mined whether these structures represent a subcompartmentof the ER that is characterized by a particular lipid compositionor rather functions to sequester specific ER proteins.What makes unsaturated fatty acids toxic in the absence of

TAG synthesis? Lack of membrane proliferations in wild-typecells exposed to oleic acid suggests a specific feedback mecha-nism on phospholipid acylation that is dependent on the flux offatty acid into TAG. A block in secretion is expected to be det-rimental, however, andwhether specific lipidmolecular speciesenriched in unsaturated FA are responsible for this defectremains to be uncovered.In summary, our results highlight the importance of TAG

synthesis in Saccharomyces cerevisiae to protect cells fromunsaturated FA-induced lipotoxicity. In the absence of TAGformation, excess FA are channeled into phospholipids, caus-ingmassivemembrane proliferations but onlymoderate induc-tion of the unfolded protein response, which is dispensable forthis process altogether. Triacylglycerol synthesis is thus a cru-cial determinant and regulator of cellular fatty acid and mem-brane homeostasis. Garbarino et al. found similar results (74).

Acknowledgments—We thank Uros Petrovic and Stephen Jesch forstrains and plasmids and Gerald Rechberger for help with fatty acidanalyses.

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Klaus Natter and Sepp D. KohlweinJulia Petschnigg, Heimo Wolinski, Dagmar Kolb, Günther Zellnig, Christoph F. Kurat,

Maintain Membrane Homeostasis in YeastGood Fat, Essential Cellular Requirements for Triacylglycerol Synthesis to

doi: 10.1074/jbc.M109.024752 originally published online July 16, 20092009, 284:30981-30993.J. Biol. Chem. 

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