Glycerol-3-Phosphate Acyltransferases Gat1p and Gat2p Are … · Overexpression system. Plasmid pYES-GAT1 was previously described (32). To make plasmid pYES-GAT2, the GAT2 open reading

EUKARYOTIC CELL, Aug. 2009, p. 1184–1196 Vol. 8, No. 81535-9778/09/$08.00�0 doi:10.1128/EC.00085-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Glycerol-3-Phosphate Acyltransferases Gat1p and Gat2p AreMicrosomal Phosphoproteins with Differential

Contributions to Polarized Cell Growth�†Martin W. Bratschi,# David P. Burrowes,# Adam Kulaga, Jing F. Cheung, Ana L. Alvarez,

Jennifer Kearley, and Vanina Zaremberg*Department of Biological Sciences, University of Calgary, Alberta, T2N 1N4 Canada

Received 15 March 2009/Accepted 28 May 2009

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in the synthesis of all glycerolipids.It is the committed and rate-limiting step and is redundant in Saccharomyces cerevisiae, mammals, and plants.GPAT controls the formation of lipid intermediates that serve not only as precursors of more-complex lipidsbut also as intracellular signaling molecules. Saccharomyces cerevisiae possesses two GPATs, encoded by theGAT1 and GAT2 genes. Metabolic analysis of yeast lacking either GAT1 or GAT2 indicated partitioning of thetwo main branches of phospholipid synthesis at the initial and rate-limiting GPAT step. We are particularlyinterested in identifying molecular determinants mediating lipid metabolic pathway partitioning; therefore, asa starting point, we have performed a detailed study of Gat1p and Gat2p cellular localization. We havecompared Gat1p and Gat2p localization by fluorescence microscopy and subcellular fractionation usingequilibrium density gradients. Our results indicate Gat1p and Gat2p overlap mostly in their localization andare in fact microsomal GPATs, localized to both perinuclear and cortical endoplasmic reticula in activelyproliferating cells. A more detailed analysis suggests a differential enrichment of Gat1p and Gat2p in distinctER fractions. Furthermore, overexpression of these enzymes in the absence of endogenous GPATs inducesproliferation of distinct ER arrays, differentially affecting cortical ER morphology and polarized cell growth.In addition, our studies also uncovered a dynamic posttranslational regulation of Gat1p and Gat2p and acompensation mechanism through phosphorylation that responds to a cellular GPAT imbalance.

The first step in the synthesis of almost all membrane phos-pholipids and neutral glycerolipids is catalyzed by glycerol-3-phosphate acyltransferases (GPATs; EC 2.3.1.15). This en-zyme transfers a fatty acid from fatty acyl coenzyme A to thesn-1 position of glycerol-3-phosphate to produce lysophospha-tidic acid (LysoPA). LysoPA is further acylated at the sn-2position by a separate acyltransferase to produce phosphatidicacid (PA). PA can be either (i) dephosphorylated to producediacylglycerol (DAG) or (ii) converted to CDP-DAG. Theselipids not only are precursors of all glycerolipids but also aredynamic components of signal transduction systems that con-trol cell physiology. Regulated interconversion of signaling lip-ids like LysoPA, PA, and DAG transmits information in partby their biophysical properties (5) and through lipid-lipid andlipid-protein interactions (18, 23, 29). The mechanisms of theregulation of PA biosynthesis, of the rate-limiting GPAT step,and of lipid metabolic pathway partitioning are not known(8, 12).

GPATs are present in bacteria, fungi, plants, and animals.We and others have previously identified a unique gene pairin Saccharomyces cerevisiae, YKR067W (GAT1/GPT2) andYBL011W (GAT2/SCT1), and demonstrated that they code

for the major GPATs in this organism (32, 34). Bioinformaticapproaches, using a region conserved between the yeastGPATs and other fatty acid acyltransferases as a query, iden-tified seven members of the GPAT family in the model organ-ism Arabidopsis thaliana (33). A substantial level of redun-dancy is also found in animals. Four mammalian GPATisoforms have been identified to date, each encoded by a dif-ferent gene. Two are localized in the mitochondria (mitochon-drial GPAT1 [mtGPAT1] and mtGPAT2) (4, 20) and two inthe endoplasmic reticulum (ER) (microsomal GPAT3 andGPAT4) (4, 24). The existence of additional genes encodingproteins with GPAT activity has been suggested (12).

Thus, the emerging picture indicates that the traditional PAbiosynthetic pathway in most eukaryotes is divided into manymore parts that were recently believed and opens the possibil-ity of each GPAT having a differential contribution to specificpools of LysoPA, PA, and DAG. In this regard, metabolicanalysis of yeast containing an inactivated GAT1 gene or aninactivated GAT2 gene indicated that Gat2p is the primarysupplier of DAG, used mainly in triacylglycerol synthesis andphosphatidylcholine synthesis through the CDP-choline path-way (32). These results indicated partitioning of the two mainbranches of phospholipid synthesis at the initial and rate-lim-iting GPAT step (Fig. 1).

We are particularly interested in identifying molecular de-terminants mediating lipid metabolic pathway partitioning.Elucidation of how lipid metabolic systems are spatiotempo-rally regulated is a major challenge for the field (29).

It is well known that within eukaryotic cells, the synthesis oflipids is restricted, and localization of biosynthetic systems is in

* Corresponding author. Mailing address: Department of BiologicalSciences, University of Calgary, 2500 University Dr., NW, Calgary,Alberta, T2N 1N4 Canada. Phone: (403) 220-4298. Fax: (403) 289-9311. E-mail: [email protected].

# These authors contributed equally to the work.† Supplemental material for this article may be found at http://ec

.asm.org/.� Published ahead of print on 12 June 2009.

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fact the first determinant of the distinct compositions of or-ganelles. One plausible explanation for the differential contri-bution of Gat1p and Gat2p to lipid metabolic pathway parti-tioning is that they are localized to different subcellularcompartments.

To explore this possibility, we have compared Gat1p and

Gat2p subcellular localization by fluorescence microscopy andsubcellular fractionation using equilibrium density gradients.Biochemical assays have previously pointed out that GPATactivity in yeast is distributed between microsomal fractionsand lipid particles (1, 2). Furthermore, a global green fluores-cent protein (GFP) localization study in yeast indicated thatGat1p and Gat2p localize primarily to the ER, but it was notdetermined whether the Gat1-GFP and Gat2-GFP proteinswere functional (1, 2, 11). Our results indicate that Gat1p andGat2p are in fact microsomal GPATs, localized to both pe-rinuclear and cortical ER in exponentially growing cells. Al-though they overlap mostly in their localization, a detailedanalysis of their distribution using equilibrium density gradi-ents suggests a differential enrichment of Gat1p and Gat2p indistinct ER fractions. Moreover, overexpression of Gat1p orGat2p in the absence of endogenous GPATs induces prolifer-ation of distinct ER arrays, differentially affecting cortical ERmorphology. Our studies also revealed a dynamic posttransla-tional regulation of Gat1p and Gat2p through phosphorylationthat responds to Gat1p/Gat2p cellular imbalance.

MATERIALS AND METHODS

Media, plasmids, and yeast strain construction. Standard molecular biologymethods, yeast genetic techniques, and transformation methods were used (13,26). Yeast complex medium was supplemented to a final concentration of 2%(wt/vol) glucose (yeast extract-peptone-dextrose) or 2% (wt/vol) galactose (yeastextract-peptone-galactose), and synthetic minimal medium using 2% (wt/vol)glucose, galactose, or raffinose was supplemented, as required for plasmid main-tenance (13). All strains used in this study are listed in Table 1.

Strains with endogenous expression of yeast GPATs. Strains expressing Gat1-GFP and Gat2-GFP from the endogenous locus and native promoter (Invitro-gen) were isogenic to strain BY4741. Gat1-TAP and Gat2-TAP, also expressedfrom the endogenous locus and native promoter (Euroscarf) were derived fromS288C. Mutant strains with Gat1-GFP/TAP or Gat2-GFP/TAP were made bymating with gat2� or gat1� deletion strains in the same background, respectively,followed by sporulation and tetrad dissection analysis. Gene disruption events aswell as tag insertions were confirmed through genomic PCR and sequencing.

FIG. 1. Differential partitioning of glycerolipids metabolized byseparate GPATs in yeast. PC, phosphatidylcholine; PE, phosphati-dylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol;TAG, triacylglycerol; LPAAT, LysoPA acyltransferase; CoA, coen-zyme A.

TABLE 1. Strains used in this study

Strain Genotype Reference/source

W303-1a MATa ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 31W303-1A MAT� ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 31CMY201 MAT� ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat2�::HIS3 31CMY202 MATa ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat2�::HIS3 31CMY203 MAT� ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat1�::TRP1 31CMY204 MATa ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat1�::TRP1 31CMY228 MAT� ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat1�::TRP1 gat2�::HIS3 �pGAL1::GAT1 URA3� 31VZY23 MAT� ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100 gat1�::TRP1 gat2�::HIS3 �pGAL1::GAT2 URA3� This studyBY4741 MATa his3 leu2 met15 ura3 EuroscarfY15983 MAT� his3 leu2 lys2 ura3 gat1�::kanMX4 EuroscarfY13037 MAT� his3 leu2 lys2 ura3 gat2�::kanMX4 EuroscarfSIK1-RFP MAT� his3 leu2 lys2 ura3 SIK1::RFP kanMX4 P. ArvidsonYKR067W-GFP MATa his3 leu2 met15 ura3 GAT1::GFP HIS3 InvitrogenYBL011W-GFP MATa his3 leu2 met15 ura3 GAT2::GFP HIS3 InvitrogenVZY40 MATa/� his3/his leu2/leu2 met15/MET15 LYS2/lys2 ura3/ura3 GAT1::GFP HIS3/GAT1 SIK1/SIK1::RFP kanMX4 This studyVZY41 MATa/� his3/his3 leu2/leu2 met15/MET15 LYS2/lys2 ura3/ura3 GAT2::GFP HIS3/GAT2 SIK1/SIK1::RFP

kanMX4This study

VZY21 MAT� his3 leu2 ura3 GAT1::GFP HIS3 gat2�::kanMX4 This studyVZY31 MAT� his3 leu2 ura3 GAT2::GFP HIS3 gat1�::kanMX4 This studySC2158 MATa ade2 arg4 leu2-3,112 trp1-289 ura3-52 GAT1::TAP K.I.URA3 EuroscarfSC0428 MATa ade2 arg4 leu2-3,112 trp1-289 ura3-52 GAT2::TAP K.I.URA3 EuroscarfVZY27 MAT� lys2 ade2 leu2 ura3 GAT1::TAP K.I.URA3 gat2�::kanMX4 This studyVZY28 MAT� trp1 his3 leu2 ura3 GAT2::TAP K.I.URA3 gat1�::kanMX4 This study

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Overexpression system. Plasmid pYES-GAT1 was previously described (32).To make plasmid pYES-GAT2, the GAT2 open reading frame (lacking its stopcodon) was amplified by PCR from genomic DNA of strain BY4741 and clonedinto pYES-TOPO (Invitrogen). The resulting plasmid contained GAT2 in framewith the coding sequence of the V5 epitope tag at its 3� end and under the controlof the GAL1 promoter, with URA3 as the selectable marker. To generate theconditional lethal strain VZY23, pYES-GAT2 was transformed into the CMY201and CMY204 strains. Ura� transformants were mated, a diploid strain bearingthe plasmid was selected and sporulated, and meiotic progeny was isolated onplates with yeast extract-peptone-dextrose containing galactose. GAT1 andGAT2 gene disruptions of cells unable to grow on glucose were confirmedthrough genomic PCR.

Subcellular fractionation. Subcellular fractionation of yeast cells (microsomalfraction preparation) was performed, as described previously (32). Fractionationusing sucrose gradients was carried out, as described previously (16), with severalmodifications. Briefly, 20 optical-density-at-600-nm units of cells grown to mid-log phase were harvested, washed once, resuspended in 10 mM dithiothreitol and10 mM Tris-HCl (pH 8.9), and incubated at 30°C for 15 min. Cells were thenresuspended in spheroplasting buffer {1 M sorbitol, 20 mM PIPES [piperazine-N,N�-bis(2-ethanesulfonic acid)]-KOH [pH 6.8]} supplemented with 0.12 mg ofzymolyase (T100) and incubated at 30°C for 30 to 45 min, with occasionalshaking. Spheroplasts were harvested and washed once in spheroplasting bufferby pelleting at 3,000 � g for 3 min. Spheroplasts were then resuspended in lysisbuffer (0.2 M sorbitol, 10 mM Tris-HCl [pH 7.5], 10 mM EDTA) with proteaseinhibitors (1� Roche complete, 10 mM phenylmethylsulfonyl fluoride [PMSF],3 �g/ml pepstatin) and lysed using a Dounce homogenizer (30 strokes). Celllysates were cleared for 5 min at 3,000 � g. Total cell lysate (150 �g protein) wasloaded onto a 20-to-60% sucrose gradient and centrifuged at 100,000 � g for 18 h(MLS-50 swinging bucket rotor, Optima MAX-E Ultracentrifuge; Beckman).Fractions (200 �l) were collected from the top of the gradient. In order to detectendogenous Gat1p and Gat2p using anti-GFP antibodies (Clontech), proteinswere acetone precipitated. Four volumes of ice-cold acetone were added to onevolume of fraction containing 10 �g bovine serum albumin as a protein carrier.Samples were precipitated overnight at 20°C and then centrifuged at maximumspeed in an Eppendorf microcentrifuge (15,700 � g) for 20 min at 4°C. Pelletswere then washed with 500 �l ice-cold acetone, dried, and resuspended in 100mM NaOH and 1% sodium dodecyl sulfate (SDS). Fractions were analyzed by8% SDS-polyacrylamide gel electrophoresis, followed by Western blotting withantibodies against GFP as well as Pma1p and Erg6p (kind gifts from RamonSerrano, Universidad Politecnica de Valencia, and G. Daum, Universitat Graz,respectively) and Dpm1p or Pgk1p (Molecular Probes) and subsequently withhorseradish peroxidase-conjugated secondary antibodies, followed by detectionusing enhanced chemiluminescence (Pharmacia). Densitometry of the blots wasperformed using ImageJ (Wayne Rasband, National Institutes of Health). Ex-periments were repeated four times.

Immunoprecipitation and phosphatase treatment. Exponentially growing cellswere harvested and resuspended in an equal volume (vol/vol) of lysis buffer (50mM Tris-HCl [pH 7.2], 0.15 M NaCl, 0.27 M sucrose, 0.1% deoxycholic acid, 1%Nonidet P-40, 1 mM dithiothreitol, 3 �g/ml pepstatin, 1 mM PMSF, and com-plete EDTA-free protease inhibitor mix [Roche]). A 1:1 dilution volume ofacid-washed glass beads was added to each sample and bead beaten (two times,1 min each) at 4°C, followed by removal of unbroken cells and debris (13,000rpm, 1 min). Cell lysates were then incubated with 15 �l of agarose-immobilizedgoat immunoglobulin G (Bethyl) per 100 �g of total protein for 2 h at 4°C.Clarified protein extracts were incubated with 10 �l of agarose-immobilized goatanti-V5 antibody (Bethyl) per 100 �g of total protein overnight at 4°C. Immu-noprecipitates were washed three times with lysis buffer and resuspended in 30�l of phage phosphatase buffer (50 mM Tris-HCl [pH 7.5], 0.1 mM EDTA, 5mM dithiothreitol, 0.01% Brij 35, and 2 mM MnCl2). After incubation for 30 minat 30°C, with or without the addition of 200 units of phosphatase (New EnglandBiolabs), reactions were stopped by addition of EDTA (50 mM). Beads werecentrifuged and resuspended in 20 �l SDS loading buffer and subjected toWestern blotting with anti-V5 antibodies (Invitrogen).

Alternatively, phosphatase treatment was performed using crude cell extracts.For this, cells were lysed in TNE buffer (50 mM Tris-HCl [pH 7.4] containing 150mM NaCl, 5 mM EDTA, protease inhibitor cocktail [Roche], 1 mM PMSF, and3 �g/ml pepstatin) using glass beads, as indicated above. For phosphatase treat-ment of samples containing endogenous levels of Gat1-GFP or Gat2-GFP, 60 �gof total protein was used, and the incubation time was reduced to 20 min at 30°C.Importantly, no dephosphorylation due to endogenous phosphatases was ob-served during this incubation time in control samples lacking phosphatase.Experiments were repeated at least three times.

Fluorescence and light microscopy. Cells were processed for indirect immu-nofluorescence microscopy for the localization of Gat1p and Gat2p, as describedpreviously (15). Cells from log-phase cultures were fixed and processed fordetection of Gat1p-V5 and Gat2p-V5 by immunofluorescence using monoclonalanti-V5 antibodies (Invitrogen) and Alexa 488 goat anti-mouse secondary anti-bodies (Invitrogen). DNA was stained using 4�,6-diamidino-2-phenylindole(DAPI; BioChemika, Sigma-Aldrich). All samples were analyzed using a ZeissAxiovert 200-M microscope fitted with a Plan Neofluor 100� oil immersionobjective lens. Images were captured using a Zeiss AxioCam HR and usingAxioVision 40 version 4.6.3.0 software. Adobe Photoshop 7.0 was used for imagealignment and labeling.

GFP fusion proteins were visualized using a GFP filter, an excitation wave-length of 489 nm, and an emission wavelength of 509 nm. DNA was visualizedusing a DAPI filter, an excitation wavelength of 359 nm, and an emissionwavelength of 461 nm.

For axial ratio determination, differential interference contrast images ob-tained with the same magnification were analyzed using AxioVision LE version4.6.3.0 software.

Quantification of Gat1-GFP or Gat2-GFP association with perinuclear orcortical ER in fluorescent images was done using ImageJ (National Institutes ofHealth). The area to be analyzed was determined on a cell-by-cell basis by hand,using the free drawing tool with the program. Only cells where the entireperimeter of the nucleus was clearly visualized were used for this analysis.

Transmission electron microscopy. The cells were fixed with 1.6% parafor-maldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, over-night. After being rinsed with the same buffer, the cells were postfixed in 2%aqueous potassium permanganate for 1 h and then stained in 1% uranyl acetatefor 1 h. The samples were dehydrated through a series of solutions containing anincreasing concentration of ethanol and then embedded in Quetol resin. Ultra-thin sections were cut in a Reichert-Jung Ultracut E microtome using a diamondknife and stained with lead citrate. The sections were observed under a HitachiH-7650 transmission electron microscope at 80 kV. The images were taken withan AMT 16000 digital camera mounted on the microscope.

Protein determination. Protein concentration was determined by the Lowry etal. method (22) in the presence of 0.09% Na deoxycholate.

Statistical analysis. GraphPad Prism 3.03 software was used for statisticalanalysis of data and preparation of figures.

RESULTS

Subcellular localization of yeast GPATs. Our first goal wasto determine the subcellular localization of yeast GPATs ex-pressed at endogenous levels in exponentially growing cells.We note that Gat1p and Gat2p are predicted to be integralmembrane proteins that are not abundant, and attempts toraise polyclonal antisera have met thus far with limited success(data not shown). Therefore, we used cells in which thegenomic GAT1 or GAT2 gene was expressed from the endog-enous locus and native promoter, with GFP fused to the Ctermini, which should yield expression levels at or close to theendogenous level.

One possible concern is that fusion of a tag to these proteinscan result in nonfunctional enzymes. To evaluate this, we thentook advantage of the fact that simultaneous loss of function ofGAT1 and GAT2 is synthetically lethal (32, 34). Thus, prior toinitiating localization studies, we have assessed the functional-ity of all tagged or fusion proteins by challenging them tosupport life in the absence of the other GPAT. For this pur-pose, yeast with genetically inactivated GAT1 or GAT2 genesbut containing the genomic version of the other GPAT fusedto GFP (or TAP tag) was generated. In all cases, progeny withthe intended genotype was obtained after sporulation of dip-loids and tetrad dissection (see Materials and Methods). Via-bility of these strains indicated that the C-terminal end GFP orTAP fusions yield functional enzymes. Western blot analysis ofthe fusion proteins in the parental and single knockout strains

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showed relative abundance of Gat1p similar to that of Gat2p,regardless of the tag, further validating the use of these strainsfor the proposed studies (see Fig. S1 in the supplementalmaterial).

We then examined Gat1p and Gat2p subcellular localizationin exponentially growing cells carrying endogenous levels ofGat1p or Gat2p fused to GFP at their carboxy terminus. Flu-orescence microscopy sections of cells expressing Gat1p-GFPor Gat2p-GFP showed the typical pattern of perinuclear andcortical ER, with sporadic cytoplasmic connections (Fig. 2Aand B). No apparent differences in the localization of the twoproteins were observed.

In order to corroborate the results observed with fluores-cence microscopy in vivo and to learn more details about theirlocalization, subcellular fractionation was performed on su-crose density gradients that resolve the ER from other intra-cellular membranes. Cells were spheroplasted and gently lysedin order to preserve organelles (see Materials and Methods).Lysis and fractionation were performed in the presence ofEDTA in order to strip the ER of ribosomes. This alters ERdensity, allowing resolution of this compartment from the

plasma membrane (PM) (9, 25). The distribution of endoge-nous Gat1p and Gat2p was compared by Western blot analysis,to that of the ER marker Dpm1p, ER/lipid particle proteinErg6p and to that of Pma1p, which marks the PM. A cytosolicmarker, Pgk1p, was also included (Fig. 3; see also Fig. S1 in thesupplemental material). Due to the low abundance of Gat1pand Gat2p combined with the diluting nature of the methodused, we were forced to precipitate proteins from the fractionsin order to detect the GPATs using anti-GFP antibodies.

This procedure revealed a unique bimodal distribution ofGat1p and Gat2p. The first peak (fractions 6 to 8) (Fig. 3A toC; see also Fig. S1A to C in the supplemental material) co-fractionated with the ER resident protein Dpm1p. The secondpeak (spanning fractions 13 to 15) (Fig. 3A to C) overlappedthe equilibrium distribution of the PM marker Pma1p and wasalso enriched in Erg6p. Although Gat1p and Gat2p completelyoverlapped in their equilibrium distribution, some highly re-producible differences were observed. We constantly foundGat1p to be most abundant in the low-density peak, whileGat2p was enriched in the second, denser peak and expandedtoward the bottom of the gradient (Fig. 3B to C). All together,

FIG. 2. Gat1p and Gat2p are localized to the ER. Live imaging of endogenous levels of Gat1-GFP and Gat2-GFP in exponentially growingcells. (A) Perinuclear ER localization of Gat1p and Gat2p (green) is observed in cells coexpressing the nucleolus marker Sik1-RFP (red). DIC,differential interference contrast images. (B and C) GAT1-GFP GAT2 and GAT2-GFP GAT1 (wild-type background) (B) or GAT1-GFP gat2� andGAT2-GFP gat1� (C) strains. Images were obtained using the same exposure time to allow for comparison of the pictures. (D) Cortical/perinuclearratio distribution of Gat1-GFP or Gat2-GFP in the wild type (wt) versus that in the gat single mutants. Central horizontal lines correspond to themedians. The asterisk in the distribution of Gat2-GFP (�gat1) denotes a significantly (P � 0.001) different distribution than that of the wild type(Dunnett’s test, n � 60).



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these results indicate that Gat1p and Gat2p are microsomalGPATs that overlap mostly in their subcellular localization.They also suggest that distinct subcompartments may exist inthe ER, which are differentially enriched in Gat1p or Gat2p.

In an attempt to provide further evidence to support thisidea, we then examined the effect that deletion of one GPATwould have on the localization of the other one. For thispurpose, yeast with genetically inactivated GAT1 or GAT2genes but containing the other GPAT fused to GFP expressedfrom its native promoter was subjected to localization studies,as described above. Analysis of live cells by fluorescence mi-croscopy revealed an enhanced cortical localization of Gat2pin the absence of Gat1p, while no significant change in Gat1pdistribution in the absence of Gat2p was detected (Fig. 2D).The differences previously observed in the equilibrium distribu-tion of Gat1p and Gat2p in sucrose density gradients were accen-tuated when the other GPAT was missing (compare Fig. 3B andD as well as Fig. 3C and E). Importantly, no changes in the levelsor distribution of the markers analyzed were observed, except foran enrichment of Pma1p in the bottom fractions when Gat1p wasmissing (see Discussion). In summary, ER distribution of Gat2p issensitive to the presence of Gat1p, suggesting cellular mecha-nisms act to adapt to a GPAT imbalance.

Cells compensate for GPAT deficit. Our localization studiesraised the possibility that a compensation mechanism in thegat1 deletion strains acts to offset the GPAT deficit. In fact,Western blot analysis of endogenous Gat1p and Gat2p fromwild-type, gat1�, and gat2� deletion cells revealed that bothGat1p and Gat2p undergo striking changes when cells lack theother GPAT (Fig. 4; see also Fig. S1 in the supplementalmaterial). Interestingly, multiple Gat1p and Gat2p mobilityshifts were resolved by SDS-polyacrylamide gel electrophore-sis, and a pronounced shift in the electrophoretic mobility ofGat1p and Gat2p was observed in gat2� and gat1� deletionstrains, respectively. These results were independent of the tagused, since similar migration patterns were observed withstrains carrying either GFP (Fig. 4B) or TAP-tagged Gat pro-teins (Fig. 4A) expressed at endogenous levels.

Therefore, deletion of one GPAT seems to be counterbal-anced by a mechanism that involves regulation through post-translational modification of yeast GPATs.

Yeast GPATs are phosphoproteins. Reduced electrophoreticmobility bands are often indicative of phosphorylation, leading usto test if the yeast GPATs were phosphorylated. For that purpose,

FIG. 3. Subcellular fractionation of Gat1p and Gat2p. The samestrains shown in Fig. 2B and C expressing endogenous levels of Gat1-GFP or Gat2-GFP (in the wild type or gat single-knockout mutants)were grown to mid-exponential phase in liquid culture. Cells werespheroplasted, and subcellular fractionation was performed, as indi-cated in Materials and Methods, using sucrose density gradients. Equi-librium distribution of Pgk1p (cytosol, black), Dpm1p (ER, red),Erg6p (ER/lipid particle, blue), and Pma1p (PM, orange) in an iso-genic wild-type strain. (A) was similar in all GFP strains analyzed (B toE), except for Pma1p in gat1� (E). Densitometry of the blots obtainedfor each protein was performed, and the amount per fraction wasexpressed relative to the value obtained for its respective peak. Arepresentative experiment of four performed under similar conditionsis shown. Corresponding blots are shown in Fig. S1 in the supplementalmaterial.



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Gat1-GFP and Gat2-GFP were subjected to treatment with phosphatase. We note that cell lysates lacking phosphataseinhibitors significantly loose the slow-migrating bands whenincubated for 20 min at 30°C (data not shown). Thus, in orderto decrease the presence of endogenous phosphatases whileenriching the samples in GPATs, we isolated microsomal frac-tions. Lysis and subcellular fractionation were performed inthe presence of phosphatase inhibitors, and proteins were pre-cipitated before resuspension in phosphatase assay buffer. Thisstrategy resulted in better preservation of slow-migratingbands during incubation of control untreated samples (Fig.5A). Importantly, the mobility of both proteins was increased

by phosphatase treatment (Fig. 5A), suggesting Gat1p andGat2p are basally phosphorylated in wild-type yeast.

We reasoned that if low levels of total GPAT induce anincrease in the electrophoretic mobility of endogenous Gat1pand Gat2p (Fig. 4), then overexpression of these proteinswould have the opposite effect, enriching our samples with themore reduced mobility forms. We therefore used the GAL1galactose-inducible promoter to drive GAT1 or GAT2 expres-sion from a high-copy plasmid (32, 34) in gat1� or gat2� de-letion strains, respectively, as well as in wild-type cells. Bothproteins were V5 tagged in their carboxy termini. Time courseexperiments of gat1�(pGAT1) and gat2�(pGAT2) transfor-

FIG. 4. Cells compensate for GPAT deficit. Analysis of endogenous Gat1p and Gat2p levels in exponentially grown wild-type or gat single-knockoutstrains. (A) TAP-tagged strains were lysed (see Materials and Methods), and 25 �g total protein samples were loaded onto an 8% denaturing gel andthen subjected to Western blot analysis using rabbit immunoglobulin G (to recognize the protein A moiety of the TAP tag). The loading controls showncorrespond to the same membranes stained with Ponceau red after protein transfer. MW, molecular weight. (B) Alternatively, GFP-tagged strains shownin Fig. 2 and 3 (50 �g total protein) were blotted with a monoclonal anti-GFP antibody. The nonspecific band (NS) serves as the loading control. Thesame samples used in on the left in panels A and B were loaded in a parallel gel that was run for an extra 30 min to allow further separation andvisualization of the multiple bands present in each sample. It is worth noting that the appearance of multiple bands and that the migration shift observedin the single-knockout strains, regardless of the tag, compared to those of wild-type cells for both Gat1p and Gat2p. Gat1p predicted molecular mass,83.6 kDa; Gat1-TAP, �104 kDa; Gat1-GFP, �114 kDa; Gat2p, 85.7; Gat2-TAP, �106 kDa; Gat2-GFP, �116 kDa.



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mants grown on raffinose revealed that both Gat1p and Gat2pwere predominantly expressed as a single band after 30 to 60min of shifting the cells to galactose (Fig. 5B). As expressionincreased, bands of more reduced electrophoretic mobility ap-

peared (2 h), and this was sustained after maximum expressionwas achieved (untreated cells) (Fig. 5C). Interestingly, therewas a significant delay in the appearance of the upper bands,for both Gat1p and Gat2p, after the shift to galactose in

FIG. 5. Gat1p and Gat2p are phosphoproteins. (A) Wild-type cells expressing endogenous levels of Gat1-GFP or Gat2-GFP were grown tomid-exponential phase in liquid culture and then lysed in the presence of phosphatase inhibitors, followed by subcellular fractionation, as indicatedin Materials and Methods. Fifteen micrograms of protein from microsomal fraction preparations were acetone precipitated, resuspended inphosphatase assay buffer, and either left untreated or incubated with phosphatase (400 units) for 20 min at 30°C. Samples were then subjectedto Western blot analysis with anti-GFP antibodies (as described in Materials and Methods). NS, nonspecific band. (B) Strains CMY203 (gat1�)and CMY201 (gat2�) were transformed with pGAL1-GAT1-V5 or pGAL1-GAT2-V5, respectively. Isogenic wild-type cells were also transformedwith the same plasmids. Transformants were grown to mid-exponential phase in selective media containing 2% raffinose (time zero), and thenexpression was induced by shifting the cells to fresh media containing 2% galactose. Samples were collected at the indicated time points after theshift and lysis was performed in the presence of phosphatase inhibitors. Expression of Gat1p and Gat2p (5 �g of total protein) was analyzed byWestern blotting using anti-V5 antibodies. (Right) Gat2p pattern in wild-type and gat2� cells after 2 h of the shift. Deliberately less total proteinwas loaded for the wild-type sample to show that the differences observed in the pattern are not due to differences in total Gat2p present in thesample. (C) Cell extracts from gat1�(pGAL1-GAT1-V5) and gat2�(pGAL1-GAT2-V5) transformants similar to the ones used in panel B weregrown on galactose to mid-exponential phase and subjected to immunoaffinity precipitation using anti-V5-agarose beads. Immunoprecipitates werewashed, as indicated in Materials and Methods, and either left untreated or incubated with phosphatase (200 units) for 30 min at 30°C. Beadswere then subjected to Western blot analysis with anti-V5 antibodies. MW, molecular weight.



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GPAT-deficient transformants compared to that in wild-typetransformants (Fig. 5B). These results further support ourfindings of endogenous levels of Gat1p and Gat2p in singleknockout strains, suggesting faster-migrating forms are favoredwhen there is a GPAT deficit.

To confirm that the mobility shift of Gat1p and Gat2p wasdue to phosphorylation, whole-cell extracts overexpressingGat1p-V5 or Gat2p-V5 were immunoaffinity precipitated andsubjected to treatment with phosphatase. The mobility ofboth Gat1p-V5 and Gat2p-V5 was increased by phosphatasetreatment, indicating that they were phosphorylated and thatthe phosphatase acted directly on them (Fig. 5C).

Based on these results, we concluded that Gat1p and Gat2pare phosphoproteins and that their phosphorylation status re-sponds to total GPAT levels in the cell. In general, slow-migrating bands predominate when a GPAT surplus is in-duced, while faster-migrating forms are associated with GPATinsufficiency.

Characterization of conditional double-knockout gat1�gat2� strains. Loss of function of Gat1p and Gat2p is maskedby the compensatory effect of their redundant partner. Toexplore common as well as unique contributions of Gat1p andGat2p to cellular function, we studied the effect that overex-pression of these proteins has in cells with genetically inacti-vated GAT1 and GAT2 genes. These cells are maintained byusing the GAL1 galactose-inducible promoter to drive GAT1or GAT2 expression from a high-copy plasmid in gat1::TRP1gat2::HIS3 yeast (Fig. 6A). We will refer to these strains asdko-GAT1 (32) and dko-GAT2, for the gat1� gat2� doubleknockout containing the pGAL1-GAT1 or pGAL1-GAT2 plas-mid, respectively. Importantly, viability of these strains indi-cates that the fusion proteins are functional. Culturing theyeast on galactose allowed for cell growth, but shifting thesecells to glucose-containing medium (which suppresses tran-scription from the GAL1 promoter) resulted in cessation ofcell growth. Compared to their isogenic wild type (2 h), dko-GAT1 and dko-GAT2 cells had a slower growth rate in richmedium containing galactose, with doubling times of 4 h and6 h, respectively.

Gat1p and Gat2p were present in multiple bands at timezero and steadily declined after 6 h in glucose to very low levelsand were no longer detected at 12 h. In the case of Gat2p,many proteolytic products were detected at time zero. Al-though dko-GAT1 and dko-GAT2 cells are in the process ofdying (viability decreased to 40% after 8 h and 6 h of growth inglucose, respectively), they are fully viable 3 h after the shift,despite a significant reduction in the remaining levels of Gat1and Gat2 proteins.

Surprisingly, the cell morphology of dko-GAT1 and dko-GAT2 yeast was strikingly different. Nomarski microscopy re-vealed that dko-GAT1 cells grown in rich medium containinggalactose (which overexpressed Gat1p) were elongated, whiledko-GAT2 cells (which overexpressed Gat2p) were morespherical (Fig. 7). These observations prompted us to analyzethe phenotype of the single-knockout strains grown under thesame conditions. Significant differences in axial ratio valuescalculated for gat1� and gat2� cells were found in comparisonto that for their isogenic wild type grown in rich mediumcontaining galactose (Fig. 7B). In agreement with the pheno-types displayed by the double-knockout strains, gat1� cells

FIG. 6. Characterization of gat1� gat2� double-knockout strains.(A) Strains simultaneously lacking GAT1 and GAT2 but maintained byusing (pGAL1-GAT1-V5, URA3) dko-GAT1 or (pGAL1-GAT2-V5,URA3) dko-GAT2 were grown on media containing 2% galactose.Cultures were serial diluted and plated on galactose-defined mediumlacking uracil (control) or same medium containing 5-fluoroorotic acid(5-FOA). (B, C) Isogenic wild-type strains transformed with the emptypYES-URA3 vector were used as a control. dko-GAT1 (B) or dko-GAT2 (C) cells were shifted to glucose-containing medium, and sam-ples were collected at the indicated time points after the shift. (Top)Western blot analysis of 10 �g total protein analyzed for each timepoint. (Bottom) Percentage of viable cells during the time course,performed as indicated in Materials and Methods. A 55% and 38%reduction in the levels of Gat1p and Gat2p, respectively, was estimated3 h after the shift (while cells are fully viable).



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were round, while gat2� cells were elongated. The single-knockout cells displayed the same phenotypes when grown onglucose-containing medium (data not shown). Interestingly, allmutant strains were significantly larger than their isogenic wildtype, regardless of their morphology. These phenotypes werevery evident in cells derived from the W303 background, al-though overexpression of Gat1p or Gat2p in wild-type strainBY4741 showed a similar trend (see Fig. S2 in the supplemen-tal material). It is worth noting that both wild-type strainsbecome more elongated when grown on galactose in definedmedium, making the analysis more difficult.

As previously seen with the GFP fusion proteins expressedat endogenous levels, GPATs in dko-GAT1 and dko-GAT2cells were localized to the perinuclear and cortical ER. Inaddition, due to their overexpression, they were also present ina membranous mesh that was positive for DAPI staining, prob-ably indicative of mitochondria (Fig. 8). Electron microscopyimages of these cells supported this idea. Double-knockoutcells overexpressing Gat1p were rich in mitochondria sur-rounded by a membrane array absent in wild-type cells. Adifferent membrane arrangement that also resulted in group-ing of mitochondria was observed in cells overexpressingGat2p (Fig. 9A and B). One of the most striking differencesbetween these cells was found when we took a closer look atthe cortical ER. While dko-GAT1 cells had a normal corticalER positioned underneath the plasma membrane, dko-GAT2

cells showed an irregular layout of membranes, with pro-nounced invaginations also affecting the plasma membranemorphology (Fig. 9C).

We then analyzed the morphology of the conditional dou-ble-knockout strains after 12 h of shifting them to glucose-containing medium. Interestingly, dko-GAT1 and dko-GAT2cells are budded when they die (Fig. 10A and B). Their mor-phology was different and this would be expected due to theremarkable differences in cell morphology already displayed bythese strains at the start point of the shift to glucose. Bothmutant strains exhibited defects in cell separation, resulting inthe display of a clumpy morphology when dead. Pseudohyphalclusters of cells were often observed in dko-GAT1, whilegrape-like clusters were observed in dko-GAT2.

Examination of these multibudded cells stained with DAPIto visualize nuclear DNA indicated nuclear division was com-pleted in 90% of the cells, although most cells displayedpyknotic, fragmented nuclei, resembling apoptotic phenotypes(Fig. 10A-B). These results indicate the complete lack ofGPAT results in cell death, affecting mostly postcytokinesisevents that lead to cell separation. EM analysis of these deadcells showed cytoplasmic vacuolization frequently surroundingthe nuclear envelope (Fig. 10).

In summary, excess Gat1p results in elongated cells, whileexcess Gat2p results in larger and round cells. On the otherhand, the complete lack of GPAT results in cell death, withmultibudded cells containing divided nuclei. All together,these results point to a critical contribution of GPATs to themorphogenesis/polarity checkpoint and cell separation.

DISCUSSION

The mechanisms of the regulation of PA biosynthesis, of therate-limiting GPAT step, and of lipid metabolic pathway par-titioning are not known. It has been generally accepted thatglycerolipid biosynthetic pathways diverge at the level of PAmetabolism to DAG or CDP-DAG. Our work on yeast Gat1pand Gat2p indicated that a significant partitioning of mem-brane biosynthetic pathways occurs at the initial GPAT-cata-lyzed step of lipid synthesis (32, 34). Likely, localization ofGat1p and Gat2p contributes to their metabolic partitioning.Thus, the first aim of this work was to carefully define thesubcellular localization of these enzymes. Furthermore, sincegene dosage is a powerful approach to studying Gat1p andGat2p function, we extended our localization studies to cellsdepleted of one GPAT, or cells simultaneously lacking endog-enous Gat1p and Gat2p, but maintained it by using the GAL1galactose-inducible promoter to drive GAT1 or GAT2 expres-sion. The data obtained from cells expressing endogenous lev-els of Gat1p or Gat2p presented in this study indicate thatalthough Gat1p and Gat2p are both localized to the perinu-clear ER as well as the cortical ER in exponentially growingcells, inspection of their distribution in sucrose density gradi-ents strongly suggests that these proteins are enriched in dis-tinct subdomains of the ER. Further evidence in support ofthis conclusion is that immunoaffinity precipitation of endog-enous (10, 19) as well as overexpressed Gat1p or Gat2p (ourunpublished results), followed by mass spectroscopy analysis,failed to detect the other GPAT in the precipitate, while otherspecific ER resident proteins were identified. Most impor-

FIG. 7. Differential contribution of Gat1p and Gat2p to polarized cellgrowth. (A) Transmission images showing the cell morphology of double-knockout strains dko-GAT1 and dko-GAT2 as well as the gat1� and gat2�single-deletion strains and their isogenic wild type (W303). At least 50cells from each strain were processed, as indicated in Materials andMethods, to measure cell length and width. (B) Box plot of the axial ratiodistributions. The box plot shows the medians (central horizontal line)with the 25th and 75th percentiles (box). The bottom line coming out ofthe box ends at the 5th percentile, whereas the top one ends at the 95thpercentile. An asterisk denotes significantly (P � 0.001) different distri-bution than that of the wild type (wt) (Dunnett’s test, n � 50).



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tantly, our study unveiled a coordinated regulation betweenthese two acylation systems, with two lines of evidence sup-porting this finding. First, in vivo imaging combined with sub-cellular fractionation analysis using sucrose density gradientsof cells expressing Gat2p fused to GFP revealed changes in itsER localization and gradient distribution when Gat1p wasmissing. Second, Gat1p and Gat2p were basally phosphory-lated in wild-type cells, but their phosphorylation statuschanged according to their own protein level and that of theother GPAT. In general, we noticed Gat1p and Gat2p arehyperphosphorylated when the proteins are in excess (overex-pression), while they are less or not phosphorylated when thereis a GPAT deficit. To our knowledge, no such regulation hasbeen described previously for these enzymes in yeast or otherorganisms.

Recently, the mammalian mitochondrial GPAT (mtGPAT1)has been shown to be regulated posttranslationally via phos-phorylation of residues Ser 632 and Ser 639 in its C-terminalend (3), but the physiological relevance of these phosphoryla-tion events remains unknown. Results from tandem mass spec-trometry phosphoresidue analysis of yeast GPATs also indicatethat Gat1p and Gat2p are phosphorylated in their C-terminalends (our unpublished results). Our results do not clarify,however, whether phosphorylation regulates GPAT localiza-

tion. No obvious differences in distribution of Gat1p- orGat2p-phosphorylated species were detected in the sucrosedensity gradients analyzed for this study. This possibility willneed to be addressed using monospecific Gat1p and Gat2pantibodies raised against phosphoresidues and immunomicro-scopic analysis of their localization. Alternatively, phosphory-lation may affect activity and/or half-life of the proteins. Inter-estingly, hyperphosphorylation of the yeast GPATs due to highlevels of expression seems to be independent of their activity,since Gat1p and Gat2p mutants predicted to be catalyticallydead (14) showed the same phosphorylation status as wild-typeproteins (data not shown). These results suggest that the sig-naling events leading to phosphorylation of these enzymes arenot directly mediated by signaling lipids generated downstreamof the reaction they catalyze.

GAT1 and GAT2 represent a duplicate gene pair that wasoriginated from a whole genome duplication event in the S.cerevisiae lineage 100 million years ago (6). It has been pro-posed that in order to persist in time, the two duplicate copiesmust lose full redundancy either by having at least one of theduplicate copies gain a new function or by partitioning theancestral function (27). Redundancy of GPATs is even higherin multicellular organisms. Do the various isoforms have dis-tinct functions? Mammalian GPAT1, -2, and -3 have been

FIG. 8. Excess Gat1p and Gat2p generate aberrant membranous arrays. (A, B) dko-GAT1 or dko-GAT2 strains were grown in liquid culturescontaining galactose to mid-log phase. Cells were then fixed and processed for indirect immunofluorescence microscopy using anti-V5 antibodies.DAPI was used to detect DNA (nuclear and mitochondrial). (B) Z-stacks of 0.2 �m were sequentially imaged to analyze both signals in more detail.Notably, the aberrant membranous mesh observed in both cell types is also stained for DAPI (arrows).



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shown to function mainly in triacylglycerol synthesis (12), andjust recently, GPAT4 has been found to be different, as it is amajor contributor of phospholipid biosynthesis (7, 24). Withthe goal of learning more about the scope of cellular processesregulated by each GPAT, we explored the effect that overex-pression of Gat1p or Gat2p had on the extreme case of cellslacking endogenous GPATs. Gat1p and Gat2p are predicted to

be transmembrane enzymes, but unlike overexpression ofHmg1p, they do not form arrays of membranes around thenucleus (known as karmellae) in order to accommodate theexcess of protein, when overexpressed (17, 31). Instead, aunique ER arrangement for dko-GAT1 or dko-GAT2 cells wasobserved at the ultrastructural level (Fig. 9C and D). Thesearrays frequently enclosed or grouped mitochondria, explain-

FIG. 9. Identification of distinct membranous arrays and cortical ER alterations in dko-GAT1 and dko-GAT2 cells by electron microsco-py.(A) Comparison of wild-type, dko-GAT1, and dko-GAT2 representative cells analyzed by electron microscopy. All cells were grown in richmedium containing galactose. (B) Enlargement of a portion of dko-GAT1 and dko-GAT2 cells showing aberrant membranous arrangementscompared to those of the wild-type cells. (C) Detailed analysis of the cortical ER in all strains analyzed shows altered PM and cortical ER structuralmorphology in dko-GAT2 cells. Boxes indicate the areas that have been enlarged on the right, asterisks denote mitochondria wrapped by ER indko-GAT1 cells, and arrows point to dilated ER in dko-GAT2 cells.



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ing the DAPI stain trapped in the membranous mesh detectedby immunofluorescence microscopy of Gat1p and Gat2p. Itwill be interesting to study how Gat1p and Gat2p affect mito-chondrial lipid synthesis, composition, and function. In fact,growth of these dko strains on galactose, a semifermentativecarbon source, is abnormally slow compared to that of itsisogenic wild type.

Interestingly, gat1� gat2� double-knockout cells overex-pressing Gat1p or Gat2p (grown on galactose) show remark-able morphological differences. Elongated cells were the hall-mark of dko-GAT1 strains, while large and round cells werethe predominant morphology of dko-GAT2 cells. Analysis ofgat1� or gat2� single-deletion strains grown on galactoseshowed morphological characteristics of dko-GAT2 and dko-GAT1 strains, respectively. Importantly, we did not detect de-fects in nuclear division. Thus, we propose the imbalance inGat1p and Gat2p levels in these cells affects the morphogen-esis checkpoint, producing a delay in the apical isotropic switch(elongated cells) or rushing through it (larger and roundercells). It is worth noting that these phenotypes were clearlyobserved in strains derived from the W303 background but notin BY4741-derived deletion strains. Overexpression of Gat1por Gat2p in wild-type BY4741 cells showed the same morpho-logical trend when grown on galactose, but the phenotype wasnot as obvious. This strain-dependent difference in cell mor-phology could be due to the known polymorphism at the SSD1locus between these two strains (30). In addition, W303 has abud4 mutation, and both Bud4p and Ssd1p have been shown tohave profound effects on the yeast morphogenesis network

(30). We have not yet explored possible genetic interactionsbetween GAT1 and GAT2 with these genes.

Despite the differences between genetic backgrounds, ourresults show unique phenotypes when an important imbalancein Gat1p and Gat2p was induced. A major structural differencebetween the two dko strains was found with the cortical ERmorphology. In the case of dko-GAT2 cells, the cortical ERwas very irregular and also affected the PM organization, pre-senting frequent invaginations with multilayered membranousstructures. Cortical ER inheritance has been shown to be re-quired for normal septin organization and polarized growth(21). It is possible that alterations in the cortical ER observedin dko-GAT1 and dko-GAT2 cells differentially affect ER com-partmentalization and, consequently, cell polarity. This effecton the PM and cortical ER could also explain the change in theequilibrium distribution observed for Pma1p when Gat2p wasthe only GPAT present in the cell. A synthetic sick interactionbetween a deletion of GAT2 and a PMA1 allele that yieldsreduced levels of this essential protein (DAmP strain) has beendescribed previously (28), further supporting a role of theacyltransferase in Pma1p localization and/or function.

Lastly, the complete lack of GPATs causes cell death, withmultibudded cells containing a nucleus. In fact, we have theimpression that these cells have recycled their organelles’membranes through an autophagy-like process in order to feedand maintain their nuclear envelopes, as judged by the electronmicroscopy images of these cells showing multivesiculated ar-rangements decorating the nuclear envelope surroundings.This terminal phenotype could be indicative of problems in

FIG. 10. Cell death caused by complete depletion of GPATs results in multibudded cells. dko-GAT1 (A) and dko-GAT2 (B) strains were grownin rich medium containing galactose and then shifted to glucose for 12 h. Cells were briefly sonicated in order to separate them, and differentialinterference contrast (DIC) images were taken in order to analyze their cell morphology. Multibudded cells were detected for both dko-GAT1 anddko-GAT2 cells. Nuclei were visualized with DAPI. The multibudded phenotype was also captured in electron microscopy images of these deadcells. Multiple vesicles surrounding their nuclei were often observed.



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postcytokinesis processes that lead to cell separation. We donot know at this point if it is related to a cell cycle defect or adeficiency in the delivery of degradative enzymes to the cellwall.

Together, these findings have several interesting implica-tions. They uncover a mechanism of cellular compensation inresponse to abnormalities in the first step of acylation, formingLysoPA that impacts not only lipid metabolism but other cel-lular processes that are coupled to it, like polarized cell growthand cell separation. Future efforts will focus on understandingthe contribution of each GPAT to these processes. They in-clude identification of phosphoresidues in Gat1p and Gat2p aswell as the signaling pathway(s) involved in their phosphory-lation. In addition, the molecular mechanisms underlying dis-tinct contributions of Gat1p and Gat2p to the formation of ERsubdomains and their protein composition are the subject offuture investigations.

ACKNOWLEDGMENTS

This work was supported by an operating grant from the NationalSciences and Engineering Research Council (NSERC), a UniversityFaculty Award from NSERC (to V.Z.), URGC (University of Calgary)and Undergraduate Student Research Awards from NSERC (toJ.F.C., D.P.B., and A.L.A.).

We thank Ramon Serrano and Gunther Daum for their kind gifts ofantibodies. The thoughtful comments of Gabriel Bertolesi as well asthe members of the Calgary Yeast Research Group are gratefullyacknowledged.

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Glycerol-3-Phosphate Acyltransferases Gat1p and Gat2p Are … · Overexpression system. Plasmid pYES-GAT1 was previously described (32). To make plasmid pYES-GAT2, the GAT2 open reading