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Originally published In Press as doi:10.1074/jbc.M702719200 on August 3, 2007

J. Biol. Chem., Vol. 282, Issue 42, 30845-30855, October 19, 2007
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SLC1 and SLC4 Encode Partially Redundant Acyl-Coenzyme A 1-Acylglycerol-3-phosphate O-Acyltransferases of Budding Yeast*Formula

Mohammed Benghezal{ddagger}1, Carole Roubaty{ddagger}, Vijayanath Veepuri{ddagger}2, Jens Knudsen§, and Andreas Conzelmann{ddagger}3

From the {ddagger}Department of Medicine, University of Fribourg, CH-1700 Fribourg, Switzerland and the §University of Southern Denmark, DK-5230 Odense M, Denmark

Received for publication, March 29, 2007 , and in revised form, August 3, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal and does not eliminate all microsomal 1-acylglycerol-3-phosphate O-acyltransferase activity, suggesting that an additional enzyme may exist. Here we show that SLC4 (Yor175c), a gene of hitherto unknown function, encodes a second 1-acyl-sn-glycerol-3-phosphate acyltransferase. SLC4 harbors a membrane-bound O-acyltransferase motif and down-regulation of SLC4 strongly reduces 1-acyl-sn-glycerol-3-phosphate acyltransferase activity in microsomes from slc1{Delta} cells. The simultaneous deletion of SLC1 and SLC4 is lethal. Mass spectrometric analysis of lipids from slc1{Delta} and slc4{Delta} cells demonstrates that in vivo Slc1p and Slc4p generate almost the same glycerophospholipid profile. Microsomes from slc1{Delta} and slc4{Delta} cells incubated with [14C]oleoyl-coenzyme A in the absence of lysophosphatidic acid and without CTP still incorporate the label into glycerophospholipids, indicating that Slc1p and Slc4p can also use endogenous lysoglycerophospholipids as substrates. However, the lipid profiles generated by microsomes from slc1{Delta} and slc4{Delta} cells are different, and this suggests that Slc1p and Slc4p have a different substrate specificity or have access to different lyso-glycerophospholipid substrates because of a different subcellular location. Indeed, affinity-purified Slc1p displays Mg2+-dependent acyltransferase activity not only toward lysophosphatidic acid but also lyso forms of phosphatidylserine and phosphatidylinositol. Thus, Slc1p and Slc4p may not only be active as 1-acylglycerol-3-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
All eukaryotes utilize glycerophospholipids (GPLs)4 to build their membranes and stock triacylglycerols as an energy reserve when nutrition is abundant. Both of these lipid classes are made from phosphatidic acid (PA), a central metabolite, which is taken by several key enzymes into various pathways (1). As shown in Fig. 1, PA can be synthesized de novo through the acylation of L-glycerol-3-phosphate by Gat1p or Gat2p and subsequent acylation of the thus generated lyso-PA by Slc1p, a lyso-PA acyltransferase (LPAAT). Small variations of this general pathway can be found in various organisms; many eukaryotes, including humans, can synthesize 1-alkenyl-2-acylglycerol-3-phosphate used for plasmalogen synthesis or 1-alkyl-2-acylglycerol-3-phosphate as alternatives to 1,2-bisacylglycerol-3-phosphate. Also, the biosynthesis can start from dihydroxyacetone phosphate instead of glycerol 3-phosphate, as depicted in Fig. 1. Furthermore, yeast can also generate PA through the breakdown of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) operated by phospholipase D (PLD) activities (2-5). PA also acts as a general regulator of lipid biosynthesis in yeast. This regulation is achieved by the ability of PA to bind and inactivate the transcriptional repressor Opi1p, thereby allowing derepression of the transcription of a large variety of lipid biosynthetic enzymes when PA accumulates (6).

SLC1 was first recognized as a 1-acylglycerol-3-phosphate O-acyltransferase in a screen for second site suppressor mutations that would allow yeast to grow in the absence of sphingolipids (7). This seminal study showed that a suppressor effect was due to a point mutation in SLC1, that this gene encoded an LPAAT, and that the suppressing gain of function SLC1-1 allele allowed the enzyme to utilize C26:0 fatty acids instead of the normal C18 species (7). The fact that SLC1 was not essential and that its deletion only eliminated about 60% of microsomal LPAAT activity suggested that a second, unrelated sn-2-specific LPAAT may exist (8). Alternatively, the possibility was not excluded that 1-acylglycerol-3-phosphate may spontaneously transmute itself into 2-acylglycerol-3-phosphate by acyl migration and that the latter could be acylated in sn-1 by Gat1p/Gat2p, thus forming PA.


Figure 1
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  		FIGURE 1.
Biosynthesis and breakdown of phosphatidic acid in Saccharomyces cerevisiae. Enzymes required for the generation of GPLs from DAG and CDP-DAG are not indicated. CL, cardiolipin; DAG-PP, DAG-pyrophosphate; DHAP, dihydroxyaceton phosphate; Gro3P, glycerol 3-phosphate; PAK, PA kinase; PGP, PG-phosphate; TG, triacylglycerol.

 
The two human SLC1 homologues LPAAT-{alpha} and LPAAT-beta have been demonstrated to convert lyso-PA to PA, and humans also contain a third homologous gene, LPAAT-{zeta}. Similar to yeast, humans also harbor two PI4, 5P2-dependent, PC-specific PLDs, PLD1 and PLD2, which generate PA and are activated by ARF, Rho GTPases, and some protein kinase Cs (9). PA functions in multiple pathways; it acts as a cofactor in Akt/mTOR and Ras/Raf/Erk pathways; it activates protein kinases (Raf-1 and PKC{epsilon}), protein phosphatases (PP1 and SHP-1), and lipid kinases, such as sphingosine kinase 1 and phosphatidylinositol 4-phosphate 5-kinase; it regulates the nonreceptor tyrosine kinase Fgr, plays an essential role in exocytosis and endocytosis, and has numerous other signaling functions (9-12). Importantly, LPAAT-beta is overexpressed in some human cancers, and specific inhibitors of LPAAT-beta trigger apoptosis or necrosis of tumor cells, indicating that LPAAT-beta plays an important role in signaling pathways critical to tumor cell survival (10).

In plants, PA increases in response to various stresses, either through the activation of PLD or of diacylglycerolkinase. Subsequently, PA acts on a multitude of downstream signaling pathways to elicit the stress response (12).

Recent work on the lipid remodeling of glycosylphosphatidylinositol anchors in yeast showed that Gup1p is required for the addition of a C26 fatty acid in the sn-2-position of glycosylphosphatidylinositol anchors (13). Gup1p harbors a membrane-bound acyl transferase motif (MBOAT motif) present in a large variety of acyltransferases of bacteria as well as eukaryotes, including humans. Yeast also harbors four other MBOAT proteins, among which Yor175c still has no known function. Here we show that Yor175c, which we here name SLC4, encodes a 1-acylglycerol-3-phosphate O-acyltransferase that is partially redundant with SLC1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains, Media, and Reagents—Strains used in this study are listed in Table 1. Cells were grown on rich medium (YPD, YPG) or defined media (SD, SG) containing 2% glucose (D) or galactose (G) as a carbon source and uracil (U), adenine (A), and amino acids at 30 °C (14, 15). Media contained inositol unless indicated. Zymolyase 20T was from Seikagaku Corp. (Tokyo, Japan), and lipids were from Avanti%20Polar%20Lipids">Avanti Polar Lipids Inc. (Alabaster, AL): 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate (catalog number 857123P); 1-stearoyl-2-hydroxy-sn-glycero-3-phosphate (catalog number 857128P); 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (catalog number 855675P); 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (catalog number 856705P); 1-oleoyl-2-hydroxy-sn-glycero-3-(phospho-L-serine) sodium salt (catalog number 858143P). [14C]Oleoyl-CoA (40-60 mCi/mmol; catalog number ARC-0527), [3H]inositol (15-20 Ci/mmol; ART-116), and [3H]serine (5-25 Ci/mmol; ART-246) were from ANAWA trading SA (Wangen, Switzerland). EDTA, triethanolamine, NaN3, sorbitol, K2HPO4, KH2PO4, NaCl, lipid-free albumin, beta-mercaptoethanol, leupeptin, chymostatin, antipain, and pepstatin were from Fluka Chemie GmbH (Buchs, Switzerland). DNase was from Seravac Biotech (Pty) Ltd. (Eppindust, South Africa). Deoxycholate sodium salt was from Merck. Calf intestinal phosphatase and NEBuffer 3 were from New England Biolabs Inc. (Frankfurt, Germany).


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  		TABLE 1
Strains

 
Construction of Plasmids and Strains (Tables 1 and 2)—The open reading frames of SLC4 and SLC1 were amplified by PCR from genomic DNA using primers containing rec1 and rec2 sequences (16). The PCR-amplified SLC4 and SLC1 open reading frames were transfected into slc4{Delta} and slc1{Delta} cells, together with SalI-digested pGREG505 and pGREG546, respectively, to generate pGAL1UAS-Yor175c and pGAL1UAS-GST-SLC1 as described (16). In these transfected strains, the remaining chromosomal SLC1 and SLC4 genes were knocked out by transfection of slc1::hphNT1 and slc4::LEU2 deletion cassettes generated through PCR on plasmids pFA6a-hphNT1 (EUROSCARF) and pRS415 as templates, respectively, thus producing strains 2{Delta}.SLC4 and 2{Delta}.SLC1. For plasmid pBF27 we generated a PCR fragment with primers SLC1-F1 5'-ccaactagtctagaatacaATGAGTGTGATAGGTAGGTTCTTGTATTACT-3' and SLC1.R1 5'-ccactcgagaattcTTAATGCATCTTTTTTACAGATGAACCTTCGTTATGTGAGG-3' on genomic DNA from strain 4R3. The fragment was double digested with SpeI and XhoI, ligated into p423Met25, and cloned in the Escherichia coli strain HB101 (17).


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  		TABLE 2
Plasmids

 
Preparation of Microsomes—100 ml of YPDUA and/or YPGUA media were inoculated with the appropriate strain to give an A600 nm of 0.01. After 24 h of growth at 30 °C, cells were again diluted to an A600 nm of 0.01. Cells were grown for an additional 24 h at 30 °C to reach an A600 nm of 4-6. Cells (500 A600 nm units of cells) were washed with 50 ml of ice-cold 10 mM NaN3, resuspended in 10 ml of zymolyase buffer (10 mM NaN3, 1.4 M sorbitol, 50 mM K2HPO4, pH 7.5, 40 mM beta-mercaptoethanol, 0.4 mg/ml zymolyase 20T), and incubated for 1 h at 25 °C without shaking. Spheroplasts were collected by centrifugation at 800 x g for 5 min at 4 °C and washed with one volume of zymolyase buffer lacking beta-mercaptoethanol and zymolyase. Spheroplasts were lysed in 10 ml of lysis buffer 1 (0.2 M sorbitol, 1 mM EDTA, 10 mM triethanolamine, pH 7.2, 1 µg/ml leupeptin, 2 µg/ml chymostatin, 2 µg/ml antipain, 2 µg/ml pepstatin) containing 0.2 mg/ml DNase by vortexing for 1 min, incubating on ice for 10 min, and vortexing for an additional 1 min. A centrifugation at 800 x g for 5 min at 4 °C was performed to remove intact spheroplasts. The supernatant was centrifuged at 30,000 x g for 30 min at 4 °C to pellet the microsomes. The supernatant was discarded, microsomes were resuspended in 3 ml of lysis buffer 1, and 0.15-ml aliquots were snap-frozen in liquid nitrogen for storage at -80 °C.

Purification of GST-Slc1p—FBY4142 cells were grown overnight in SGaa to an A600 of 4, and 2000 A600 units of cells were digested with 0.2 mg/ml zymolyase as described above. Washed spheroplasts were solubilized at 4 °C in 40 ml of lysis buffer 2 (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and protease inhibitors as in lysis buffer 1) supplemented with 1% Tween 20; lysis was promoted by vortexing and rotation on a wheel for 1 h. Insoluble material was removed by centrifugation at 4 °C (Sorvall SS-34 rotor; 48,000 x g for 15 min). The supernatant was added to 0.5 ml of MagneGST glutathione particles (Promega, Madison, WI) and left on a rotating wheel for 1 h at 4 °C. The particles were then washed three times with lysis buffer 2 containing 0.1% of Tween 20 and eluted twice with 0.75 ml of the same supplemented additionally with 10 mM reduced glutathione. Glycerol was added to 25%, and 10 aliquots of 0.2 ml of eluate were frozen at -20 °C. The eluate contained 0.0125% of the protein loaded onto the affinity column, and the purification increased the specific activity 400-fold.

LPAAT Assay (Acyl-coenzyme A: 1-Acylglycerol-3-phosphate O-Acyltransferase Assay)—Stock solutions (1 mM) of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate, 1-stearoyl-2-hydroxy-sn-glycero-3-phosphate, lyso-PC, lyso-PE, lyso-PI, or lyso-PS were prepared in lipid buffer (150 mM NaCl, 10 mM K2HPO4/KH2PO4, pH 7.5, containing 1% lipid-free albumin). Lyso-PI was generated by treating PI with phospholipase A2 (PLA2). For the standard assay, reagents were added into a 1.5-ml plastic tube on ice to a final volume of 200 µl in the following order: 20 µl of Tris/HCl 200 mM, pH 7.5, water, 10 µl of lyso-PA (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate) or lyso-GPL or lipid buffer, 2 µl of 2 mM sodium meta-vanadate, sorbitol to a final concentration of 0.2 M, microsomes (corresponding to 20 µg of proteins unless stated otherwise), and, after a preincubation on ice for 10 min, 1 µl (0.02 µCi = 0.4 nmol) of [14C]oleoyl-CoA in 10 mM sodium acetate/ethanol (1:1). Tubes were incubated at 25 °C for the indicated times. The concentration of [14C]oleoyl-CoA in the standard assay amounts to ~1.2 mol %. To stop the reaction, 520 µl of CHCl3 and 260 µl of methanol were added to the 200-µl reaction mixture. After vigorous shaking and 1-min centrifugation at 12,000 x g, the lower organic phase was extracted three more times with upper phase from an identical mixture of CHCl3/CH3OH/H2O (26:13:10). The lower organic phase was evaporated and resuspended in 30 µl of CHCl3/CH3OH/H2O (10:10:3), and 15-µl aliquots were spotted onto a TLC plate or subjected to analytical treatments.

Microsomal Assay of PI, PS, and PE Biosynthesis—Frozen microsomes (20 µg of protein/assay), prepared as described above, were incubated in 1.5-ml plastic tubes, in a final volume of 200 µl for 1 h at 25 °C with either [3H]inositol (15 µCi/tube) or [3H]serine (34 µCi/tube), conditions being very similar to the LPAAT assay. The standard assay buffer contained 20 mM Tris/HCl, pH 7.5, 200 mM sorbitol, 1 mM CTP, 2 mM Mg2+, 0.1 mM oleoyl-CoA, and 50 µM lyso-PA (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate) but no vanadate. Lipids were extracted, desalted, and analyzed by TLC in solvent 1 (see below).

Assaying Acyltransferase Activity of Purified GST-Slc1p—Unless indicated otherwise, Tris/HCl (200 mM), lyso-PA or lyso-GPLs or only lipid buffer, and vanadate were added into a 1.5-ml plastic tube on ice to a final volume of 200 µl in the same order and quantities as for microsomal assays (see above). Further additions were Tween 20 in water (0.08% final concentration), 10 µl of 100 mM MgCl2, and purified protein corresponding to five A600 units of cells. After 5 min on ice, 2.5 µl (0.05 µCi = 1.0 nmol) of [14C]oleoyl-CoA were added, and tubes were incubated at 25 °C for 120 min. Reactions were stopped as in microsomal assays.

Analytical Procedures and Thin Layer Chromatography—Lipid extracts were dried, resuspended, and sonicated for 30 s in 100 µl of NEBuffer 3 supplemented with 0.1% sodium deoxycholate. 20 units of calf intestinal phosphatase were added, and the mixture was incubated at 37 °C for 2 h. Phospholipase A2 treatment was done with 5 units of PLA2 for 2 h as described (18). GPLs were deacylated in NaOH as described (19) for 1 h at 37 °C and desalted using CHCl3/CH3OH/H2O partitioning as described above. Ascending TLC on silica 60 gel glass plates was performed with the following solvents: solvent 1, CHCl3, CH3OH, 0.25% KCl (55:45:5); solvent 2, CHCl3, CH3OH, acetic acid, 5% sodium bisulfite (67:26:4.4:2.6). Radioactivity was detected and quantified by two-dimensional radioscanning using a Berthold radioscanner or by exposing TLC plates to an image plate, which was developed by the Bio-Rad molecular Imager FX. Smith Waterman scores were calculated using the World Wide Web.

Mass Spectroscopy Analysis of Lipid Extracts—Exponentially growing cells were harvested when they reached an A600 of 2.0, and lipids were extracted as described (20), using procedure IIIB. At this stage, an aliquot of a similar lipid extract from WT cells grown in [13C]glucose, supplemented with [12C]inositol, was added as an internal standard. Samples were dried and resuspended in CHCl3/CH3OH/H2O (16:16:5), heated to 65 °C, and stirred with a rotating pistil (30,000 rpm). Aliquots corresponding to 1 A600 unit of cells were immediately injected into a PVA SIL HPLC column (1 x 150 mm; YMC Europe GmbH, D-46514 Scherbeck, Germany), which was eluted at 45 µl/min. For elution, solvents A (hexane-isopropyl alcohol; 98:2), B (CHCl3-isopropyl alcohol; 65:35), C (CH3OH), all solvents containing 0.1% triethylamine (FLUKA 90335), and an equimolar amount of formic acid were changed linearly over time to give ratios A/B/C as follows: 0 min, 70:30:0; 4 min, 12:88:0; 10 min, 9.8:74.2:16; 12 min, 7.6:61.4:31; 14-20 min, 0:0:100; 22 min, 0:100:0; 26-33 min, 70:30:0. Ions in the effluent were ionized by electrospray ionization with an electrode potential of 3500 V, and the masses of negative ions were detected by a Bruker Esquire-LC ion trap mass spectrometer. The spectrometer automatically fragments ions and records secondary ions. This information is not used routinely but has been used to confirm the identity of signals while the method was being set up. The sum of the signals of selected internal 13C standard GPLs in each lipid class (PI, PE, PS, PC, and phosphatidylglycerol (PG)) was utilized to normalize the sum of signals from the corresponding lipid class in the test sample in order to make the ion counts for a given lipid class (e.g. PI) obtained in different cell lines directly comparable. (On the other hand, due to different ionization efficiency, ion counts obtained for different lipid classes (e.g. PI and PC) do not allow us to estimate the relative abundance of the different lipid classes with regard to each other.)


Figure 2
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  		FIGURE 2.
slc1{Delta} slc4{Delta} cells are not viable. WT (BY4742), slc1{Delta}, slc4{Delta}, and four different slc1{Delta} slc4{Delta} double mutant strains harboring either GST-SLC1 on a URA3 vector or SLC1-1 on a HIS3 vector were plated at 10-fold dilutions on SDaa or SDaa plus 5'-fluoroorotic acid (FOA) and incubated at 30 °C for 3 days.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Synthetic Effects of yor175c{Delta}—In an attempt to find new LPAATs, we tried to delete SLC1 in an are1{Delta} are2{Delta} gup1{Delta} gup2{Delta} yor175c{Delta} quintuple mutant lacking all five MBOAT proteins of yeast. Although the quintuple mutant grew reasonably well, it was impossible to delete SLC1 in this background. Further investigation showed that gup1{Delta} gup2{Delta} slc1{Delta} cells were perfectly viable but that it was impossible to delete SLC1 in a yor175c{Delta} mutant. To test this further, we crossed a haploid slc1::KanMX4 with a haploid yor175c::KanMX4 strain, sporulated the resulting diploid, and analyzed the progeny by dissecting tetrads (Fig. S1A). In many tetrads, less than four spores were germinating; in all tetrads producing three colonies, only two of them were kanamycin-resistant; and in all tetrads producing two colonies, none was kanamycin-resistant (Fig. S1A). At no stage did we observe a difference in colony size between WT and slc1{Delta} or slc4{Delta} single mutants (not shown). Microscopic inspection of plates showed that spores having failed to produce colonies had germinated and produced either two or four cells but no more. These data are entirely compatible with the view that the simultaneous presence of both deletions in a haploid cell renders the cell nonviable (i.e. that the two deletions are synthetically lethal). Very bad growth of slc1{Delta} yor175c{Delta} double mutants (i.e. strong synthetic sickness between these two deletions) has also been found in a recent global genetic interaction study (21). In the following, we will therefore use SLC4 to designate Yor175c. To generate viable double mutants, we transfected slc4{Delta} and slc1{Delta} strains with centromeric plasmids containing SLC4 and SLC1 under the GAL1 promoter, a promoter that initiates transcription at a high rate when galactose is present in the medium but which is repressed in the presence of glucose. In slc4{Delta} and slc1{Delta} strains containing SLC4 and SLC1, we deleted remaining chromosomal SLC1 or SLC4 genes. The resulting slc1{Delta} slc4{Delta} (2{Delta}) strains harboring a plasmid containing either SLC1 or SLC4 (named 2{Delta}.SLC1 and 2{Delta}.SLC4) grew well not only in galactose- but also in glucose-containing liquid media (Fig. S1B). The 2{Delta}.SLC1 strain also grew well on glucose-containing agar plates (Fig. 2). Data suggest that low amounts of either Slc1p or Slc4p made on glucose media are sufficient for a relatively normal cell growth. However, it was impossible to remove the GST-SLC1-containing plasmid by counterselection on 5-fluoroorotic acid, a compound that will kill cells unless they lose the SLC1-bearing plasmid (Fig. 2). This again demonstrates that slc1{Delta} is synthetically lethal with slc4{Delta}. The data in Fig. 2 further show that the gain of function allele Slc1-1p can functionally replace Slc1p and allows slc1{Delta} slc4{Delta} cells to grow at a normal rate.

Microsomes of slc1{Delta} Cells Efficiently Transfer Oleic Acid to Lysophosphatidic Acid—About 95-98% of the cellular glycerol-3-phosphate acyltransferase, as measured by the generation of lyso-PA from C18:1-CoA and [14C]glycerol-3-phosphate, resides in microsomes, the remainder being present in lipid bodies (8), and this activity is encoded by GAT1 and GAT2 (22). The incubation of microsomes with C18:1-CoA and [14C]glycerol-3-phosphate allows for the production not only of lyso-PA but also of PA, and such an assay thus simultaneously measures glycerol-3-phosphate acyltransferases and LPAATs (8). Using this assay, we found that microsomes from 2{Delta}.SLC1 and 2{Delta}.SLC4 grown for 2 days on glucose produced strongly reduced amounts of PA in comparison with WT cells but that lyso-PA was still made (not shown). To be able to measure sn-2-specific acyltranferase independently from Gat1p/Gat2p activity, we incubated microsomes with [14C]C18:1-CoA in the absence or presence of lyso-PA (1-palmitoyl-glycerol-3-phosphate or 1-stearoyl-glycerol-3-phosphate) (23). In the absence of lyso-PA, microsomes incorporated radioactivity into lipids that comigrated either with PE, PI, PC, and PA standards or with free fatty acids and diacylglycerol (DAG) standards (Fig. 3, A and B, lanes 1, 2, 5, and 6). Interestingly, the appearance of PA, PI, and DAG was strong in microsomes containing SLC1, whereas the synthesis of PC and PE was strongly dependent on Slc4p. The assay does not contain CTP, serine, or inositol, and this suggests that the labeled PI, PE, and PC are not made from endogenous lyso-PA, which would get acylated to [14C]PA and then further processed, but that these GPLs arise through direct acylation of lyso-GPLs, a notion that is confirmed by another experiment shown below (Fig. 6). The addition of lyso-PA to the assay strongly increased the accumulation of PA and completely blocked the incorporation of label into other lipids in slc4{Delta} (Fig. 3A, lanes 1-4) and 2{Delta}.SLC1 (Fig. S2), whereas GPLs continued to be made in the presence of lyso-PA in cells containing Slc4p (Fig. 3, A (lane 7) and B (lanes 3 and 7) and S2). The addition of lyso-PA blocked the incorporation of label into other lipids (PC, PI, and PE), possibly by acting as competitive inhibitor of potential lyso-GPL substrates, since a large part of [14C]oleate (>70%) is consumed during the assay. As can be seen in Fig. 3, A and B, the band considered to be PA was sensitive to alkaline phosphatase, whereas other species identified as PE, PI, and PC were not, as expected. Alkaline phosphatase shifted the label of [14C]PA to a position corresponding to DAG (Fig. 3, A and B, lanes 4 and 8). As shown in Fig. 3C, the lipids getting labeled in microsomes of 2{Delta}.SLC4 were susceptible to mild alkaline hydrolysis, whereby the released label migrated at a position corresponding to [14C]oleic acid and a second, uncharacterized band of higher Rf (Fig. 3C, lanes 1, 2, 5, and 6). From lipids labeled in the absence of lyso-PA, PLA2 released the bulk of the radioactivity as [14C]oleic acid, but two relatively minor lyso-GPLs carrying [14C]oleic acid presumably in the sn-1-position were also generated (Fig. 3C, lanes 3 and 4). In assays done in the presence of exogenous lyso-PA, almost all of the label was released by PLA2 (lanes 7 and 8) and thus had been incorporated into the sn-2-position of lyso-PA or lyso-GPL.


Figure 3
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  		FIGURE 3.
Microsomes from WT (BY4742), slc1{Delta}, slc4{Delta}, and 2{Delta}.SLC4 cells make phosphatidic acid from 1-acylglycerol-3-phosphate. Microsomes were prepared from cells growing exponentially in YPGUA. A and B, microsomes were incubated with [14C]oleoyl-CoA in the presence (+) or absence (-) of lyso-PA (50 µM) for 120 min. After incubation, lipids were extracted and were incubated in the presence (+) or absence (-) of calf intestinal alkaline phosphatase (AP). The combined effect of incorporation into lipids and hydrolysis of [14C]oleoyl-CoA led to disappearance of 72 ± 4.6% of [14C]oleoyl-CoA during incubation in all four cell lines, whether or not lyso-PA was added. C, lipids made by microsomes from 2{Delta}.SLC4 cells either in the presence or absence of lyso-PA (2 µM) were deacylated with mild base (NaOH)(+) or treated with PLA2 (+) or mock-treated (-). Lipids were desalted and analyzed by TLC in solvent 1 followed by radioimaging. The identity of the labeled lipids is indicated and was obtained from the position of standards run in parallel, which could be revealed by iodine vapor, or were elaborated in separate metabolic labeling experiments using [3H]inositol and [3H]serine (not shown). No lipids got labeled when boiled microsomes were used (not shown). The experiment is representative of several experiments showing similar results.

 
SLC4 Expression Correlates with the 1-Acylglycerol-3-phosphate Acyltransferase Activity in slc1{Delta} Microsomes—To show that overexpression or depletion of Slc4p influenced the LPAAT activity, the kinetics of PA synthesis in microsomes from 2{Delta}.SLC4 grown in galactose or glucose were investigated. As shown in Fig. 4A, when cells had been on glucose, one could observe a linear increase in [14C]PA up to 32 min. A parallel increase of lipids migrating in the region of GPLs containing PI, PS, and PC and of very apolar lipids migrating to the top of the TLC plate (not shown) was equally observed. However, when the cells had been grown in galactose, the LPAAT activity was so very strong that it had consumed most [14C]oleoyl-CoA already at time 0 (i.e. during the time elapsing between the addition of radioactivity and the addition of organic solvent to stop the reaction (about 1 min), a time during which reaction tubes were on ice). Indeed, >70% of added radioactivity was incorporated into PA at time 0. Thus, overexpression of SLC4 on galactose media led to much a higher activity than expression on glucose media. As seen in Fig. 4B, lanes 1-3, decreasing the amount of microsomal proteins from 200 to 2 µg increased overall incorporation of radioactivity into lipid (by 23%), but high amounts of protein favored the incorporation into GPLs. Nonsaturating concentrations of lyso-PA were also tested. As seen in Fig. 4B, lanes 5-16, and the corresponding quantification in Fig. 4C, linear incorporation of radioactivity into PA was observed during 15 min with WT microsomes and lyso-PA concentrations of 0.05 and 0.5 µM, whereas again, rapid saturation was achieved at 50 µM lyso-PA. The incorporation of label into GPLs and DAG was not dependent on the concentration of lyso-PA (Fig. 4B, lanes 5-13; data not shown). This can be rationalized by assuming that the GPLs are made by the acylation of endogenous lyso-GPLs. 50 µM lyso-PA seemed to be inhibitory for GPL biosynthesis (Fig. 4, B (lanes 14-16) and C).


Figure 4
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  		FIGURE 4.
Assay of O-acyltransferase activity in microsomes of 2{Delta}.SLC4. 2{Delta}.SLC4 were grown either on glucose (YPDUA) or galactose (YPGUA), and microsomes were prepared. A, microsomes, lyso-PA (50 µM final concentration), and [14C]oleoyl-CoA were placed into reaction vials on ice, and vials were rapidly placed into a 25 °C heating block and incubated for 0-64 min as indicated, whereupon the reactions were stopped by the addition of organic solvent. The 0 min vials received organic solvent after sitting for 1 min on ice. The TLCs of this figure were developed with solvent 2, so that PI, PC, and PS are not well resolved and migrate in the zone denoted GPL. o, origin. B, acyltransferase assays were set up with microsomes from 2{Delta}.SLC4 grown on YPGUA. Lanes 1-4, assays containing 2, 20, or 200 µg of microsomal protein and 50 µM lyso-PA were incubated for 30 min; a control with boiled microsomes was run in lane 4. Lanes 5-16, lyso-PA concentrations of 0.05, 0.5, 5, or 50 µM were tested, and assays were incubated for 1, 4, or 15 min. C, counts present in PA and GPLs on TLC plates shown in B were determined by radioscanning.

 


Figure 5
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  		FIGURE 5.
Expression levels of Slc4 protein correlate with 1-acylglycerol-3-phosphate O-acyltransferase activity in microsomes of 2{Delta}.SLC4. A, microsomes were prepared from WT and 2{Delta}.SLC4 cells grown on YPDUA (Glc) or YPGUA (Gal). Microsomes plus limiting amounts of lyso-PA (1 µM final concentration = 0.6 mol %) were incubated for 0-64 min, and lipid extracts were analyzed by TLC in solvent 2. Only the zone of the TLC plate containing PA is shown. B, TLC plates of the experiment shown in A as well as of an independent second experiment were scanned, and counts present in PA and in mature GPLs (PC, PI, and PS) were determined by radioscanning.

 
In the following, we chose to compare activities of different strains at 1 µM lyso-PA corresponding to about 0.6 mol %. As shown in Fig. 5A and the corresponding quantification in Fig. 5B, the LPAAT activity in 2{Delta}.SLC4 microsomes was drastically reduced when cells had been grown on glucose during 48 h for depletion of Slc4 protein. It is worth noting that 2{Delta}.SLC4 cells grown on glucose had much less microsomal LPAAT activity than WT cells but nevertheless grew normally (Figs. 5 and S1B). This suggests that LPAAT in normal cells is not rate-limiting.

On the other hand, when grown on galactose, the acyltransferase activity of 2{Delta}.SLC4 microsomes was as strong as that of microsomes from WT cells grown on glucose. Significant amounts of radioactivity were also incorporated into GPLs (PI, PS, and PC), and this incorporation was significantly enhanced when SLC4 was induced (Fig. 5B).


Figure 6
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  		FIGURE 6.
PA generated by Slc1p and Slc4p is metabolized to PI. The same microsomes as used in Fig. 3 were incubated with [3H]inositol in the standard assay mix containing CTP/Mg2+, oleoyl-CoA, and lyso-PA (complete) or in a mix where the ingredient indicated at the top was lacking. TLC of the extracted lipids in solvent 1 showed a single radioactive band (for illustration, see Fig. S3). The counts obtained in PI when a reagent was omitted relative to the counts obtained in the complete assay are indicated below each lane (percentage of complete). About 5% of added [3H]inositol was incorporated into PI in all microsomes when the complete assay mixture was used.

 
Phosphatidic Acid Generated by Slc1p and Slc4p Is Utilized with Similar Efficiency in Microsomal PI, PS, and PE Biosynthesis—To see if PA generated by Slc1p and Slc4p could be further metabolized in various biosynthetic pathways, we modified the microsomal system in such a way as to contain CTP and either [3H]inositol or [3H]serine. As shown in Fig. 6, [3H]inositol was incorporated efficiently into PI, whereby the incorporation was totally dependent on CTP and significantly enhanced by lyso-PA and also somewhat better in the presence of oleoyl-CoA. This suggests that the assay allows for the incorporation of lyso-PA into PI along the physiological route via PA and CDP-diacylglycerol (CDP-DAG) (Fig. 1). Similarly, when [3H]serine was added instead of [3H]inositol, microsomes made PS and PE in a CTP-, oleoyl-CoA-, and lyso-PA-dependent way (Fig. S4, A and B). Again, the synthesis of PS and PE was dependent on CTP and strongly enhanced by the presence of lyso-PA and oleoyl-CoA (Fig. S4, A and B). The absolute CTP dependence of these assays demonstrates that the PE, PI, and PC species observed in Fig. 3 could not be derived from the processing of endogenous or exogenous lyso-PA via CDP-DAG, because these assays did not contain any CTP. We also found that the addition of CDP-ethanolamine or CDP-choline to microsomal assays containing [14C]oleoyl-CoA did not enhance the labeling of PE and PC. We therefore conclude that the PE, PI, and PC species observed in Fig. 3 were generated through direct incorporation of [14C]oleate into lyso-PE, lyso-PI, and lyso-PC. Moreover, the data shown in Fig. 6 and Fig. S4, A and B, argue that PA for microsomal PE, PI, and PS biosynthesis via CDP-DAG can originate from either Slc1p or Slc4p, since microsomes from all different cell lines generated comparable amounts of these lipids.

SLC4 and SLC1 Generate Almost the Same Fatty Acid Profile on Glycerophospholipids—Lipid extracts were prepared from WT, slc1{Delta}, slc4{Delta}, and 2{Delta}.SLC1, and 2{Delta}.SLC4 cells and analyzed by HPLC-electrospray ionization-MS/MS. As shown in Fig. 7A, total GPLs were slightly decreased in slc1{Delta} and slc4{Delta} mutants, but overexpression of either Slc1p or Slc4p in slc1{Delta} slc4{Delta} cells brought total ion counts back to normal. Moreover, when all PA was made by a single LPAAT, all cell types still made each individual GPL species in quantities amounting to at least 70% of the normal amount present in WT (Fig. 7B). We also found that all of the lines shown in Fig. 7 made similar amounts of triacylglycerols (Fig. S5), indicating that PA made through both Slc1p and Slc4p was metabolized to neutral fat. PG, PI, and PS are all derived from CDP-DAG (Fig. 1), and, as seen in Fig. 8A, the fatty acid profile of PI and PG is quite similar. However, the number of double bonds varies between PI and PG on the one hand and other kinds of GPLs on the other hand, since PI and PG mostly contain one double bond, PE and PC predominantly contain two double bonds, and PS contains about equal amounts of species with one and two double bonds in their fatty acids (Fig. 8A). Our findings, made in cells grown on galactose (YPGUA at 30 °C), are in close agreement with the observations made in a similar HPLC-electrospray ionization-MS/MS analysis of the X2180 strain, grown on glucose (YPD) at 24 °C, results which are reproduced for comparison in Fig. 8B (24). In particular, in the biosynthetic pathway leading from PS to PE and to PC (depicted with thick arrows in Fig. 1), we see the same relative decrease of 34:1 and parallel increase of 32:2 species described before (24). These findings are also in agreement with the observation that the "normal" PE and PC mostly contain an unsaturated fatty acid in sn-1, especially when cells are grown in rich medium, as was the case here (25). The different fatty acid content between species, which of course all derive from PA, suggests a species-selective metabolism or exchange of fatty acids on GPLs. Importantly, the varying number of double bonds in different GPL species observed in WT cells (Fig. 8, A and B) is equally seen in cells depending exclusively on Slc4p (slc1{Delta}, 2{Delta}.SLC4; Fig. 8, C and D) or exclusively on Slc1p (slc4{Delta}, 2{Delta}.SLC1; Fig. 8, E and F). Furthermore, the BY4742 as well as X2180 WT cells show a tendency to have more PI, PS, and PE species with 34 carbon atoms than with 32 carbon atoms, whereas in PC, the tendency is reversed (Fig. 8, A and B), and this feature again suggests some species-selective metabolism or fatty acid exchange. With regard to this phenomenon, a slight difference is observed, depending on the LPAAT that is used by cells. Cells entirely depending on Slc4p for PA biosynthesis (slc1{Delta}, 2{Delta}.SLC4; Fig. 8, C and D) show a relatively higher percentage of PI, PS, and PE species with 32 carbon atoms than either WT cells (Fig. 8, A and B) or cells depending exclusively on Slc1p (slc4{Delta}, 2{Delta}.SLC1; Fig. 8, E and F). This minor difference suggests that Slc4p may have a better affinity for or better access to C16:1-CoA than Slc1p. Overall, the fatty acid profiles in GPLs of all of the strains are surprisingly similar.


Figure 7
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  		FIGURE 7.
Normal amounts of all major glycerophospholipid species are made in cells lacking Slc1p or Slc4p. BY4742 WT cells, slc1{Delta}, slc4{Delta}, 2{Delta}.SLC1, and 2{Delta}.SLC4 cells were grown in YPGUA at 30 °C. Exponentially growing cells were harvested, and lipid extracts were prepared, were mixed with a fixed amount of lipid extract from WT cells grown in [13C]glucose, and were analyzed by HPLC-electrospray ionization-MS/MS. A, in BY4742 WT cells, the total ion counts were 4.999 x 106 for PC, 0.302 x 106 for PE, 0.832 x 106 for PI, 0.103 x 106 for PG, and 0.338 x 106 for PS. After normalization using the internal standard, the corresponding figures for all lipid species in the mutant cell lines were summed up and expressed as a percentage of total ion counts in GPLs of WT, which were set as 100%. B, in the same data set, the ion counts for each individual GPL species of WT cells was set as 100%, and the corrected ion counts for corresponding GPL species in mutant cells are depicted as a percentage of ion counts in the WT sample.

 
Characterization of GST-Slc1p—We tried to purify GST-tagged versions of Slc1p and Slc4p. Purified GST-Slc4p had only very low LPAAT activity. On the other hand, purified GST-Slc1p was very active and stable in storage at -20 °C. The partially purified material contained a major band at 62 kDa, the expected molecular mass of GST-Slc1p upon SDS-PAGE and staining of gels with silver nitrate (Fig. 9A, lanes 1 and 2). This band was below the detection level in the starting microsomal extract (Fig. 9A, lanes 3 and 4) and could be stained with anti-GST antibody (Fig. 9A, lanes 5 and 6). The purified GST-Slc1p generated PA from [14C]oleoyl-CoA, from which [14C]oleate could be released with PLA2 or mild base treatments (not shown). As can be seen in Fig. 9B, the activity of the purified enzyme was dependent on the addition of lyso-PA (lane 1 versus lane 4) and was enhanced by the presence of Mg2+ (lane 4 versus lane 6), whereas other divalent cations (Ca2+, Mn2+, and Cu2+) had no effect (not shown). Short term assays allowed us to calculate that Mg2+ enhanced the LPAAT activity of GST-Slc1p 7-20-fold (Fig. 9C; data not shown). However, the acyl-coenzyme A:1-acylglycerol-3-phosphate O-acyltransferase activity of Slc1p present in microsomes from slc4{Delta} cells was not enhanced by the addition of Mg2+ (not shown). GST-Slc1p was slightly more active with palmitoyl-sn-glycerol-3-phosphate than with 1-stearoyl-sn-glycerol-3-phosphate as a substrate (Fig. 9D, lanes 2 and 5). Boiling for 5 min destroyed the enzymatic activity (Fig. 9B, lanes 4 and 5).

GST-Slc1p Can Transfer Acyls from CoA onto Lysoglycerophospholipids—As mentioned, although all GPLs of yeast are derived from PA, they contain different fatty acids (24, 25), and metabolic labeling studies have suggested that fatty acids on GPLs are turning over with half-lives that are significantly shorter than the half-life of GPLs as measured in Formula pulse-chase experiments (25-27). In view of the findings of Figs. 3, 6, and S4, we were interested to see if purified GST-Slc1p was able to transfer fatty acids onto lyso-GPLs (i.e. if the presence of a head group on the phosphate of lyso-PA would hinder the interaction of the substrate with the enzyme). As seen in Fig. 9D, in the presence of lyso-PA, the purified GST-Slc1p made PA and no other product, but it made PC, PE, PS, and PI in the presence of the corresponding lyso-GPLs. The activity was relatively weak with lyso-PC and lyso-PE but quite significant with lyso-PI and lyso-PS (Fig. 9D). In the absence of Mg2+, the acyltransferase activity with lyso-PI, lyso-PS, and lyso-PA was comparable (not shown). In the presence of Mg2+, the activity was enhanced only 2-fold with lyso-PI and lyso-PS but 7-20-fold with lyso-PA (Fig. 9, C and D; data not shown). Mg2+ may have to shield the strong negative charge of lyso-PA and therefore be more important for the binding of this substrate than for binding of lyso-GPLs to the enzyme. The data suggest that Slc1p may be involved in the exchange of fatty acids on GPLs in vivo and offer an explanation for the labeling of GPLs with [14C]C18:1-CoA in the absence of CTP in slc4{Delta} cells (Fig. 3A).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Microsomes were reported to contain an uncharacterized LPAAT, not related to SLC1 (8, 28). Here, we show that Slc4p can account for this latter activity. The slc1{Delta} and slc4{Delta} cells grow at normal rates, and mass spectrometric analysis reveals a quite normal GPL profile in both, cells depending solely on Slc1p and cells depending solely on Slc4p (Fig. 8). A previous study also indicated that slc1{Delta} cells have the same GPL profile as WT apart from a reduction of a minor C12:0-containing PI species (29). This finding argues that PA originating from either Slc1p or Slc4p feeds into all lipid biosynthetic pathways and suffices for all of the essential functions of a cell. Despite this functional redundancy, Slc1p and Slc4p are not homologous to each other and have very different structures. Indeed, Slc1p contains a single transmembrane domain, whereas 12 such domains are predicted for Slc4p.


Figure 8
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  		FIGURE 8.
The fatty acid profile of major glycerophospholipid species in cells depending on only Slc1p or only Slc4p are highly similar. From the experiment shown in Fig. 7, corrected ion counts were utilized to calculate the relative abundance of the various forms defined by the number of carbon atoms and double bonds present in the two fatty acids for all detectable GPL species having a given head group. In each cell line, the total of all different forms of a given GPL was taken as 100%. Data in B show X2180 WT cells grown at 24 °C in YPD and are taken from Fig. 6 of the report by Schneiter et al. (24) but are plotted in such a way as to be comparable with other data in this figure.

 
It is likely that Slc4p itself harbors the enzymatic activity, since it contains an MBOAT motif, which has been found in many ascertained acyltransferases (30). SLC4 also contains the proposed active site residue, which is His382. This His residue is the only strictly conserved residue throughout this large gene family and was recently shown to be required for acyltransferase activity of Gup1p, a yeast homologue of SLC4, involved in lipid remodeling on glycosylphosphatidylinositol-anchored proteins (13, 30).

To date, all of the known mammalian as well as bacterial LPAATs are homologous to SLC1. However, homologues of SLC4 are found in all eukaryotes. The human genome harbors four genes predicting proteins with about 30% identity to SLC4, having Smith Waterman scores between 500 and 660 and for which no function is known. In comparison, the four yeast homologues of SLC4 have scores of 105 and below. Thus, it will be interesting to investigate if the mammalian SLC4 homologs also have LPAAT activity. However, the Blast server at NCBI finds no homologues of SLC4 in bacteria.

Genetic data indicate that Slc1p and Slc4p are not functionally exchangeable in all respects. Indeed, slightly different roles for Slc1p and Slc4p are suggested by the fact that the slc1{Delta} and slc4{Delta} deletion strains influence the effect of other gene deletions differently (i.e. that they display different synthetic interactions). When combined with either ric1{Delta} or rgp1{Delta} deletions, slc1{Delta} shows synthetic sickness, whereas slc4{Delta} shows synthetic enhancement (alleviation) (21). The ric1{Delta} and rgp1{Delta} deletions affect nucleotide exchange on Ypt6p, a G protein involved in the vesicular retrotransport from the late endosome to the Golgi.

Deletion of SLC4 eliminated the microsomal biosynthesis of PE and PC, whereas deletion of SLC1 reduced the biosynthesis of PA, PI, and DAG (Fig. 3, A and B). Due to the absolute CTP dependence of the synthesis of these GPLs in the microsomal system (Figs. 6 and S4), we conclude that the labeling of PE, PI, and PC observed in the absence of CTP (Fig. 3, A and B) proceeds from direct acylation of lyso-GPLs. In confirmation, direct acyl transfer onto lyso-PI and lyso-PS could be demonstrated with purified Slc1p (Fig. 9D). In an earlier study, acyltransferase activity in microsomes from slc1{Delta} cells, overexpressing SLC1 or not, was measured using [14C]oleoyl-CoA and lyso-PI, lyso-PE, or lyso-PC as substrates (28). The incorporation of [14C]oleic acid into lipids in these slc1{Delta} microsomes was strongly dependent on the addition of exogenous lyso-GPLs but was only slightly stronger (10-20%) when SLC1 was overexpressed. Thus, the bulk of activity was SLC1-independent. Based on this and on the analysis of products, the authors concluded that it was unlikely that Slc1p itself was an acyltransferase for lyso-PI, lyso-PE, or lyso-PC (28). The use of different assay conditions may have prevented the detection of the lyso-GPL acyltransferase activity of Slc1p in this former study. Since the lyso-GPL acyltransferase activity of Slc1p seems to be able to use lyso forms of PI and PS but no other lyso-GPLs as substrates (Fig. 9D), we are offered a reasonable explanation for the relatively strong labeling of PI but weaker labeling of PC and PE in microsomes of slc4{Delta} cells (containing only Slc1p) (Fig. 3A, lane 1 versus lane 5). The specificity of Slc1p for PI and PS may also provide part of the explanation as to why in lcb1{Delta} SLC1-1 cells C26:0 fatty acids get incorporated specifically into PI and not into all other GPLs as well (7).


Figure 9
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  		FIGURE 9.
Characterization of purified GST-Slc1p. A, the indicated amounts of protein of starting yeast lysate (lanes 3, 4, 7, and 8) and partially purified GST-Slc1p (lanes 1, 2, 5, and 6) were analyzed by SDS-PAGE/silver staining (lanes 1-4) and by Western blotting with antibody against GST (lanes 5-8). The most prominent band in the purified fraction is stained by anti-GST and is close to the expected molecular mass of 61,180 Da. B, acyltransferase activity of purified GST-Slc1p was assayed with native (n) or boiled (b) enzyme in the presence or absence of lyso-PA (50 µM) and Mg2+ (5 mM). In lane 7, Mg2+ was added after incubation. C, GST-Slc1p was assayed with short incubations ranging from 3 to 30 min. Quantitation of PA in the radiograms is shown in the graph below. D, acyltransferase activity of purified GST-Slc1p was assayed with native (n) or boiled (b) enzyme (E) in the presence of MgCl2 and 50 µM lyso-PA, lyso-PC, lyso-PS, lyso-PE or lyso-PI. In lanes 2 and 5, 1-palmitoyl- and 1-stearoyl-2-hydroxy-sn-glycero-3-phosphate were used, respectively. Only 25 and 50% of the reactions were spotted for samples containing lyso-PA and lyso-PS, respectively. Radioscanning allowed us to determine that the total amounts of product made from different substrates were as follows: lyso-PA-C16, 19,200 cpm; lyso-PA-C18, 15,500 cpm; lyso-PC, 460 cpm; lyso-PE, 400 cpm; lyso-PS, 3,100 cpm; lyso-PI, 3,300 cpm. Lipids were analyzed by TLC in solvents 2 (B) and 1 (C and D).

 
Exchange of fatty acids of GPLs (lipid remodeling) is a well established phenomenon in yeast (27). In plants and yeast, the acylation of lyso-GPLs may be important to metabolize the lyso-PE and lyso-PC, which are generated by phospholipid: DAG acyltransferases (PDAT), such as scLRO1 or AtPDAT1. These enzymes transfer fatty acids from PE and PC onto DAG to generate triacylglycerols. Similar enzymes transfer fatty acids from GPLs onto sterols or act as phospholipase A1 and thereby also generate lyso-GPLs (31-33). Several studies have reported on enzymatic activities or specific enzymes of yeast able to reacylate the sn-2-position of lyso-PC (28, 34-37). Purified Plb1p, a phospholipase B, was shown to be capable of transferring [14C]C16:0 from 1-[14C]palmitoyl-sn-glycerol-3-phosphorylcholine onto the sn-2-position of the same lipid in a CoA-independent manner (34, 35). A similar activity has been described for Taz1p. Taz1p is homologous to the human tafazzin, the loss of which causes a congenital myo- and neuropathy (Barth syndrome) (37). Plb1p and Taz1p, however, are located in the plasma membrane and the outer mitochondrial membrane, respectively, and do not need acyl-CoA. Acyl-CoA-dependent lyso-GPL acyltransferase activities have been measured in yeast microsomes before (28, 36). Some of our data suggest that not only Slc1p but also Slc4p may contribute to this lyso-GPL acyltransferase activity. Indeed, the generation of labeled PE, PI, and PC in the presence of [14C]oleoyl-CoA was significantly enhanced in microsomes overexpressing Slc4p as compared with microsomes carrying physiological levels of Slc4p (Fig. 3A, lanes 7 and 8, versus Fig. 3B, lanes 7 and 8). This suggests that Slc4p, as Slc1p, has the capacity to transfer oleate from [14C]oleoyl-CoA onto certain lyso-GPLs. The fact that not exactly the same lyso-GPLs are acylated in Slc1p- and Slc4p-containing microsomes may be due to differences in substrate specificity or subcellular localization of the two enzymes. Although Slc1p has been localized to the endoplasmic reticulum and lipid bodies, two global proteome localization studies have failed to produce any data for Slc4p (8) (available on the World Wide Web). Further studies are required to determine if the acyl-CoA:1-acyl-sn-glycerol-3-phosphorylcholine acyltransferase activity of slc1{Delta} microsomes is provided by Slc4p and if this function is physiologically relevant.


   FOOTNOTES
 
* This work was supported by Swiss National Science Foundation Grant 31-67188.01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4. Back

1 Present address: Helicobacter Research Laboratory, Microbiology M502, University of Western Australia, QEII Medical Centre, Nedlands 6009, Western Australia. Back

2 Present address: H.No: 11-9/5, New Gaddiannaram, Hyderabad 500060, India. Back

3 To whom all correspondence should be addressed: Division of Biochemistry, Chemin du Musée 5, CH-1700 Fribourg, Switzerland. Tel.: 41-26-300-8630; Fax: 41-26-300-9735; E-mail: andreas.conzelmann{at}unifr.ch.

4 The abbreviations used are: GPL, glycerophospholipid; DAG, diacylglycerol; LPAAT, lysophosphatidic acid acyltransferase; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PLA2, phospholipase A2; PLD, phospholipase D; PS, phosphatidylserine; WT, wild type; HPLC, high pressure liquid chromatography; MS, mass spectrometry; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We thank Hans Kristian Hannibal-Bach and Cécile Knöpfli for excellent technical help.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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