A Lecithin Cholesterol Acyltransferase-like Gene Mediates Diacylglycerol Esterification in Yeast*

The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases. Four of these genes are similar to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor. The residual triglyceride biosynthesis that persists in theLRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol acyltransferase. These mechanisms may also exist in mammalian cells.

Triglyceride (TG) 1 biosynthesis is a common method of energy storage and thus has an important role in energy balance. In humans, overaccumulation of TG, either as obesity or elevated serum triglyceride, has been shown to be an independent risk factor for a variety of diseases including diabetes (1) and atherosclerosis (2,3). In the pathway described by Kennedy (4) for glyceride and glycerophosphatide synthesis, a branch point is reached at diacylglycerol (DG) that can serve as a precursor for several phospholipid species and as a substrate for acyl-CoA, diacylglycerol O-acyltransferase (DGAT) (EC 2.3.1.20), which catalyzes the terminal step in TG synthesis. Expression of a recently identified mammalian DGAT cDNA in insect and mammalian cells conferred elevated triglyceride synthesis but did not change incorporation of fatty acids into sterol ester (5)(6)(7). The DGAT gene belongs to the acyl-CoA cholesterol acyltransferase (ACAT) gene family that includes two mammalian ACATs (ACAT1, ACAT2) and two yeast ACAT-related enzymes (ARE1, ARE2) that catalyze intracellular sterol esterification (8). Whereas yeast can synthesize TG from oleoyl-CoA and DG, deletion mutants in ARE1 and ARE2 do not reduce [ 3 H]oleate incorporation into TG (9). Therefore, a conspicuous absence from the ACAT gene family is a yeast DGAT.
Further examination of the Saccharomyces cerevisiae genome data base revealed two DGAT-like genes in addition to the ARE genes. We show here that these four genes do not have a major role in TG synthesis in yeast. By contrast, a yeast gene with sequence similarity to mammalian lecithin-cholesterol acyltransferase (LCAT) (EC 2.3.1.43) catalyzes the esterification of DG using phosphatidylcholine as the acyl donor. This novel enzymatic reaction mediates the majority of TG synthesis in the yeast cell during exponential growth.
LRO1 Expression Plasmid Construction-A PCR product including the LRO1 open reading frame, 941 bp of the 5Ј flanking sequence and 547 bp of the 3Ј flanking sequence, was generated using the primers 008Wfow, 5Ј GGTAGCTTTTCAATGGTGGCTG; 008Wrev, 5Ј CTGGCA-CAAGCCATACCTCCG; and W303-1B genomic DNA. The 3.5-kilobase pair PCR product was treated with T4-DNA polymerase and ligated into SmaI-digested pRS413, or digested with NheI, to remove all but 232 bp of the 5Ј flanking sequence and 393 bp of the 3Ј flanking sequence, and ligated into SpeI-digested pRS426-GP, downstream of the GAL1/10 promoter.
Pulse Labeling-A dilute inoculation (ϳ1/1000 of a saturated culture) of each strain into YPD or SC ϩ 2% galactose, 1% raffinose, lacking the appropriate nutrient for plasmid maintenance when necessary, was grown overnight at 30°C into logarithmic (log) phase (OD 660 ϭ 0.55-0.80). 8.5 ml of cells were added to 2.5 Ci of [ 3 H]oleate or [ 3 H]palmitate (final concentration 59 nM) or 2.5 Ci of [2-3 H]glycerol (final concentration 1.5 M) and incubated at 30°C for 30 min. with shaking, washed twice with 0.5% tergitol and once with dH 2 O, and lyophilized. Lipids were extracted from dried cell pellets by cell wall hydrolysis (lyticase) and organic extraction and were resolved by TLC in hexane:diethyl ether:acetic acid (70:30:1) as described. 2 Each lane was cut according to lipid standards and counted by liquid scintillation. Assays were performed on a minimum of two independent strains of each genotype on two different days for n ϭ 8 -19. Statistical analysis was performed using t tests.
TG Accumulation-Normal or lro1⌬ haploids were labeled to steady state by dilute inoculation into 8.5 ml of YPD with 0.25 Ci of [ 3 H]oleate (final concentration 5.9 nM) and overnight growth at 30°C into log phase. The cells were washed and lipids were extracted and analyzed as described above. TG mass was assessed by charring with copper sulfate/ phosphoric acid as described (19).
Western Blot Analysis-pYES2/GS containing the LRO1 open reading frame and a COOH-terminal V5 epitope (Invitrogen) was transformed into a lro1⌬ strain. Cells were subfractionated (9), and 1.8 g of microsomes, cytosol, and crude lipid droplets were resolved by SDS polyacrylamide gel electrophoresis, transferred to nitrocellulose, and detected with 1:5000 anti-V5 antibody (Invitrogen) and enhanced chemiluminescence.
Isolation of Microsomes-A dilute inoculation of normal or lro1⌬ haploids into 500 ml of YPD was grown overnight at 30°C into log phase. The cells were washed and lysed, and microsomes prepared from a 100,000 ϫ g spin as described previously (20). Protein concentrations were determined as described (21).
In Vitro Assays-All triglyceride synthesis assays were performed in a final volume of 200 l for 15 min. at 23°C. DGAT activity was assayed as described (22), with the addition of phospholipid liposomes (23 C]arachidonyl-sn-glycerol; 20,000 dpm/nmol) was included. For the lecithin/diacylglycerol transferase assay, oleoyl-CoA was omitted, and 7.5 g of purified nonspecific lipid transfer protein was added to facilitate transfer of phosphatidylcholine (1-palmitoyl-2-[ 14 C]oleoyl L-3phosphotidylcholine; 20,000 dpm/nmol) between liposomes and microsomes (25). All reactions were stopped by the addition of chloroform/ methanol (2:1), 15 g of [ 3 H]triolein was added as an internal standard/ carrier, and the lipids were separated by TLC in hexane:diethyl ether: acetic acid (170:30:1).

RESULTS AND DISCUSSION
Deletion of Candidate Yeast DGATs-We screened the S. cerevisiae genome using the human DGAT sequence with the hypothesis that this enzyme would be conserved across kingdoms. As expected, the yeast sterol esterification enzymes, Are1 and Are2 (9), which share 25 and 28% overall identity with DGAT, respectively, were identified. In addition, two uncharacterized open reading frames, YGL084c and YPL189w, were identified with a lower degree of conservation. The identity (26 and 21%, respectively) was restricted to a COOHterminal region of 146 amino acids where the maximal conservation among Are1, Are2, and DGAT occurs. The YGL084c and YPL189w predicted proteins are 51% identical to each other and were not identified when human ACAT1 was used to screen the yeast genome. To assess the role of these four DGATlike yeast genes in triglyceride synthesis, we used are1⌬ and are2⌬ mutants created previously (9) and generated YGL084c and YPL189w deletion mutants. To address the possibility of redundant enzymes, a yeast strain (4X⌬) was created where all four DGAT-like genes were deleted.
Characterization of Deletion Mutants-Growth of the mutants at a variety of temperatures (15,30, and 37°C) showed the ygl084c⌬ and 4X⌬ strains to be temperature-sensitive at 37°C and to reach mid-log phase about 4 h later than normal strains after dilute inoculation into YPD at 30°C. The other mutations caused no detectable growth defects. The ability of these strains to synthesize triglyceride was assayed by pulse labeling cells in log phase with either [ 3 H]oleate or [ 3 H]glycerol, as shown in Tables I and II, respectively. Only the are1⌬ mutant showed a reduction in glycerol incorporation into TG. This difference was not observed with oleate labeling or palmitate labeling (data not shown), suggesting Are1 to be, at most, a minor contributor to TG synthesis. By contrast, deletion of ygl084c resulted in an increase in TG synthesis with a concomitant decrease in phospholipid (PL) synthesis. The nature of this biochemical effect is under further investigation.
A Candidate Diacylglycerol Acyltransferase with Identity to Human LCAT-Because abundant TG synthesis remained in the absence of the four yeast DGAT-like enzymes, we sought alternate candidates. Under experimental conditions, porcine LCAT has been shown to esterify diacylglycerol in addition to cholesterol (26,27). The yeast genome contains only one gene, LRO1 (LCAT-related open reading frame, YNR008w), with distinct sequence similarity to human LCAT. Aligning the LRO1 predicted protein with human LCAT (28) shows 27% overall identity with conservation of Ser 181 and Asp 345 , two members of the LCAT catalytic triad (29,30). The third amino acid of the triad, His 377 , is aligned within four amino acids of a histidine in the Lro1 protein. Lro1, like LCAT, also contains a serine lipase motif (V . L(I/V)GHS . G) at amino acids 318 -326. However, Lro1 is unlikely to be a functional LCAT homolog, because sterol esterification is abrogated upon deletion of ARE1 and ARE2 (9,31).
Analysis of LRO1 by Deletion-LRO1 deletion strains showed no detectable growth defects at 15, 30, and 37°C. Strains harboring a deletion of LRO1 were grown into log phase and pulse-labeled as above (Tables I and II). LRO1 deletion mutants showed a 65% decrease in oleate incorporation into TG and a 75% decrease in glycerol incorporation into TG compared with the normal strain. The concomitant 65% increase of glycerol incorporation into DG is consistent with Lro1 mediating esterification of DG. Oleate incorporation into DG was not statistically different in the LRO1 deletion strain. To address whether the Lro1-independent TG synthesis activity was mediated by the four DGAT-like genes, a quintuple deletion strain, 5X⌬, with LRO1, ARE1, ARE2, YGL084c, and YPL189w deleted was also assayed and showed no further decrease in TG synthesis (Tables I and II).
To determine whether LRO1 deletion also leads to a decreased cellular TG mass, TLC plates of lipids isolated from normal and lro1⌬ strains, grown to log phase, were either charred or stained with iodine vapors. Both assays showed a visually marked decrease in TG mass (not shown). This was supported by steady state labeling experiments where lro1⌬ strains accumulated 75% less [ 3 H]oleate into TG than normal strains. These observations are comparable with a previous study that found yeast with a deletion of LRO1 to have a 40% reduction in triglyceride mass (32).
Analysis of LRO1 by Overexpression-The role of Lro1 in TG synthesis was examined by overexpression. LRO1 was expressed in normal or lro1⌬ yeast in either a low copy plasmid using the endogenous LRO1 promoter or a high copy plasmid using the GAL1/10 promoter, which should result in significantly greater expression levels. The transformed yeast were grown into log phase and assayed for [ 3 H]oleate incorporation into TG (Table III). Expression of LRO1 driven by its own promoter rescued the TG synthesis deficit in lro1⌬ and did not increase TG synthesis in a normal strain. Increased expression of LRO1 from the GAL1/10 promoter leads to significantly increased TG synthesis and decreased DG and PL labeling, consistent with a rate-limiting role of this enzyme in DG esterification.
In Vitro Microsomal Assays of DG Esterification-A V5 epitope-tagged form of Lro1 localized to microsomes (data not shown) that are the major site of TG synthesis in mammalian cells (33). We thus performed a series of in vitro assays with microsomes isolated from normal and lro1⌬ haploids to investigate the mechanism of LRO1-mediated DG esterification. Supplying microsomes with [ 14 C]oleoyl-CoA and unlabeled diacylglycerol showed lro1⌬ to have equivalent or greater DGAT activity than normal microsomes (Fig. 1A). However, when [ 14 C]diacylglycerol was supplied in the absence of oleoyl-CoA, the lro1⌬ microsomes synthesized 5-fold less TG than normal microsomes (Fig. 1B). Further, when supplied with 1-palmitoyl-2[1-14 C]oleoyl phosphatidylcholine and unlabeled DG, normal microsomes incorporated radiolabeled fatty acid into TG at a rate of 120 pmol/min/mg (Fig. 1C). This activity was absent in lro1⌬ microsomes, indicating that Lro1 mediates esterification of DG using the sn-2 acyl group of phosphatidylcholine as the acyl donor. Moreover, normal microsomes showed no incorporation of fatty acid from PC into sterol ester (data not shown), indicating that ergosterol is not a substrate for Lro1 under these assay conditions. When 5X⌬ microsomes were assayed, their DG esterification activities matched those of lro1⌬ strains (not shown). Thus, the 4 DGAT-like genes do not mediate the Lro1-independent activities shown in Fig. 1, A and B.
Described here are our studies in yeast of the terminal step in triglyceride synthesis, diacylglycerol esterification. We analyzed yeast undergoing exponential growth to avoid the complex metabolic changes associated with the transition into sta- We have shown that yeast mainly utilize enzymes without sequence similarity to mammalian DGAT to catalyze the terminal step in TG synthesis. DGAT-independent TG synthesis also occurs in mammals in that a deletion mutant mouse for DGAT retains significant triglyceride synthesis activity (34). Two qualitatively different DGAT activities identified in rat liver (35,36) and an oleoyl-CoA-independent DG transacylase in rat intestine (24) further support the presence of more than one mammalian enzyme, with more than one mechanism, that can catalyze the terminal step in TG synthesis. Lro1 may provide a paradigm for such an enzyme. Our in vitro assays identified a novel mechanism of synthesizing TG from lecithin that was absent in lro1⌬ strains. The sequence similarity to mammalian LCAT and the use of a phospholipid substrate suggest that Lro1 uses an LCAT-like mechanism involving a phospholipase and an acyltransferase activity. This reaction accounts for the majority of TG synthesis in yeast but plays no detectable role in sterol esterification. Because the amount of TG synthesis was not further decreased when the four DGAT-like genes were deleted along with LRO1, there clearly exists uncharacterized acyl-CoA-dependent and -independent mechanism(s) for TG synthesis in yeast. The amount of radioactivity incorporated into TG was measured as described under "Experimental Procedures." Assays were performed in duplicate on three different days. Asterisks denote significant differences (p Ͻ 0.01) compared with normal strains.