Abstract Intracellular triacylglycerol (TAG) is a ubiquitous energy storage lipid also involved in lipid homeostasis and signaling. Comparatively, little is known about TAG’s role in other cellular functions. Here we show a pro-longevity function of TAG in the budding yeast Saccharomyces cerevisiae. In yeast strains derived from natural and laboratory environments a correlation between high levels of TAG and longer chronological lifespan was observed. Increased TAG abundance through the deletion of TAG lipases prolonged chronological lifespan of laboratory strains, while diminishing TAG biosynthesis shortened lifespan without apparently affecting vegetative growth. TAG-mediated lifespan extension was independent of several other known stress response factors involved in chronological aging. Because both lifespan regulation and TAG metabolism are conserved, this cellular pro-longevity function of TAG may extend to other organisms.

Author Summary Triacylglycerol (TAG) is a ubiquitous lipid species well-known for its roles in storing surplus energy, providing insulation, and maintaining cellular lipid homeostasis. Here we present evidence for a novel pro-longevity function of TAG in the budding yeast, a model organism for aging research. Yeast cells that are genetically engineered to store more TAG live significantly longer without suffering obvious growth defects, whereas those lean cells that are depleted of TAG die early. Yeast strains isolated from the wild in general contain more fat and also display longer lifespan. One of the approaches taken here to force the increase of intracellular TAG is to delete lipases responsible for lipid hydrolysis. Energy extraction from TAG thus is unlikely an underlying cause of the observed lifespan extension. Our results are reminiscent of certain animal studies linking higher body fat to longer lifespan. Potential mechanisms for the connection of TAG and yeast lifespan regulation are discussed.

Citation: Handee W, Li X, Hall KW, Deng X, Li P, Benning C, et al. (2016) An Energy-Independent Pro-longevity Function of Triacylglycerol in Yeast. PLoS Genet 12(2): e1005878. https://doi.org/10.1371/journal.pgen.1005878 Editor: Valter D. Longo, University of Southern California, UNITED STATES Received: August 17, 2015; Accepted: January 27, 2016; Published: February 23, 2016 Copyright: © 2016 Handee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: WH was supported by the Royal Government of Thailand Scholarship for Development and Promotion of Science and Technology and subsidized by NSF MCB1050132; XL was partially supported by a fund from the GEDD Focus Group, MSU. The work on TAG biosynthesis in the Benning lab was supported by a Department of Energy–Great Lakes Bioenergy Research Center Cooperative Agreement DE-FC02-07ER6449. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors declare that no competing interests exist.

Introduction Lipid is essential for all life forms on earth. Polar lipids, most notably phospholipids, are the primary components of biological membranes, whereas neutral lipids such as triacylglycerols (TAG; or triglycerides, TG), are long believed to store excessive energy and to provide thermal and physical insulation for animals. By esterifying three molecules of fatty acids to the glycerol backbone, TAG packs the highest density of chemical energy among major biomolecules, consistent with a role in storing surplus energy. In addition, TAG metabolism is linked to the overall lipid homeostasis in cells and the organism [1]. This is commonly achieved via diacylglycerol, DAG, which is a shared precursor for TAG and phospholipids biosynthesis. Interestingly, in many organisms, TAG accumulation is not a response of surplus nutrients, but of different stresses. For example, starvation for nitrogen, phosphorus, or sulfur causes a model photosynthetic microalga Chlamydomonas reinhardtii to accumulate TAG [2, 3]. Even the oleaginous marine alga Nannochloropsis species increase the TAG content by 50% when starved of nitrogen, and to a lesser extent when stressed by high light and salinity [4]. In animals, mild (5%) calorie restriction administered to laboratory mice shifted the relative abundance of fat and muscle by increasing the fat mass by 68%, and reducing the lean mass by 12%, with total bodyweight remained unchanged [5]. In budding yeast Saccharomyces cerevisiae, a minimal amount of TAG is synthesized by vegetatively growing cells. When glucose becomes limited and that cells enter the stationary phase, TAG synthesis rises sharply [6]. One apparent reason for these organisms to maintain a larger storage of TAG under stress is to cope with the uncertainties of the environments. By storing chemical energy and materials for membrane lipid biosynthesis, TAG helps the underlying cells quickly resume robust metabolism and growth when conditions improve. On the other hand, whether the existence of TAG in cells affords other benefits for survival through the environmental stress remains an unaddressed question. TAG is composed of a glycerol backbone esterified to three fatty acids by acyl coenzyme A:diacylglycerol acyltransferases, DGATs, and phospholipid:diacylglycerol acyltransferases, PDATs. In budding yeast, Dga1p and Lro1p are the major DGAT and PDAT, respectively [7]. Of these two, Lro1p appears to be responsible for TAG synthesis in vegetatively growing cells, whereas Dga1p contributes more significantly to the post-diauxic shift accumulation of TAG [8–10]. In addition to TAG, fatty acids can be esterified to sterols to form steryl esters (SE), another class of storage neutral lipids [10]. The two major enzymes responsible for SE biosynthesis are Are1p and Are2p [10]. In contrast to the stark differentiation of TAG abundance in log and stationary phase cells, SEs are maintained at a constant level at different phases of the growth curve [11], suggesting a function unique to TAG in the stationary phase. Intriguingly, deleting the four major neutral lipid biosynthetic genes (DGA1, LRO1, ARE1, ARE2), while causing yeast cells to lose practically all storage neutral lipids, does not result in significant deleterious effects in vegetatively growing cells [10]. However, these lean cells are hypersensitive to exogenous fatty acids and die with a phenotype of membrane over-proliferation [12], indicating that maintaining the capacity of incorporating excessive free fatty acids in the form of TAG or SE affords an important means to prevent lipotoxicity of free fatty acids. Accumulation of TAG and SE in the ER membrane causes expansion of the membrane, which eventually buds out to form lipid droplets (LD), a dynamic phospholipid monolayered organelle that has gained increasing research interests [13, 14]. A variety of proteins have been found associated with LD, including multiple TAG and SE hydrolases and signaling proteins that together play important roles in lipid homeostasis [15]. The yeast TGL3 and TGL4 genes encode the two major TAG lipases in yeast; additional lipases Tgl5p, Ayr1p and Lpx1p appear to be less robust enzymatically [16]. Tgl4p, which is thought to be the functional orthologue of the mammalian ATGL [17, 18], has been shown to be regulated by Cdk1-mediated phosphorylation in G1-to-S transition of dividing cells [19]. Blocking this phosphorylation event delays bud emergence. Deleting either or both major TAG lipases causes accumulation of TAG in stationary phase cells without a clear growth defect [18]. Similar to TAG, SE can be broken down by functionally redundant lipases Tgl1p, Yeh1p, and Yeh2p [20, 21]. SE lipase triple knockout cells, aside from possessing a significantly larger pool of SE, are phenotypically indistinguishable from the wildtype counterpart [20]. Together, these studies demonstrated clearly that yeast cells have the capacity of metabolizing neutral storage lipids. However, these lipids are not essential for cellular viability. Budding yeast has been a model for two modes of aging [22, 23], chronological lifespan (CLS) and replicative lifespan (RLS). CLS refers to the overall viability of stationary-phase cells over time. RLS examines the number of daughters that each mother cell can produce before ceasing division. These two modes simulate, respectively, the senescence of post-mitotic (e.g., muscles and neurons) and stem cells in metazoans. Chronological aging in yeast has been linked closely to the nutrient status. When glucose is depleted, yeast cells exit from the log phase to enter diauxic shift, then into the stationary phase, a mitotically inactive yet metabolically active state [24]. The population viability is maintained in cells that enter the quiescent state [25, 26]. However, over time, the number of viable quiescent cells diminishes as well, resulting in a progressive increase of population mortality, a condition similar to metazoans including humans [22]. While there are yeast-specific chronological senescence and longevity factors (e.g., acetic acid and glycerol, respectively) [27, 28], a number of pathways are conserved [22, 23]. Some of the most notable pro-aging pathways include the Target Of Rapamycin (TOR)/S6 kinase (Sch9p in yeast) [29] and the Ras/adenylate cyclase/PKA pathways [30]. Intriguingly, these pathways control both CLS and RLS [23]. These two pathways are activated in response to the intake of selective nutrients, and are suppressed by caloric restriction, consistent with many reports that different organisms extend their lifespan when subjected to calorie restriction [31, 32]. Other pro-aging factors include oxidative stresses [33], mitochondrial dysfunction [34, 35], defective autophagy [36], DNA damages and replication stresses [37], and metabolic alterations [38]. Yeast genome-wide studies have identified a variety of gene mutations that extend chronological lifespan [39–42]. Many of these genes are involved in the metabolism of amino acids, nucleotides, or alternative carbon source, further underscoring the important roles played by metabolites in the control of lifespan. However, despite these relatively unbiased screens, very little is known regarding the role of lipids in the control of CLS. Here we present evidence for a novel energy usage-independent, anti-senescence function of TAG in yeast.

Discussion Here we present evidence for a novel pro-longevity function of intracellular TAG in yeast. Deleting TAG lipases or overproducing a DGAT increased TAG accumulation and extended chronological lifespan. Deleting the two TAG biosynthetic enzymes practically eliminated TAG and significantly shortened the chronological lifespan, as well as the median and maximum replicative lifespan. The fact that chronological lifespan extension is seen in different lipase knockout (i.e., tgl3Δ, tgl4Δ, and tgl3Δ tgl4Δ) and in DGAT overexpression strains argues strongly that the accumulation of TAG was the contributing factor for lifespan extension. This conclusion is consistent with the observation that deleting TGL3 cannot rescue the early death phenotype of dga1Δ lro1Δ lean cells (rows 5 and 6, S5 Fig). Unlike other lifespan extension regimes such as SCH9 knockout and rapamycin treatment that also retard mitotic growth [60, 61], we have yet to detect obvious growth defects in the three lipase deletion strains. For example, aside from normal, or even faster growth rates (Fig 2E), mating and sporulation efficiency of these fat cells was essentially identical to their wildtype mother strain (S3A and S3B Fig). There were no significant differences in cellular sensitivity to heat (55°C), 260 nm UV, high concentrations of NaCl, or to H 2 O 2 (S4 Fig). It is perceivable that a negative phenotype would be linked to TAG lipase null cells if they were grown without any fatty acid supplement, and with concomitant presence of a fatty acid synthase inhibitor such as cerulenin [62]. These cells would suffer from the lack of energy and fatty acid building blocks for growth and division. TAG hydrolysis is thus by and large dispensable as long as fatty acids are available from the environment or can be synthesized by de novo activities. It should be noted that Daum and colleagues reported that tgl3- homozygous knockout cells were unable to form spores [18]. Possible causes for this discrepancy may include differences in the genetic background of the strains, and the protocols for sporulation. Chronological lifespan of S. cerevisiae is regulated by both yeast-specific and conserved factors and drugs. Experimental results shown in Figs 4 to 7 strongly suggest that TAG preserves viability during chronological senescence in a manner that is independent of those factors tested herein, including caloric restriction, high osmolarity, medium pH, rapamycin and paraquat responses, and conserved pathways involving TOR1, RAS2, and SOD2 genes. Results from genetic interaction tests also suggest a novel TAG pathway in CLS control. Both fat and lean strains responded similarly to the deletion of TOR1, RAS2, or SOD2 (Fig 7), indicating that changes in TAG metabolism does not affect the function of these conserved CLS regulators. Intriguingly, deleting TOR1 shortens the lifespan of both lipase-null and DGAT-deficient cells (Fig 7) but, as expected, protects those cells possessing the normal TAG metabolic capacity. We suggest that tor1Δ cells that survive on the caloric restriction pathway through chronological aging [23, 28] need to tap into the energy depot of TAG. Without TAG biosynthetic enzymes or TAG hydrolytic lipases perturbs this energy flux, thus resulting in early death of tor1Δ cells. In addition to TOR1 and RAS2, we have also combined tgl3Δ and sch9Δ mutations. Deleting SCH9, the ribosome S6 kinase homologue, has been shown to activate the Rim15-Msn2/4 and superoxide dismutase (SOD) stress pathways and prolongs lifespan significantly [29, 63]. Our tests of the genetic interaction between TGL3 and SCH9 were inconclusive. Independent tgl3Δ sch9Δ isolates showed mixed results, ranging from longer CLS to synthetic sickness (S6 Fig). The reason for the stochastic phenotypes is unclear. Taking into account the observations presented above as well as from previous reports, we hypothesize that TAG has a role in stress response that underlies the observed phenotypes in chronological aging. Firstly, TAG accumulates when yeast cells enter stationary phase in which nutrients are becoming progressively limited [6, 47]. Starvation and stress-induced TAG accumulation appears to be a widespread response in different organisms, including photosynthetic algae [2–4, 64] and animals as well [65–67]. Dietary restriction has been suggested to prolong lifespan by eliciting cellular stress response [31]. Intriguingly, laboratory mice [5] and developing Caenorhabditis elegans [68] have an increased body fat mass when subjected to dietary restriction. Secondly, wild yeast strains in general exhibit higher TAG content and longer chronological lifespan (Fig 1). Food shortage is a common environmental crisis in the wild, but rarely a relevant factor for lab strains. A systematic phenotypic and transcriptomic survey of wild and laboratory strains showed that the latter are less tolerant of many environmental stresses [44]. Certain traits, including stress responses and high levels of TAG, might have lost during the domestication of S. cerevisiae in laboratory environments, in which the selection pressure for long-living, stress-tolerant stationary phase cells is low. It seems plausible that besides preserving energy to cope with uncertainties in food supply, the increased fat content in stressed cells may confer an additional, energy-independent function that helps sustain longevity. The presumptive stress antagonized by TAG in post-mitotic cells remains to be identified. One candidate is fatty acid-solicited lipotoxicity [69]. Sequestering fatty acids in the form of TAG may prevent lipotoxicity that erodes replicative potential and chronological viability. Disabling TAG biosynthesis results in surplus fatty acids, which may arise from de novo synthesis, uptake from environment, or from lipolysis, that may disrupt membrane lipid homeostasis [9]. Indeed, in an extreme situation where the ability to incorporate fatty acids to TAG and SE is altogether eliminated, yeast cells become hypersensitive to fatty acids and die with membrane hyper-proliferation [12]. Similarly, the fission yeast Schizosaccharomyces pombe dga1+ and plh1+ double knockout cells (equivalent to the dga1Δ lro1Δ strain of S. cerevisiae) also die upon entering stationary phase, and are hypersensitive to exogenous fatty acids during vegetative growth [70]. While this lipotoxicity model explains the early death phenotype of dga1Δ lro1Δ cells, total lipid analysis of our long-living fat cells failed to detect significant changes in free fatty acids or DAG (see, for example, Fig 3A). While we cannot rule out the possibility that a small but critical change in certain lipid species contributes more critically to lifespan extension, other hypotheses are worth considering. One frequently cited cause of aging is mitochondrial dysfunction that also involves oxidative damages [71]. While subcellular compartmentalization confines TAG synthesis and storage to ER and lipid droplets, respectively, a number of reports have demonstrated physical association of mitochondria with ER and LD [72], lending support for functional crosstalk between neutral lipid metabolism and mitochondria biogenesis [73]. Moreover, mitochondria also possess a type II fatty acid synthesis pathway [74]. Deleting enzymes within this pathway causes mouse embryonic lethality and yeast respiratory defects [75, 76]. Fatty acids trafficking between mitochondria and LD may help achieve mitochondrial lipid homeostasis. Importantly, mitochondria are a major source for reactive oxygen species. Free radicals that would otherwise escape from mitochondria and cause pleiotropic cellular damages might enter LD and attack the fatty acyl chains of the storage TAG molecules [77]. It is possible that the high density of peroxidated fatty acids in LD facilitates crosslinking of neighboring radicalized molecules, hence terminating the vicious propagation of radicals. This “radicals sink” model appears to be consistent with the experimental findings presented above. TAG metabolism and many aspects of cellular aging are conserved. It is thus possible that the cytoprotective role of TAG also exists in higher organisms. For example, Bailey et al. recently reported an anti-oxidant role of lipid droplets in the stem cell niche of Drosophila during neurodevelopment by limiting the levels of reactive oxygen species and inhibiting the oxidation of polyunsaturated fatty acids [78]. In transgenic mice, overexpression of DGAT1 in the skeletal muscle and heart increased intracellular TAG abundance as well as insulin sensitivity of the underlying animals [79, 80]. Similarly, deleting adipose triglyceride lipase (ATGL) protected animals from high-fat diet-induced insulin resistance [81]. However, excessive TAG in heart muscle resulting from ATGL knockout also was associated with cardiac dysfunction [73, 82]. These transgenic animal studies underscore the complexity of mammalian metabolism and the interdigitating relationships between triglycerides (dietary, circulating, and in different tissues) and other nutrients. While the positive influence of intracellular TAG on chronological lifespan in yeast is reminiscent of the so-called obesity paradox in humans, that is, the overweight population has the lowest mortality under a number of medical conditions [83, 84], we caution that the comparatively simple yeast may not be immediately applicable to the complex human system. An integrative strategy combining metabolomics, lipidomics, and transcriptomics of representative yeast strains will help elucidate the molecular basis of this novel function of TAG, which might provide a toolbox for a better understanding of the benefits of intracellular TAG in humans.

Materials and Methods Strains and media Yeast strains used in this study are shown in Table 1. YPD medium contained 2% glucose (Sigma-Aldrich) (unless otherwise stated in the text), 2% peptone (BD Difco), and 1% yeast extract (BD Difco). SC medium (synthetic complete) contained 2% glucose, 5 g/l ammonium sulfate (Sigma-Aldrich), 1.7 g/l yeast nitrogen base without amino acids or ammonium sulfate (BD Difco), and complete amino acids as described in [85]. Auxotrophic nutrients were supplied at four-fold excess as recommended [23]. Citrate phosphate buffering was done as described [27]. Rapamycin (Sigma-Aldrich) and paraquat dichloride (Fluka) were added from 10 μM and 250 mM stocks to SC medium to make final concentrations of 10 nM and 10 mM, respectively. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Yeast strains used in this study. https://doi.org/10.1371/journal.pgen.1005878.t001 Yeast methods Yeast transformation was performed using the lithium acetate method [86]. Deletion of TGL3 and TGL4 was described in [2]. To delete DGA1, PCR reactions using primers ATGTCAGGAACATTCAATGATATAAGAAGAAGGAAGAAGGAAGATCCCCGGGTTAATTAA and TTACCCAACTATCTTCAATTCTGCATCCGGTACCCCATATTTATTCGAGCTCGTTTAAAC and template pFA6a-KanMX6 [87] were conducted. To delete LRO1, primers TATCCATATGACGTTCCAGATTACGCTGCTCAGTGCGGCCGCATGTCAGGAACATTCAAT and GAATTTCGACGGTATCGGGGGGATCCACTAGTTCTAGCTAGATTACCCAACTATCTTCAA were used with pBS1539 as the template to amplify K. lactis URA3 gene as the selective marker [88]. To delete TOR1, primers GAACCGCATGAGGAGCAGATTTGGAAGAGTAAACTTTTGAAATGAAGCTTGATATCGAAT and CCAGAATGGGCACCATCCAATATAATGTTGACATAACCTTTCTACGACTCACTATAGGGC were used with pBS1539 to amplify K. lactis URA3. For RAS2 deletion, primers CCTTTGAACAAGTCGAACATAAGAGAGTACAAGCTAGTCGTCTGAAGCTTGATATCGAAT and ACTTATAATACAACAGCCACCCGATCCGCTCTTGGAGGCTTCTACGACTCACTATAGGGC were used to amplify K. lactis URA3 on pBS1539. For SOD2 deletion, primers TTCGCGAAAACAGCAGCTGCTAATTTAACCAAGAAGGGTGGTTGAAGCTTGATATCGAAT and GATCTTGCCAGCATCGAATCTTCTGGATGCTTCTTTCCAGTTTACGACTCACTATAGGGC were used to amplify K. lactis URA3 on pBS1539. The PCR products were gel-purified for yeast transformation. Genomic PCR was used to verify the correct insertion. To overproduce Dga1p, a yeast genomic PCR product was co-transformed with Not I-linearlized pMK595 [89] for ADH1-controlled expression (pMK595-DGA1). A second multicopy DGA1 overexpression plasmid with DGA1 under its own promoter control (pMK595 P DGA1 -DGA1) was constructed similarly, except that the DGA1 promoter (961 bp) was included in the transforming PCR DNA, using primers ATCCATATGACGTTCCAGATTACGCTGCTCAGTGCGGGTAAAGAATCTAAATCGAGCTAC and atcggggggatccactagttctagctagagcggccTAGATAGGTACAATCGACTTAAAGC. The tgl3Δ /tgl3Δ diploid strain yWH74 was generated by first transforming yXL004 with YCp50-HO [90] to induce mating type switch and subsequently spontaneous mating of cells in the same colony. Homozygous diploid cells were identified by their inability to mate as either a MATa and MATalpha strain. Diploid cells were then grown in YPD for two days to allow for the loss of YCp50-HO, resulting in 5-FOA resistant, ura3- cells. For growth curve analyses, yeast cells were seeded at an initial concentration of 0.1 OD 600 in 150 μl of YPD medium in 96-well plates with biological and technical duplicates. The plates were examined by measuring OD 630 every 30 minutes via a BioTex PowerWave XS plate reader until cells reached saturation. The machine was programmed to shake at the high-speed setting and to control temperature at 30°C. Lifespan analyses Chronological lifespan was measured by the outgrowth method as described in [85]. Briefly, yeast from stab cultures were inoculated to YPD broth and grown at 30°C overnight or until late log phase. Cultures were then diluted into 5 ml SC medium at 0.1 OD 600 . The seeded cultures, in 15-ml glass tubes with a loose metal cap, were incubated in a rotator drum at 30°C. At selected time points, 5 μl of stationary phase cultures were sampled out and mixed with 145 μl of fresh liquid YPD in a 96-well plate. The plates were sealed with parafilm to prevent evaporation, and incubated in the BioTex PowerWave XS plate reader. Cell density (OD 630 ) was monitored every 30 minute for 48 hours. Growth curves, doubling times, and survival fractions were calculated according to [85]. To compare the quantitative differences in lifespan of different strains, the time by which each culture reached 10%, 1%, and 0.1% survival fractions was obtained from three independent survival curves. The cultures which survived beyond 30 days before reaching the specified survival fractions were not included in the statistical calculation and represented as >30 days. For spot assays, stationary phase cultures at selective time points were adjusted to 1 OD 600 with sterile water in 96-well plates and ten-fold serially diluted. 5 μl of cells from each well were spotted to a YPD plate and grown at 30°C for two days. The culture viability was quantified by counting visible colonies at the most diluted spot. The number of colonies multiplied by the dilution factor of that spot was regarded as the viability. This method was adapted from the Tadpole assay as described in [91]. Replicative lifespan was performed according to previously described [92] by counting number of progeny produced by 60–90 virgin cells from young mother of each strain. Daughter cells were removed every 90 minutes by a micromanipulator. The lifespan analyses were performed by using R software, version 3.0.3 with survival and KMsurv packages. Breslow test was used for statistic analysis. Lipid analysis and Nile red staining Total lipid extraction was conducted essentially as described before [70] with modifications. Briefly, 3 OD 600 cells from selected time points were harvested by centrifugation at 5,000 rpm for 5 minutes at room temperature, followed by washing once with 1 ml water, and were kept at -80°C if lipid extraction was not done immediately after cell collection. To extract total lipids, cells, if frozen, were removed from the freezer and mixed directly with 300 μl of glass beads (425–600 μm, Sigma-Aldrich) and 1 ml of chloroform: methanol (17:1, v/v) (JT Baker) by vortexing twice for 90 seconds at 4°C. The mixtures were briefly spun and the supernatant was moved to a new glass tube. The remaining cell debris and glass beads were vortexed with an additional 1 ml of chloroform: methanol (2:1, v/v). The supernatant was collected as above and pooled with the previous fraction. 1 ml of 0.2 M phosphoric acid and 1 M KCl, was added to the pooled organic fraction and vortexed vigorously for 30 seconds, and spun at 3,000 rpm, 4°C for 5 minutes to separate the organic and aqueous phases. The chloroform phase at the bottom was collected and dried under nitrogen gas. This lipid extract served as the total lipid fraction. To purify TAG, total lipids were developed by thin-layer chromatography (TLC) on a G60 silica plate (EMD Chemicals). The mobile phase was composed of petroleum ether (35–60°C, Macron): diethyl ether (JT Baker): acetic acid (JT Baker) = 80: 20: 1 (v/v/v). Following development, the TLC plates were briefly stained with iodine vapor to reveal the position of TAG. Spots co-migrating with a TAG control (olive oil, Dante) were isolated and converted to fatty acyl methyl esters (FAME) by reacting with 1 ml of 1 N Methanolic HCl (Sigma-Aldrich) at 80°C for 25 minutes [93]. A universal internal control of 5 μg of pentadecanoic acid (Sigma-Aldrich) was included in all samples for FAME derivatization and gas chromatography (GC). 1 ml of 0.9% NaCl was added to stop the FAME reaction, followed by the addition of 1 ml of hexane, and vortexed for 30 sec to extract FAME. Centrifugation at 3,000 rpm for 5 min at 4°C was conducted before the hexane layer was aspirated to another glass tube, and the volume reduced to 30 μl under nitrogen blowing. 2 μl of the FAME in hexane was injected to a gas chromatography system (Agilent Technology, 7890A) for quantification. To visualize lipid droplets in yeast cells, approximately 0.5 OD 600 cells were collected by centrifugation (14,000 rpm for 1 min in a microfuge) and washed once by 1 ml TE buffer (pH 7.4). Cell pellets were suspended in 100 μl of TE buffer and stained with 1 mg/ml Nile red in the presence of 3.7% formaldehyde for concomitant fixation, and let sit in the dark for 20 min. Cells were collected again by microcentrifugation and re-suspended in 100 μl TE buffer and stored in the dark for no more than two days. For microscopy, an Olympus BX51 station with a Exfo X-cite 120 UV light fixture and a DP30-BW CCD camera were used. A GFP filter was used for lipid droplet fluorescence detection. For semi-quantitative comparison of lipid droplets, a fixed exposure (typically 2 seconds) for fluorescence was applied to all samples. Sporulation and mating analysis Sporulation efficiency was determined by microscopically examining the percent of diploid cells forming asci. Overnight YPD cultures of diploid strains were transferred to PSP2 medium (Potassium phthalate (Sigma-Aldrich) 8.3 g/l, yeast extract 1 g/l, 1.7 g/l yeast nitrogen base without amino acids or ammonium sulfate, ammonium sulfate 5 g/l, potassium acetate 10 g/l, pH 5.4 at 0.1 OD 600 and grown at 30°C under vigorous shaking (200–250 rpm). After 24 hours, cells were harvested by centrifugation (14,000 rpm, 30 sec in a microfuge), washed once with sterile water, and re-suspended in 1 ml SPM medium (potassium acetate 3 g/l, raffinose 0.2 g/l). Cultures were shaken vigorously at 200–250 rpm at 30°C for 48–72 hours. A small amount of cells were removed from the culture, spun, and suspended in 1 mg/ml DAPI (4,6-diamidino-2-phenylindole) in a mounting medium (1 mg/ml p-phenylenediamine, 0.9% glycerol, 2.25 μg/ml DAPI). Percent of cells forming asci with four DAPI foci (i.e., four spores) were counted. Mating efficiency was quantified by mixing 0.1 OD 600 of “tested” strains with 0.5 OD 600 of tester strains of the opposite mating type (227a or 70 ‹), all in early log phase, in 1 ml of YPD. Cell mixtures were let sit at 30°C for 5 hours. The tested and tester stains were also incubated separately as the negative control. Cells were washed once with sterile water and plated on SD medium (2% glucose, 1.7 g/l nitrogen base with amino acids or ammonium sulfate, 5 g/l ammonium sulfate, 20 g/l agar). Mating between the tested and tester strains generated prototroph diploid cells that were able to form colonies on the SD medium plate. Wild strains segregation The strains B454, B756, B779, B359, B370, B357 and B390 were segregants of isolates collected in the wild (see Table 1 for source and reference). In order to generate isogenic haploid strains, the heterozygous wild isolates were sporulated and self diploidized due to homothallism. We randomly selected one of the four homozygous segregants from each tetrad. Next, strains were made heterothallic via removal of the HO gene, using homologous gene replacement with either a Hgh cassette (pAG32) [94] (strain B454 due to a natural kanamycin resistance) or a KanMX6 cassette (pFA6a) [95] (strains B756, B779, B359, B370, B357 and B390). Transformed strains were sporulated and dissected to verify 2:2 segregation of either hygromycin or kanamycin resistance. One MATa and MATalpha haploid transformant was retained from each strain. Proper integration at the HO locus was verified via PCR. Strain B653 was obtained from John McCusker.

Supporting Information S1 Fig. Lab and wild yeast strains used in this work were examined by microscopy. 5-day old stationary phase cultures were harvested for neutral lipid staining with Nile red. DIC (differential interference contrast) and fluorescence microscopy were done using an Olympus BX51 station equipped with a Exfo X-cite 120 UV light fixture and a DP30-BW CCD camera. Fluorescence micrographs were taken with a fixed, 2-second exposure to aid comparison of the fluorescence intensity. Scale bars: 5 μm. All pictures were re-sized identically for presentation. https://doi.org/10.1371/journal.pgen.1005878.s001 (TIF) S2 Fig. Deleting either or both TAG lipase genes resulted in similar upregulation of TAG in yeast. Total TAG, expressed as percentage of total cellular lipids, of wildtype, tgl3Δ, tgl4Δ, and tgl3Δ tgl4Δ strains were isolated and quantified by gas chromatography. Cells were from 3-day old YPD cultures. *, P<0.05; **, P<0.01 https://doi.org/10.1371/journal.pgen.1005878.s002 (TIF) S3 Fig. TAG accumulation does not affect mating efficiency, sporulation, or medium acidification. (A) Haploid strains, as indicated, were tested for their mating efficiency with a tester strain. (B) TGL3 +/+ and -/- diploid strains were subjected to sporulation. 3 days after transferring cells to the sporulation medium, cells were examined under a microscope to quantify for the number of tetrads. https://doi.org/10.1371/journal.pgen.1005878.s003 (TIF) S4 Fig. TAG-rich and–deficient cells do not exhibit significant differential susceptibility to common stresses. Day 3 early-stationary phase cultures were exposed to the shown stresses before plating to YPD. For the test of salt sensitivity, NaCl (0.7 or 1.4 M) was included in the YPD plate. Heat and H 2 O 2 sensitivity were conducted by exposing cell suspension (after water wash for H 2 O 2 ) to the stress before serially diluted for plating. For UV exposure, serially diluted cells were spotted to YPD before UV exposure. The plates were incubated in dark afterwards. https://doi.org/10.1371/journal.pgen.1005878.s004 (TIF) S5 Fig. Deleting TGL3 does not rescue the early death phenotype of the dga1Δ lro1Δ lean cells. The indicated strains (on the right) were grown in SC with 2% glucose and sampled at the indicated time for semi-quantitative comparison of colony forming units. 10-fold serially diluted cell suspension was spotted to YPD. https://doi.org/10.1371/journal.pgen.1005878.s005 (TIFF) S6 Fig. Combining tgl3Δ and sch9Δ null alleles caused stochastic growth defects. SCH9 was deleted from TGL3+ and tgl3Δ backgrounds for chronological lifespan assessment. Two independent tgl3Δ sch9Δ transformation colonies were isolated and tested. Both were ›+, but one apparently was synthetic sick while the other exhibited normal growth and extended lifespan. Shown are representative results of two independent SCH9 knockout attempts. https://doi.org/10.1371/journal.pgen.1005878.s006 (TIFF)

Acknowledgments We thank Zhiying You for assisting statistical analysis; Eve Gardner, Jaikishan Prasad, Rachel Green, Hao Nguyen, Jan Stoltin, and Rebecca Roston for technical assistance; and Kelly Tatchell and Paul Siliciano for tracing the history of yMK839 and EG123. Barbara Sears, Astrid Vieler, and Neil Bowlby are thanked for their advice on TAG analysis. We acknowledge Erik Martinez-Hackert, David Arnosti, Tom Sharkey, and John Wang for their critical reading and suggestions for this manuscript.

Author Contributions Conceived and designed the experiments: WH XL MHK. Performed the experiments: WH XL KWH XD PL. Analyzed the data: WH XL KWH XD BLW CB. Contributed reagents/materials/analysis tools: WH XL KWH CB MHK. Wrote the paper: MHK. Collaboratively coordinated this project: CB BW MHK.