Lipid droplets (LDs) are emerging cellular organelles that are of crucial importance in cell biology and human diseases. In this study, we present our screen of ∼4,700 Saccharomyces cerevisiae mutants for abnormalities in the number and morphology of LDs; we identify 17 fld (few LDs) and 116 mld (many LDs) mutants. One of the fld mutants (fld1) is caused by the deletion of YLR404W, a previously uncharacterized open reading frame. Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1Δ cells demonstrate significantly enhanced fusion activities both in vivo and in vitro. Interestingly, the expression of human seipin, whose mutant forms are associated with Berardinelli-Seip congenital lipodystrophy and motoneuron disorders, rescues LD-associated defects in fld1Δ cells. Lipid profiling reveals alterations in acyl chain compositions of major phospholipids in fld1Δ cells. These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

Lipid droplets (LDs) are important cellular organelles that govern the storage and turnover of lipids. Little is known about how the size of LDs is controlled, although LDs of diverse sizes have been observed in different tissues and under different (patho)physiological conditions. Recent studies have indicated that the size of LDs may influence adipogenesis, the rate of lipolysis and the oxidation of fatty acids. Here, a genome-wide screen identifies ten yeast mutants producing “supersized” LDs that are up to 50 times the volume of those in wild-type cells. The mutated genes include: FLD1, which encodes a homologue of mammalian seipin; five genes (CDS1, INO2, INO4, CHO2, and OPI3) that are known to regulate phospholipid metabolism; two genes (CKB1 and CKB2) encoding subunits of the casein kinase 2; and two genes (MRPS35 and RTC2) of unknown function. Biochemical and genetic analyses reveal that a common feature of these mutants is an increase in the level of cellular phosphatidic acid (PA). Results from in vivo and in vitro analyses indicate that PA may facilitate the coalescence of contacting LDs, resulting in the formation of “supersized” LDs. In summary, our results provide important insights into how the size of LDs is determined and identify novel gene products that regulate phospholipid metabolism.

LDs (lipid droplets) are cellular organelles which can be found in nearly all eukaryotic cells. Despite their importance in cell biology, the mechanism underlying LD biogenesis remains largely unknown. In the present study we report that conditions of ER (endoplasmic reticulum) stress stimulate LD formation in Saccharomyces cerevisiae. We found that LDs accumulated in yeast mutants with compromised protein glycosylation or ER-associated protein degradation. Moreover, tunicamycin and Brefeldin A, agents which induce ER stress, were found to stimulate LD formation. In contrast, the restoration of protein glycosylation reduced LD accumulation. Interestingly, enhanced neutral lipids synthesis and LD formation under conditions of ER stress was not dependent on Ire1p. Lastly, we demonstrated that the absence of LDs did not compromise cell viability under ER stress. Our results suggest that although more LDs are produced, LDs are not essential to cell survival under ER stress.

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The isoprenoid squalene is an important precursor for the biosynthesis of sterols. The cellular storage of squalene and its impact on membrane properties have been the subject of recent investigations. In a screen for abnormal lipid droplet morphology and distribution in the yeast Saccharomyces cerevisiae, we found significant lipid droplet clustering (arbitrarily defined as an aggregation of six or more lipid droplets) in a number of mutants (e.g. erg1) that are defective in sterol biosynthesis. Interestingly, these mutants are also characterized by accumulation of large amounts of squalene. Reducing the level of squalene in these mutants restored normal lipid droplet distribution. Moreover, inhibition of squalene monooxygenase in two mammalian cell lines (CHO-K1 and 3T3-L1) by terbinafine also resulted in lipid droplet clustering. These results indicate that the level of squalene may affect the growth and distribution of lipid droplets. IntroductionLipid droplets (LDs) are energy storage organelles that comprise a neutral lipid core of triacylglycerols (TAG) and sterol esters (SE), surrounded by a monolayer of phospholipids with proteins embedded [1][2][3][4][5]. Formation of LDs is thought to occur in the endoplasmic reticulum (ER) [6,7]. The most favored model of LD biosynthesis [6][7][8][9] proposes that newly synthesized neutral lipids accumulate between two leaflets of the endoplasmic reticulum (ER) membrane before budding into the cytosol [10,11]. Varying in size and composition, LDs are found in nearly all eukaryotic cells [12][13][14][15], and provide a store of energy and of bioactive lipids, such as fatty acids and sterols [16]. For instance, although mature adipocytes usually contain one or a few huge TAG-rich LDs (25-300 lm in diameter) [16], other mammalian cell types contain smaller neutral LDs with different TAG : SE ratios. Plant seeds usually store abundant TAG-rich LDs, and some seed tissues may contain as much as 76% lipid w/w [17]. Depending on the growth phase, cells of the budding yeast Saccharomyces cerevisiae contain up to a dozen lipid particles/droplets (~0.4 lm in diameter) per cell [1,18], which are made up of equal amounts of TAG and SE [1,19,20].LDs may grow in size in response to increased lipid synthesis and/or uptake [7]. Giant or supersized LDs represent the most efficient form of lipid storage in terms of surface to volume ratio. Small LDs, on the other hand, provide more surface area for LD-associated proteins, such as lipases. Giant LDs may also affect the cell structure and cytoskeleton in a negative way, given their sheer volume. Therefore, the growth and final size of LDs have important implications in cell biology and function. Recent studies have suggested that LDs may grow through fusion, and the clustering of LDs may be a prerequisite for LD growth and fusion [21]. Despite rapid progress in LD research, the fundamental mechanisms that govern the formation, size and distribution of cellular LDs are still unclear. Little is Abbreviations ER, endoplasmic reticulum; LD, ...

The pandemic of lipid-related disease necessitates a determination of how cholesterol and other lipids are transported and stored within cells. The first step in this determination is the identification of the genes involved in these transport and storage processes. Using genome-wide screens, we identified 56 yeast (Saccharomyces cerevisiae) genes involved in sterol-lipid biosynthesis, intracellular trafficking, and/or neutral-lipid storage. Direct biochemical and cytological examination of mutant cells revealed an unanticipated link between secretory protein glycosylation and triacylglycerol (TAG)/steryl ester (SE) synthesis for the storage of lipids. Together with the analysis of other deletion mutants, these results suggested at least two distinct events for the biogenesis of lipid storage particles: a step affecting neutral-lipid synthesis, generating the lipid core of storage particles, and another step for particle assembly. In addition to the lipid storage mutants, we identified mutations that affect the localization of unesterified sterols, which are normally concentrated in the plasma membrane. These findings implicated phospholipase C and the protein phosphatase Ptc1p in the regulation of sterol distribution within cells. This study identified novel sterol-related genes that define several distinct processes maintaining sterol homeostasis.Both cholesterol biosynthesis and storage are controlled in response to levels and localization of regulatory pools of sterols (33,37,54,65). In response to high cholesterol levels in the endoplasmic reticulum (ER) membrane, the enzyme acyl coenzyme A (CoA):sterol O-acyltransferase (ASAT) initiates sterol esterification and storage by covalently coupling fatty acids to cholesterol. Through an active process, the esterified cholesterol is amalgamated with other neutral lipids into lipid storage droplets that are released from the ER membrane (42, 88). The trafficking of unesterified sterols also affects the sterol distribution in regulatory pools. Although cholesterol is synthesized in the ER, the highest level of unesterified cholesterol is found in the plasma membrane (33) and maintenance of normal sterol levels requires the efficient transport of cholesterol from the ER membrane to the plasma membrane. The maintenance of cholesterol levels in the plasma membrane is affected by sorting from endosomal compartments and recycling back to the cell surface (33, 54), and feedback regulation of cholesterol on its own biosynthesis and storage also controls levels of cellular sterols (16, 68). These findings suggest that the maintenance of cellular cholesterol homeostasis requires the regulatory integration of cholesterol synthesis, storage, and transport pathways.As in mammalian cells, the budding yeast Saccharomyces cerevisiae synthesizes its own cholesterol-like lipids but, under normal aerobic conditions, yeast does not internalize exogenous sterol lipids. Apart from this difference, other elements of sterol homeostasis, including lipid storage and transport pathways, app...

Lipid droplets (LDs) are highly dynamic organelles that play a central role in mammalian energy storage. LDs are also involved in many cellular functions, including protein storage and degradation, and membrane and lipid trafficking (1-4). Each LD contains a hydrophobic neutral lipid core enclosed by a phospholipid monolayer (5). In mammals, the lipid core comprises mainly triacylglycerols (TAGs) and cholesteryl esters. The molecular events underlying the biogenesis of LDs remain to be determined (6). While several models have been proposed, the prevailing view is that LDs originate and bud from the endoplasmic reticulum (ER), followed by expansion and maturation (7). A recent study suggests that the fat storage-inducing transmembrane proteins are required for proper budding of LDs from the ER (8). The growth of LDs is not fully understood, as the size of LDs varies within different tissues and even within the same cell type (9). Recent studies have established the Cide family proteins, Cidec/FSP27 in particular, as key regulators of LD growth/fusion in adipocytes (10, 11). Phospholipids, especially phosphatidylcholine (PC) and phosphatidic acid (PA), also appear to be important regulators of LD growth and proliferation, as a decrease in PC or an increase in PA can both result in the formation of giant or "supersized" LDs (1, 9, 12, 13). LDs can also grow by lipid synthesis in situ, as TAG synthesis enzymes can relocate from the ER to the LD surface to mediate LD growth (14). Despite these important findings, Abstract The expansion of lipid droplets (LDs) and the differentiation of preadipocytes are two important aspects of mammalian lipid storage. In this study, we examined the role of CDP-diacylglycerol (DAG) synthases (CDSs), encoded by CDS1 and CDS2 genes in mammals, in lipid storage. CDS enzymes catalyze the formation of CDP-DAG from phosphatidic acid (PA). Knocking down either CDS1 or CDS2 resulted in the formation of giant or supersized LDs in cultured cells. Moreover, depleting CDS1 almost completely blocked the differentiation of 3T3-L1 preadipocytes, whereas depleting CDS2 had a moderate inhibitory effect on adipocyte differentiation. The levels of many PA species were significantly increased upon knocking down CDS1. In contrast, only a small number of PA species were increased upon depleting CDS2. Importantly, the amount of PA in the endoplasmic reticulum was dramatically increased upon knocking down CDS1 or CDS2. Our results suggest that the changes in PA level and localization may underlie the formation of giant LDs as well as the block in adipogenesis in CDS-deficient cells. We have therefore identified CDS1 and CDS2 as important novel regulators of lipid storage, and these results highlight the crucial role of phospholipids in mammalian lipid storage.-Qi, Y., T.