Chapter 14 - Genome-Wide Screens for Gene Products Regulating Lipid Droplet Dynamics

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Abstract

Lipid droplets (LDs) are emerging as dynamic cellular organelles that play a key role in lipid and membrane homeostasis. Abnormal lipid droplet dynamics are associated with the pathophysiology of many metabolic diseases, such as obesity, diabetes, atherosclerosis, fatty liver, and even cancer. Understanding the molecular mechanisms governing the dynamics of LDs, namely, their biogenesis, growth, maintenance, and degradation, will not only shed light on the cellular functions of LDs, but also provide additional clues to treatment of metabolic diseases. Genome-wide screen is a powerful approach to identify genetic factors that regulate lipid droplet dynamics. Here, we summarize recent genome-wide studies using yeast and Drosophila cells to understand the cellular dynamics of LDs. The results suggest that the genome-wide screens should be carried out in multiple organisms or cells, and using different nutritional conditions.

Introduction

Lipid droplets (LDs) are found in almost all eukaryotic cells and also in some prokaryotes. LDs consist of a hydrophobic core of neutral lipids, mainly triacylglycerol (TAG) and/or sterol ester (SE), which is delimited by a monolayer of polar lipids with attached or embedded proteins. It is believed that LDs are synthesized from the endoplasmic reticulum (ER) in all eukaryotic cells, including yeast, though the exact mechanism remains an enigma (Murphy and Vance, 1999, Martin and Parton, 2006, Ploegh, 2007, Robenek et al., 2006, Walther and Farese, 2009a, Ohsaki et al., 2009). So far only the synthesis of neutral lipids has been found to be essential in the formation of LDs. The yeast mutant strain dga1Δlro1Δare1Δare2Δ, which is deficient in the formation of TAG and SE, does not produce LDs (Sandager et al., 2002, Oelkers et al., 2002). Interestingly, the adipocytes differentiated from DGAT1−/− DGAT2−/− mouse embryonic fibroblast cells also lack LDs (Harris et al., 2011).

The structure of yeast LDs is similar to that of their mammalian and plant counterparts. In addition, molecular mechanisms that govern the dynamics of LDs, including their formation, growth, maintenance, and degradation, appear to be conserved from yeast to human (Walther and Farese, 2009b). Therefore, the study of LDs using yeast as a model may contribute to a better understanding of the cell biology and physiology of human LDs.

It has long been recognized that under laboratory culture conditions exponentially growing yeast cells maintain low levels of storage lipids, both TAG and SE, but their synthesis increases dramatically upon entry into late exponential or early stationary phase (Bailey and Parks, 1975, Taylor and Parks, 1979). The rise of cellular TAG and SE content in stationary phase is partly, if not all, attributable to conversion from pre-synthesized phospholipids and free sterols, respectively (Taylor and Parks, 1979, Taylor and Parks, 1978). When cell growth resumes in fresh media, TAG is then utilized for synthesis of membrane phospholipids (Taylor and Parks, 1979), while SE serves as a supply of free sterols (Taylor and Parks, 1978). In line with this, Tgl4, a yeast TAG lipase, was recently found to be phosphorylated and activated by Cdk1 to provide precursors for membrane phospholipid synthesis when stationary phase cells were inoculated into fresh media (Kurat et al., 2009). Given that the requirement for membrane lipid synthesis oscillates as the nutrient availability of yeast cells fluctuates, storage lipids appear to function as a temporary reserve for future membrane biosynthesis when conditions become favorable. The yeast dga1Δlro1Δare1Δare2Δ mutant underwent massive proliferation of membranes and cell death when challenged with exogenous unsaturated fatty acids, reflecting the importance of the formation of neutral lipids and LDs as a buffering system for membrane synthesis (Petschnigg et al., 2009, Garbarino et al., 2009).

The metabolism of storage lipids is inextricably interwoven with that of membrane lipids. First, TAG and phospholipid synthesis shares a common intermediate, phosphatidic acid (PA) (Fig. 1A). In yeast, through CDP-DAG, PA can be converted into any major phsopholipid species including phosphatidylserine (PS) and its derivatives phosphatidylethanolamine (PE) and phosphatidylcholine (PC), phosphatidylinositol (PI), and cardiolipin. It should be noted that synthesis of PS from CDP-DAG is missing from higher eukaryotic cells including mammals (Dowhan, 1997). PA can also be hydrolyzed by PA phosphatases to diacylglycerol (DAG), which is used either for synthesis of PC and PE via the Kennedy pathway or for synthesis of TAG. It appears that PA is preferentially channeled into phospholipids by yeast cells in exponential phase but into TAG in stationary phase. Consistent with this notion, PA phosphatase activity was found to be markedly elevated in stationary phase (Hosaka and Yamashita, 1984).

Moreover, membrane phospholipids undergo rapid turnover. In addition to remodeling of acyl chains by deacylation–reacylation at the sn-1 or sn-2 position, phospholipids can be deacylated at both sn-1 and sn-2 positions by phospholipase B enzymes (Plb1, Plb2, Plb3, and Nte1), generating glycerophosphodiesters (Merkel et al., 1999, Fernandez-Murray and McMaster, 2007). Hydrolysis of glycerophosphodiesters by phosphodiesterase such as Gde1 produces glycerol-3-phosphate, which can be further converted to PA by stepwise acylations (Fig. 1A). Phospholipids can also be hydrolyzed by phospholipase D enzyme, Pld1/Spo14, resulting in the formation of PA (Rose et al., 1995). As a result, phospholipid turnover provides substrates for TAG synthesis.

On the other hand, accumulated cellular TAG, when hydrolyzed by lipases (Tgl3, Tgl4, and Tgl5), generates DAG. As mentioned above, DAG can be used for PC and PE synthesis via the Kennedy pathway if choline and ethanolamine is available. DAG can also be converted by the DAG kinase (Dgk1) into PA, while PA can be synthesized into all major phospholipids through CDP-DAG. Evidence has been shown very recently that Dgk1 is required to convert TAG-derived DAG to PA for the synthesis of membrane phospholipids (Fakas et al., 2011).

Yeast sterols comprise ergosterol and its upstream precursors including, but not limited to, those shown in Fig. 1B. Ergosterol is the predominant sterol in plasma membranes and secretory vesicles, the fractions with the highest sterol levels; yet its precursors are present at higher percentages in other subcellular membranes which exhibit lower sterol contents (Zinser et al., 1993). Both ergosterol and precursors can be esterified to neutral SEs. The percentage of esterified precursors among all SEs gradually rises in stationary phase, accompanied by a decrease in the percentage of ergosterol esters (Bailey and Parks, 1975). Esterified ergosterol precursors cannot be directly synthesized into ergosterol (Bailey and Parks, 1975); instead they have to be hydrolyzed in order to be converted to the final product, ergosterol (Wagner et al., 2009).

The yeast acyl-CoA:sterol acyltransferases (Are1 and Are2) catalyze the esterification of sterols (Yang et al., 1996, Yu et al., 1996). The yeast acyl-CoA: diacylglycerol acyltransferase (DGAT), Dga1, and phospholipid:diacylglycerol acyltransferase (PDAT), Lro1, catalyze the esterification of DAG by using acyl-CoA and phospholipid as the acyl donor, respectively (Oelkers et al., 2002, Dahlqvist et al., 2000, Sorger and Daum, 2002, Oelkers et al., 2000). Are1 and Are2 catalyze the minute formation of TAG when Dga1 and Lro1 are absent (Sandager et al., 2002). Dga1 localizes both to the ER membrane and lipid droplet surface, and the majority of DGAT activity is associated with the lipid droplet fraction (Sorger and Daum, 2002). In contrast, Lro1, Are1, and Are2 localize to the ER membrane only (Zweytick et al., 2000, Sorger and Daum, 2003). Therefore, SE synthesis takes place at the ER, and TAG formation at both the ER and LDs.

The budding yeast has three TAG lipases (Tgl3, Tgl4, and Tgl5) and three SE hydrolases (Tgl1, Yeh1, and Yeh2). All three TAG lipases and two main SE hydrolases (Tgl1 and Yeh1) localize to LDs (Athenstaedt and Daum, 2003, Athenstaedt and Daum, 2005, Jandrositz et al., 2005, Koffel et al., 2005), except that Yeh2 resides in the plasma membrane (Koffel et al., 2005, Mullner et al., 2005). As a result, the breakdown of TAG and hydrolysis of most SE occurs at the LDs to generate DAG, free sterols, and fatty acids.

In addition to how LDs are formed at the ER, many other questions remain unanswered regarding the dynamics of LDs. For instance, how is the synthesis of neutral lipids and LDs controlled at the cellular and molecular level? Do LDs undergo fission or fusion? How do LDs mature? What factors determine the spatial relationship between LDs and the ER, mitochondria, peroxisomes, plasma membrane, and vacuoles? How do cells sense the need of membrane lipid synthesis and hence supply substrates from breaking down TAG and SE? To answer some of these questions, genome-wide screens in genetically amenable systems such as yeast may represent a viable and strategic approach.

Section snippets

Genome-Wide Screen of Yeast Deletion Mutants for Changes in the Dynamics of Lipid Droplets

Three genome-wide studies were recently carried out to identify genetic factors that affect LD dynamics in yeast (Fei et al., 2008, Mullner et al., 2005, Szymanski et al., 2007, Fei et al., 2011a). The first two studies used rich YPD media to culture yeast cells; while our group focused on the changes in the number and size of LDs, Dr. Goodman and colleagues examined more parameters. Aiming to identify additional mutants that synthesize supersized LDs (which will be discussed later), our second

Additional Insights From Genome-Wide Studies in Drosophila Cells

Recently two independent genome-wide RNA interference (RNAi) screens were carried out in Drosophila cells to identify key genes involved in the regulation of LD dynamics. Guo et al. used S2 cells and examined alterations in droplet number, size and dispersion, whereas Beller et al. used Kc167 cells and examined differences in cellular lipid storage by computational analysis of ratio of LD-to-nucleus cross-sectional area (Guo et al., 2008, Beller et al., 2008). The phenotype of LDs in S2 cells

Concluding Remarks

The importance of LDs has been increasingly recognized, yet the molecular mechanisms underlying their biogenesis, growth, and catabolism remain largely unknown (Farese and Walther, 2009). Genetic screen was fundamental in many research areas such as vesicular trafficking and cell cycle control. Undoubtedly this approach will again be highly valuable to our understanding of the cell biology of LDs.

So far, genome-wide screens in yeast and Drosophila cells have successfully identified several key

Acknowledgments

This work is supported by a research grant from the Australian Research Council (DP0984902). H. Yang is a Future Fellow of the Australian Research Council.

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