Lipid droplet formation on opposing sides of the endoplasmic reticulum

In animal cells, the primary repositories of esterified fatty acids and alcohols (neutral lipids) are lipid droplets that form on the lumenal and/or cytoplasmic side of the endoplasmic reticulum (ER) membrane. A monolayer of amphipathic lipids, intermeshed with key proteins, serves to solubilize neutral lipids as they are synthesized and desorbed. In specialized cells, mobilization of the lipid cargo for delivery to other tissues occurs by secretion of lipoproteins into the plasma compartment. Serum lipoprotein assembly requires an obligate structural protein anchor (apolipoprotein B) and a dedicated chaperone, microsomal triglyceride transfer protein. By contrast, lipid droplets that form on the cytoplasmic face of the ER lack an obligate protein scaffold or any required chaperone/lipid transfer protein. Mobilization of neutral lipids from the cytosol requires regulated hydrolysis followed by transfer of the products to different organelles or export from cells. Several proteins play a key role in controlling droplet number, stability, and catabolism; however, it is our premise that their formation initiates spontaneously, solely as a consequence of neutral lipid synthesis. This default pathway directs droplets into the cytoplasm where they accumulate in many lipid disorders. In animal cells, the primary repositories of esterified fatty acids and alcohols (neutral lipids) are lipid droplets that form on the lumenal and/or cytoplasmic side of the endoplasmic reticulum (ER) membrane. A monolayer of amphipathic lipids, intermeshed with key proteins, serves to solubilize neutral lipids as they are synthesized and desorbed. In specialized cells, mobilization of the lipid cargo for delivery to other tissues occurs by secretion of lipoproteins into the plasma compartment. Serum lipoprotein assembly requires an obligate structural protein anchor (apolipoprotein B) and a dedicated chaperone, microsomal triglyceride transfer protein. By contrast, lipid droplets that form on the cytoplasmic face of the ER lack an obligate protein scaffold or any required chaperone/lipid transfer protein. Mobilization of neutral lipids from the cytosol requires regulated hydrolysis followed by transfer of the products to different organelles or export from cells. Several proteins play a key role in controlling droplet number, stability, and catabolism; however, it is our premise that their formation initiates spontaneously, solely as a consequence of neutral lipid synthesis. This default pathway directs droplets into the cytoplasm where they accumulate in many lipid disorders. cholesteryl ester cytoplasmic lipid droplet diacylglycerol diacylglycerol acyltransferase endoplasmic reticulum lipoprotein particle lipid transfer protein microsomal triglyceride transfer protein phospholipid-diacylglycerol acyltransferase perilipin steryl ester triacylglycerol Lipids are critical determinants of membrane integrity, important sources of energy, and in some cells, substrates for the synthesis of hormones. Endogenous lipid synthesis consumes significant chemical energy and is therefore tightly regulated and coordinated with frugal transport processes to assimilate them from the environment and/or store them safely. Lipids enter the cytoplasm as acids (free fatty acids) or alcohols (e.g., free cholesterol). High concentrations of free fatty acids and sterols are injurious to cells, whereas alcohols such as diacylglycerol are bioactive at low concentrations as signaling molecules. Consequently, efficient systems have evolved to limit their concentrations but retain their availability by coesterification of the acids and alcohols into neutral lipids. Neutral lipids confer several selective advantages to the cell and organism in which they reside. They provide a conduit for detoxification of free fatty acids and a key reservoir of membrane components and energy. Esterification of sterols with FA to form steryl esters (SE) [e.g., cholesteryl ester (CE)] provides for future membrane rebuilding and remodeling. Triacylglycerol (TG), a fatty acyl ester derivative of glycerol, represents the major energy depot of all eukaryotic and some bacterial cells. The energy of complete oxidation of the alkyl chains of TG (38 KJ/g) is more than twice the same weight of carbohydrate or protein, and unlike polysaccharide, TG carries no extra weight as water of solvation. Similarly, esterified long- and short-chain alcohols, such as wax esters and diesters, form an important water repellant permeability barrier in the skin and fur of mammals and the cuticle of plants. The majority of neutral lipid synthesis is completed at the endoplasmic reticulum (ER). However, neutral lipid synthesis poses two problems: neutral lipids have limited solubility in the ER membrane bilayer, and they are immiscible with the hydrophilic intracellular environment. These quandaries are solved by lipid sequestration into cytoplasmic lipid droplets (CLD), a process that nullifies any impact on the osmolarity of the cytosol. The formation of CLDs is a phenomenon common to all eukaryotes (for examples, see Fig. 1) that has been maintained for over 2 billion years of evolution, presumably to confer protection against toxic free fatty acids and sterols. In metazoans with circulatory systems, neutral lipids are actively transported between organs, from the sites of dietary acquisition (i.e., the intestine) to distribution (e.g., the liver) for either storage (e.g., adipose) or catabolism (e.g., skeletal and cardiac muscle cells) via emulsion particles called “lipoproteins.” Intracellular assembly of neutral lipid-rich lipoproteins begins in the lumen of the ER of specialized cells. These lipoprotein particles exhibit a marked resemblance to the aforementioned CLDs (Fig. 1). Indeed, lipid droplets and lipoprotein particles both have a neutral lipid core surrounded by a phospholipid/sterol monolayer into which key proteins are interdigitated (Fig. 2). The role of these proteins and the means by which they are incorporated into the particle has been carefully scrutinized with significantly more success with respect to lipoprotein assembly than with CLDs. Lipoprotein formation requires an obligate scaffold of related proteins [lipophorins, vitellogenins, or in mammals, apolipoprotein B (apoB)] and a dedicated chaperone [microsomal triglyceride transfer protein (MTP)]. Interestingly, an obligate scaffolding protein or chaperone has yet to be identified for CLD biogenesis. In 1892, in a series of lectures at Columbia University, NY, E. B. Wilson characterized cytoplasmic lipid droplets as “lifeless bodies (metaplasm) suspended in the cytoplasmic reticulum” (1Wilson, E. B., 1896. The Cell in Development and Inheritance Macmillan, New York.Google Scholar). We now have a different perspective; this organelle is by no criteria inert or innocuous. The pathophysiological accumulation of neutral lipids, both within cells and in serum, is a clear harbinger of ill-health. In terms of health risk in Western societies, elevated cellular deposition of neutral lipid, such as CE in macrophages and smooth muscle cells or TG in adipocytes, typifies the twin “epidemics” of atherosclerosis and obesity, respectively. Similarly, elevated plasma levels of CE or TG in low-density lipoprotein particles represent independent risk factors for atherosclerosis. Moreover, as specialized fat storage tissues become saturated beyond capacity, other tissues start storing lipids, leading to diseases such as diabetes and insulin resistance due to lipotoxicity at pancreatic β-cells and hepatocytes/myocytes. The fate of excess lipids is thus a critical component of lipid homeostasis that provokes some of the most severe diseases confronting our society. Here we present our perspective that the thermodynamic properties of neutral lipids suffice to promote droplet formation and that the evolutionary constraint of lipotoxicity then promoted stabilization and regulated catabolism of the CLD. This process was likely coopted by certain animal cells to create circulatory lipoproteins for organ-to-organ lipid distribution and hydrolysis. The major purpose of CLDs and lipoprotein particles is to retain or release their cargo in response to cellular need. Triacylglycerols and cholesteryl esters represent valuable commodities that are lipolyzed by highly regulated pathways involving phosphorylation/dephosphorylation of several key lipases and their cofactors. The breakdown products are then transported by poorly characterized mechanisms most likely involving protein-facilitated processes. By contrast, specialized tissues such as intestine and liver have evolved a sophisticated system to mobilize neutral lipids en masse by the intracellular assembly of lipoproteins. Numerous enzymatic reactions located in several organelles direct the incorporation of acetate into neutral lipids; however, the terminal and committed step of conjugation of alcohols (e.g., diacylglycerol and sterols) with free fatty acids is primarily performed at the ER membrane. Metabolic labeling studies describe the appearance of newly synthesized TG on both lumenal and cytosolic sides of the membrane (2Chao F.F. Stiers D.L. Ontko J.A. Hepatocellular triglyceride synthesis and transfer to lipid droplets and nascent very low density lipoproteins.J. Lipid Res. 1986; 27: 1174-1181Abstract Full Text PDF PubMed Google Scholar). To date, three distinct acyltransferase gene families have been identified that accomplish the esterification reactions (Fig. 3) (3Turkish A. Sturley S.L. Regulation of triglyceride metabolism. I. Eukaryotic neutral lipid synthesis: “Many ways to skin ACAT or a DGAT”.Am. J. Physiol. Gastrointest. Liver Physiol. 2007; 292: G953-G957Crossref PubMed Scopus (50) Google Scholar). The esterification of sterol is mediated by members of the acyl-CoA cholesterol acyltransferase (ACAT) family. ACAT1-mediated esterification is ubiquitous, whereas expression of ACAT2 is restricted to lipogenic tissues such as liver and intestine. Diacylglycerol (DAG) esterification using an acyl-CoA donor is primarily the activity of DGAT1 (also a member of the ACAT family based on sequence conservation) or DGAT2 (a member of the evolutionarily unrelated diacylglycerol acyltransferase 2 gene family). DGAT2 mRNA expression is highest in the liver and adipose tissue, whereas DGAT1 mRNA expression is relatively low in these tissues (4Stone, S. J., Myers, H. M., Watkins, S. M., Brown, B. E., Feingold, K. R., Elias, P. M., Farese, R. V., Jr, . 2003. Lipopenia and skin barrier abnormalities in DGAT2-deficient mice. J. Biol. Chem. 279:11767–11176.Google Scholar). Loss of DGAT2 in induced mutant mice results in a failure to thrive that is not compensated by expression of DGAT1, suggesting that the DGAT2 enzyme is responsible for the majority of TG synthesis (4Stone, S. J., Myers, H. M., Watkins, S. M., Brown, B. E., Feingold, K. R., Elias, P. M., Farese, R. V., Jr, . 2003. Lipopenia and skin barrier abnormalities in DGAT2-deficient mice. J. Biol. Chem. 279:11767–11176.Google Scholar). Further, combined deletion of DGAT1 and DGAT2 does not obviate TG synthesis in murine cells, indicating the presence of alternative DAG esterification pathways (5Harris C.A. Haas J.T. Streeper R.S. Stone S.J. Kumari M. Yang K. Han X. Brownell N. Gross R.W. Zechner R. et al.DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes.J. Lipid Res. 2011; 52: 657-667Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). In humans, the DGAT2 gene family is complex, comprising six additional members, all of which demonstrably synthesize TG in vitro (6Turkish A.R. Henneberry A.L. Cromley D. Padamsee M. Oelkers P. Bazzi H. Christiano A.M. Billheimer J.T. Sturley S.L. Identification of two novel human acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily.J. Biol. Chem. 2005; 280: 14755-14764Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Turkish A.R. Sturley S.L. The genetics of neutral lipid biosynthesis: an evolutionary perspective.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E19-E27Crossref PubMed Scopus (30) Google Scholar). Within each gene family, it is likely that the multiple gene duplication events have facilitated divergence of active sites and thus alternative substrate preferences. Indeed, members of the DGAT2 family of TG synthases also direct esterification of mono­acylglycerol (8Yen C.L. Stone S.J. Cases S. Zhou P. Farese Jr, R.V. Identification of a gene encoding MGAT1, a monoacylglycerol acyltransferase.Proc. Natl. Acad. Sci. USA. 2002; 99: 8512-8517Crossref PubMed Scopus (151) Google Scholar–10Cheng D. Nelson T.C. Chen J. Walker S.G. Wardwell-Swanson J. Meegalla R. Taub R. Billheimer J.T. Ramaker M. Feder J.N. Identification of acyl coenzyme A:monoacylglycerol acyltransferase 3, an intestinal specific enzyme implicated in dietary fat absorption.J. Biol. Chem. 2003; 278: 13611-13614Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), long-chain alcohols (6Turkish A.R. Henneberry A.L. Cromley D. Padamsee M. Oelkers P. Bazzi H. Christiano A.M. Billheimer J.T. Sturley S.L. Identification of two novel human acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily.J. Biol. Chem. 2005; 280: 14755-14764Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and long-chain dialcohols (Turkish et al., unpublished data). The latter two reactions are particularly important in the mammalian sebocyte and meibocyte, where wax monoesters and diesters comprise key components of the epidermal and corneal permeability barrier. Similarly, the cuticle of insects and plants is neutral lipid rich; accordingly, the genomes of many model systems (e.g., Drosophila, Arabidopsis, and Nicotiana) predict orthologs that encode these reactions (12Bouvier-Navé P. Benveniste P. Oelkers P. Sturley S.L. Schaller H. Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase.Eur. J. Biochem. 2000; 267: 85-96Crossref PubMed Scopus (219) Google Scholar). In model systems ranging from the unicellular eukaryote Saccharomyces cerevisiae to multicellular systems, such as nematodes and insects, ACAT-related enzymes (in yeast, Are1p and Are2p) are responsible for the esterification of sterols (13Yang H. Bard M. Bruner D.A. Gleeson A. Deckelbaum R.J. Aljinovic G. Pohl T.M. Rothstein R. Sturley S.L. Sterol esterification in yeast: a two-gene process.Science. 1996; 272: 1353-1356Crossref PubMed Scopus (224) Google Scholar–16Sandager L. Dahlqvist A. Banas A. Stahl U. Lenman M. Gustavsson M. Stymne S. An acyl-CoA:cholesterol acyltransferase (ACAT)-related gene is involved in the accumulation of triacylglycerols in Saccharomyces cerevisiae.Biochem. Soc. Trans. 2000; 28: 700-702Crossref PubMed Scopus (26) Google Scholar). Diacylglycerol esterification is mediated by members of the DGAT2 gene family [in yeast, the DGA1 gene product (15Oelkers P. Cromley D. Padamsee M. Billheimer J.T. Sturley S.L. The DGA1 gene determines a second triglyceride synthetic pathway in yeast.J. Biol. Chem. 2002; 277: 8877-8881Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar)] and DGAT1 of the ACAT family (Fig. 3A). Additionally, yeast, algae, and green plants use an acyl-CoA-independent, phospholipid diacylglycerol acyltransferase (PDAT, encoded by the LRO1 gene in yeast) reaction that derives the acyl group from phospholipids for esterification of diacylglycerol (Fig. 3B) (17Oelkers P. Tinkelenberg A. Erdeniz N. Cromley D. Billheimer J.T. Sturley S.L. A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast.J. Biol. Chem. 2000; 275: 15609-15612Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). PDAT gene family members (Fig. 3B) are typified by the mammalian lecithin cholesterol acyltransferase (LCAT) enzyme, the predominant product of which is cholesteryl ester. This reaction resides primarily in the plasma compartment and uses lipoprotein-associated sterols as substrates. The mammalian LCAT enzyme also directs the production of TG in vitro (18Subbaiah P.V. Subramanian V.S. Liu M. Trans unsaturated fatty acids inhibit lecithin: cholesterol acyltransferase and alter its positional specificity.J. Lipid Res. 1998; 39: 1438-1447Abstract Full Text Full Text PDF PubMed Google Scholar), although its contribution to TG levels in vivo is undetermined. By contrast, the major physiological role of fungal and plant PDATs (17Oelkers P. Tinkelenberg A. Erdeniz N. Cromley D. Billheimer J.T. Sturley S.L. A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast.J. Biol. Chem. 2000; 275: 15609-15612Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 19Dahlqvist A. Stahl U. Lenman M. Banas A. Lee M. Sandager L. Ronne H. Stymne S. Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants.Proc. Natl. Acad. Sci. USA. 2000; 97: 6487-6492Crossref PubMed Scopus (634) Google Scholar) is to synthesize triacylglycerol from DAG and phospholipids as an alternative acyl-donor. Thus, neutral lipid synthesis in various organisms occurs in the ER by various enzymes belonging to three distinct gene families. The precise role of these individual enzymes remains to be determined. Particularly, why have so many genes (at least 12 genes and three distinct reactions in humans) evolved for the same step in neutral lipid synthesis? Subsequent to synthesis, neutral lipids are rapidly and efficiently deposited into cytoplasmic lipid droplets (CLD) in all cells (Fig. 4A) and into lipoprotein particles in specialized cells, such as hepatocytes, enterocytes, and cardiomyocytes (Fig. 4B). It remains to be determined whether CLD formation is a spontaneous process that is stabilized or whether proteins are needed to assemble them immediately after neutral lipids are synthesized. The biophysical and thermodynamic properties of neutral lipids have prompted the suggestion that an oil droplet spontaneously forms as a “lens” in the ER bilayer. Alternatively, the neutral lipid may diffuse freely throughout the ER membrane until it reaches a critical concentration that requires an amphipathic shell of phospholipids for solubility. This process in itself likely deforms the membrane of the ER, and as a consequence, the droplets “bud” from the organelle. This “bilayer to emulsion” transition was first proposed by Donald Small (20Small, D. M., 1981. Membrane and plasma lipoproteins bilayer-to-emulsion and emulsion to bilayer transitions. In Membranes, Molecules, Toxins and Cells. K. Bloch, L. Bolis, and D. C. Tosteson, editors. PSG Publ. Co., Boston. 11–34.Google Scholar) to be the initiating step in the biogenesis of neutral lipid-rich serum lipoproteins. Transport of neutral lipids into the ER lumen is dependent on MTP activity and the presence of a scaffold such as apolipoprotein B (21Hussain M.M. Rava P. Walsh M. Rana M. Iqbal J. Multiple functions of microsomal triglyceride transfer protein.Nutr. Metab. (Lond). 2012; 9: 14Crossref PubMed Scopus (184) Google Scholar). MTP acts as both a chaperone by physically interacting with nascent apoB, as well as a lipid transfer protein that loads lipids onto the nascent apoB polypeptide. The MTP reaction catalyzes transfer of several lipids but is functionally pertinent because of its ability to transfer neutral lipids. It has been suggested that MTP can also help in the formation of lumenal lipid droplets that lack apoB (21Hussain M.M. Rava P. Walsh M. Rana M. Iqbal J. Multiple functions of microsomal triglyceride transfer protein.Nutr. Metab. (Lond). 2012; 9: 14Crossref PubMed Scopus (184) Google Scholar, 22Hussain M.M. Shi J. Dreizen P. Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly.J. Lipid Res. 2003; 44: 22-32Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). This function could be related to its ability to interact with lipid vesicles as well as its ability to transfer lipids (23Bakillah A. Hussain M.M. Binding of microsomal triglyceride transfer protein to lipids results in increased affinity for apolipoprotein B: evidence for stable microsomal MTP-lipid complexes.J. Biol. Chem. 2001; 276: 31466-31473Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Thus, MTP activity creates a concentration gradient that is essential for transferring neutral lipids into the lumen of the ER that is then further stabilized by apoB for secretion. The directionality of this process is reversible, in that liver-specific genetic ablation or chemical inhibition of MTP impedes VLDL formation and enhances accumulation of CLDs in the cytoplasm, resulting in hepatic steatosis (24Raabe M. Veniant M.M. Sullivan M.A. Zlot C.H. Bjorkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (359) Google Scholar). In contrast to lipoprotein formation at the lumenal face of the ER, the deposition of neutral lipids in the cytoplasm is more obscure but more prevalent. Neutral lipid accumulation drives a “bilayer to emulsion” transition (20Small, D. M., 1981. Membrane and plasma lipoproteins bilayer-to-emulsion and emulsion to bilayer transitions. In Membranes, Molecules, Toxins and Cells. K. Bloch, L. Bolis, and D. C. Tosteson, editors. PSG Publ. Co., Boston. 11–34.Google Scholar) that forces the formation of the CLD (25Walther T.C. Farese R.V. The life of lipid droplets.Biochim. Biophys. Acta. 2009; 1791: 459-466Crossref PubMed Scopus (376) Google Scholar, 26Murphy D.J. Vance J. Mechanisms of lipid-body formation.Trends Biochem. Sci. 1999; 24: 109-115Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). Other models propose that neutral lipid may leave the ER through a pore as it coalesces. Alternatively, a preexisting shell of phospholipid associates with the ER and is then filled with neutral lipid as it is synthesized (25Walther T.C. Farese R.V. The life of lipid droplets.Biochim. Biophys. Acta. 2009; 1791: 459-466Crossref PubMed Scopus (376) Google Scholar). None of these models are mutually exclusive; certainly no “smoking gun” to any of them is currently available. Surprisingly, very little is known about the role of the neutral lipid biosynthetic reactions in formation of the oil droplet in eukaryotes. The reaction is definitely required; strains harboring deletions of all four acyltransferase genes (are1Δ, are2Δ, dga1Δ, and lro1Δ) are viable but completely lack neutral lipids or CLDs (27Garbarino J. Padamsee M. Wilcox L. Oelkers P.M. D'Ambrosio D. Ruggles K.V. Ramsey N. Jabado O. Turkish A. Sturley S.L. Sterol and diacylglycerol acyltransferase deficiency triggers fatty acid-mediated cell death.J. Biol. Chem. 2009; 284: 30994-31005Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Loss of the CLD compartment in such mutant strains results in marked sensitivity to excess fatty acids, a common phenomenon known as lipotoxicity (27Garbarino J. Padamsee M. Wilcox L. Oelkers P.M. D'Ambrosio D. Ruggles K.V. Ramsey N. Jabado O. Turkish A. Sturley S.L. Sterol and diacylglycerol acyltransferase deficiency triggers fatty acid-mediated cell death.J. Biol. Chem. 2009; 284: 30994-31005Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Similarly, loss of murine DGAT1 and DGAT2 markedly diminishes cellular CLD content (5Harris C.A. Haas J.T. Streeper R.S. Stone S.J. Kumari M. Yang K. Han X. Brownell N. Gross R.W. Zechner R. et al.DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes.J. Lipid Res. 2011; 52: 657-667Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Importantly, this demonstrates the necessity of neutral lipid synthesis for CLD formation. Unlike, for example, peroxisome biogenesis (28Yamasaki M. Hashiguchi N. Fujiwara C. Imanaka T. Tsukamoto T. Osumi T. Formation of peroxisomes from peroxisomal ghosts in a peroxisome-deficient mammalian cell mutant upon complementation by protein microinjection.J. Biol. Chem. 1999; 274: 35293-35296Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), there is no detectable “CLD phospholipid ghost” awaiting neutral lipid deposition. Whether neutral lipid synthesis is sufficient for CLD formation remains to be determined; it clearly is necessary. The majority (>90%) of the neutral lipid cargo in CLDs and mammalian lipoproteins is TG. Diacylglycerol esterification at the ER is predominantly acyl-CoA-dependent, directed by DGAT1 (13Yang H. Bard M. Bruner D.A. Gleeson A. Deckelbaum R.J. Aljinovic G. Pohl T.M. Rothstein R. Sturley S.L. Sterol esterification in yeast: a two-gene process.Science. 1996; 272: 1353-1356Crossref PubMed Scopus (224) Google Scholar) and DGAT2 (6Turkish A.R. Henneberry A.L. Cromley D. Padamsee M. Oelkers P. Bazzi H. Christiano A.M. Billheimer J.T. Sturley S.L. Identification of two novel human acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily.J. Biol. Chem. 2005; 280: 14755-14764Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In 1997, Zammit and coworkers (29Owen M.R. Corstorphine C.C. Zammit V.A. Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion.Biochem. J. 1997; 323: 17-21Crossref PubMed Scopus (110) Google Scholar) described two classes of DGAT activities, latent and overt, perhaps reflecting the disposition of the active sites of these enzymes. Overt activity was proposed to represent enzymes with their active sites facing the cytoplasm, whereas latent activity faced toward the lumen of the ER. This activity was recently implicated as arising from two populations of DGAT1 with alternate topologies with respect to active site orientation (30Wurie H.R. Buckett L. Zammit V.A. Evidence that diacylglycerol acyltransferase 1 (DGAT1) has dual membrane topology in the endoplasmic reticulum of HepG2 cells.J. Biol. Chem. 2011; 286: 36238-36247Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). This might suggest that different sources of TG are used for CLD and lipoprotein formation, however, it is also clear that TG from CLDs is incorporated into lipoproteins after liberation of fatty acids from the CLDs and resynthesis of TG at the ER (31Gibbons G.F. Wiggins D. The enzymology of hepatic very-low-density lipoprotein assembly.Biochem. Soc. Trans. 1995; 23: 495-500Crossref PubMed Scopus (31) Google Scholar). Therefore, whether TG is synthesized by overt or latent DGAT activity, it is ultimately incorporated with nascent lipoproteins. Similarly, the identification of the mammalian ACAT2 enzyme (14Oelkers P. Behari A. Cromley D. Billheimer J.T. Sturley S.L. Characterization of two human genes encoding acyl coenzyme A:cholesterol acyltransferase-related enzymes.J. Biol. Chem. 1998; 273: 26765-26771Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 32Cases S. Novak S. Zheng Y.W. Myers H.M. Lear S.R. Sande E. Welch C.B. Lusis A.J. Spencer T.A. Krause B.R. et al.ACAT-2, A second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization.J. Biol. Chem. 1998; 273: 26755-26764Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 33Anderson R.A. Joyce C. Davis M. Reagan J.W. Clark M. Shelness G.S. Rudel L.L. Identification of a form of acyl-CoA:cholesterol acyltransferase specific to liver and intestine in nonhuman primates.J. Biol. Chem. 1998; 273: 26747-26754Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) and subsequent elucidation of its expression profile and active site topology has prompted models in which the individual ACAT isoforms are hypothesized to form steryl esters on either side of the ER. A similar model for TG deposition into the lumen of the ER of yeast was suggested when the active site of Lro1p, the sole yeast PDAT, was oriented to the lumenal side of the ER membrane (34Choudhary V. Jacquier N. Schneiter R. The topology of the triacylglycerol synthesizing enzyme Lro1 indicates that neutral lipids can be produced within the luminal compartment of the endoplasmatic reticulum: implications for the biogenesis of lipid droplets.Commun. Integr. Biol. 2011; 4: 781-784Crossref PubMed Google Scholar). However, the physiological relevance of this observation is unclear given the absence of secreted neutral lipid in yeast. An integral role of specific acyltransferases in CLD and lipoprotein assembly has been further implicated by unexpected observations regarding viral particle biogenesis. The subcellular hydrophobic environment provided by neutral lipids has been hijacked for hepatitis C virus propagation and secretion. Hepatitis C virus assembly and its secretion requires lipoprotein biogenesis and intact DGAT1 activity (35Herker E. Harris C. Hernandez C. Carpentier A. Kaehlcke K. Rosenberg A.R. Farese Jr, R.V. Ott M. Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1.Nat. Med. 2010; 16: 1295-1298Crossref PubMed Scopus (248) Google Scholar). In addition, the hepatitis C virus is physically associated with the CLD compartment. To explain the effects of hepatitis C virus on both lumenal and cytosolic lipid droplet metabolism, we propose that hepatitis C virus associates with lipids in the ER membrane where it becomes part of apoB lipoproteins as they assemble. When apoB lipoproteins are desorbed from the ER membrane, the virus becomes part of