Structural and Biochemical Properties of Lipid Particles from the Yeast Saccharomyces cerevisiae

Institute of Biochemistry, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2008; 283(25):17065-74. DOI: 10.1074/jbc.M800401200
Source: PubMed


The two most prominent neutral lipids of the yeast Saccharomyces cerevisiae, triacylglycerols (TAG) and steryl esters (SE), are synthesized by the two TAG synthases Dga1p and Lro1p and the two SE synthases
Are1p and Are2p. In this study, we made use of a set of triple mutants with only one of these acyltransferases active to elucidate
the contribution of each single enzyme to lipid particle (LP)/droplet formation. Depending on the remaining acyltransferases,
LP from triple mutants contained only TAG or SE, respectively, with specific patterns of fatty acids and sterols. Biophysical
investigations, however, revealed that individual neutral lipids strongly affected the internal structure of LP. SE form several
ordered shells below the surface phospholipid monolayer of LP, whereas TAG are more or less randomly packed in the center
of the LP. We propose that this structural arrangement of neutral lipids in LP may be important for their physiological role
especially with respect to mobilization of TAG and SE reserves.

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Available from: Tibor Czabany, Jan 14, 2016
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    • "Across eukaryotes, evidence suggests that the monolayer phospholipid membrane of LDs and their neutral lipid contents emanate from the ER [23], [24]. In S. cerevisiae, LDs are composed of near equal molar ratios of triacylglycerols (TAGs) and sterol esters (SE) and proteomic studies reveal that upwards of 40 different proteins can localize to LDs, many of which also localize to the ER [25]–[27]. A number of LD association mechanisms are known [23]: amphipathic helices can directly bind to the monolayer LD membrane; terminal hydrophobic domains can embed into LDs; and, internal hydrophobic hairpin loops can anchor to the LD membrane. "
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    ABSTRACT: In the yeast Saccharomyces cerevisiae two alcohol acetyltransferases (AATases), Atf1 and Atf2, condense short chain alcohols with acetyl-CoA to produce volatile acetate esters. Such esters are, in large part, responsible for the distinctive flavors and aromas of fermented beverages including beer, wine, and sake. Atf1 and Atf2 localize to the endoplasmic reticulum (ER) and Atf1 is known to localize to lipid droplets (LDs). The mechanism and function of these localizations are unknown. Here, we investigate potential mechanisms of Atf1 and Atf2 membrane association. Segments of the N- and C-terminal domains of Atf1 (residues 24-41 and 508-525, respectively) are predicted to be amphipathic helices. Truncations of these helices revealed that the terminal domains are essential for ER and LD association. Moreover, mutations of the basic or hydrophobic residues in the N-terminal helix and hydrophobic residues in the C-terminal helix disrupted ER association and subsequent sorting from the ER to LDs. Similar amphipathic helices are found at both ends of Atf2, enabling ER and LD association. As was the case with Atf1, mutations to the N- and C-terminal helices of Atf2 prevented membrane association. Sequence comparison of the AATases from Saccharomyces, non-Saccharomyces yeast (K. lactis and P. anomala) and fruits species (C. melo and S. lycopersicum) showed that only AATases from Saccharomyces evolved terminal amphipathic helices. Heterologous expression of these orthologs in S. cerevisiae revealed that the absence of terminal amphipathic helices eliminates LD association. Combined, the results of this study suggest a common mechanism of membrane association for AATases via dual N- and C-terminal amphipathic helices.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "The lipids which accumulated in DGAT expressing yeast were different from the WT strain and lacked sterol esters. As previously suggested by Czabany [56], lipid composition affects LD protein content and as a consequence, the interactions between LDs and their environment could be modified. Nevertheless the objects purified from the yeast which had been induced for 18 h still had a typical LD hydrodynamic diameter. "
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    ABSTRACT: Diacylglycerol acyltransferases (DGATs) catalyze the final and only committed step of triacylglycerol synthesis. DGAT activity is rate limiting for triacylglycerol accumulation in mammals, plants and microbes. DGATs belong to three different evolutionary classes. In Arabidopsis thaliana, DGAT1, encoded by At2g19450, is the major DGAT enzyme involved in triacylglycerol accumulation in seeds. Until recently, the function of DGAT2 (At3g51520) has remained elusive. Previous attempts to characterize its enzymatic function by heterologous expression in yeast were unsuccessful. In the present report we demonstrate that expression of a codon-optimized version of the DGAT2 gene is able to restore neutral lipid accumulation in the Saccharomyces cerevisiae mutant strain (H1246), which is defective in triacylglycerol biosynthesis. Heterologous expression of codon-optimized DGAT2 and DGAT1 induced the biogenesis of subcellular lipid droplets containing triacylglycerols and squalene. Both DGAT proteins were found to be associated with these lipid droplets. The fatty acid composition was affected by the nature of the acyltransferase expressed. DGAT2 preferentially incorporated C16:1 fatty acids whereas DGAT1 displayed preference for C16:0, strongly suggesting that these enzymes have contrasting substrate specificities.
    Full-text · Article · Mar 2014 · PLoS ONE
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    • "The final step in this pathway is esterification of diacylglycerol into triacylglycerol conducted by DGAT1 and DGAT2 (Dga1p in yeast; Sturley and Hussain, 2012), which accounts for nearly all triacylglycerol synthesis in mouse cells (Harris et al., 2011). Nevertheless, alternative mechanisms have been proposed (Harris et al., 2011; Sturley and Hussain, 2012), and in plants and yeast, phospholipid:diacylglycerol O-acyltransferase (PDTA) and Lro1p can also produce triacylglycerol through trans-esterification of a fatty acid from phospholipids (Chapman et al., 2012; Czabany et al., 2008). In addition, LDs accumulate free cholesterol in adipocytes (Krause and Hartman, 1984) and cholesterol esters in most cells, especially in macrophages and steroidogenic cells. "
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    ABSTRACT: Lipid droplets (LDs) are ubiquitous dynamic organelles that store and supply lipids in all eukaryotic and some prokaryotic cells for energy metabolism, membrane synthesis, and production of essential lipid-derived molecules. Interest in the organelle's cell biology has exponentially increased over the last decade due to the link between LDs and prevalent human diseases and the discovery of new and unexpected functions of LDs. As a result, there has been significant recent progress toward understanding where and how LDs are formed, and the specific lipid pathways that coordinate LD biogenesis.
    Full-text · Article · Mar 2014 · The Journal of Cell Biology
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