No abstract

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.

The biosynthesis and storage of triglyceride (TG) is an important cellular process conserved from yeast to man. Most mammalian cells accumulate TG in lipid droplets, most prominent in adipocytes, which are specialized to store large amounts of the TG over long periods. In this study, we followed TG biosynthesis and targeting by fluorescence imaging in living 3T3-L1 adipocytes and COS7 fibroblasts. Key findings were (i) not only TG but also its direct metabolic precursor diacylglycerol, DG, accumulates on lipid droplets; (ii) the essential enzyme diacylglycerol acyltransferase 2 associates specifically with lipid droplets where it catalyzes the conversion of DG to TG and (iii) individual lipid droplets within one cell acquire TG at very different rates, suggesting unequal access to the biosynthetic machinery. We conclude that at least part of TG biosynthesis takes place in the immediate vicinity of lipid droplets. In vitro assays on purified lipid droplets show that this fraction of the biosynthetic TG is directly inserted into the growing droplet. All organisms store metabolic energy to satisfy needs when nutrient levels decrease. Mammals store most of this energy as triglyceride (TG) in the lipid droplets of adipocytes (1). Adipocytes are specialists with high rates of metabolite uptake and synthesis of TG, which is stored in large lipid droplets (10-to 100-mm diameter) and mobilized upon demand by the co-ordinated action of several lipases (2). Although essentially all cell types contain small lipid droplets, most non-adipocytes store only little TG and do not form large droplets. However, a basal ability to esterify fatty acids and glycerol is important for all cells because even low amounts of free fatty acid are toxic (3).Little is known about mechanisms by which cells package newly synthesized TG into storage droplets (1). Lipid droplets in animals, plants and yeast consist of a hydrophobic core made from neutral lipids surrounded by a phospholipid monolayer with proteins attached both integrally and peripherally (4,5). Biogenesis of neutral lipids is assumed to occur at the endoplasmic reticulum, ER. In mammals, the pathway starts by acylation of glycerol-3-phosphate with two fatty acids to give phosphatidic acid, PA. Subsequent dephosphorylation yields diacylglycerol (DG), the precursor for synthesis of both phospholipids and, by a third acylation, TG. Two enzymes diacylglycerol acyltransferase (DGAT)1 (6) and DGAT2 (7) catalyze the TG synthesis. Recently, microscopic analysis of plant cells overexpressing tagged DGAT1 and DGAT2 pinpointed their localization to distinct regions of the ER, excluding each other (8). Also in mammalian cells, overexpressed, tagged DGAT2 localized to the ER (9). Analysis of phenotypes of knockout mice point to a complex physiological organization of TG synthesis. Mice lacking the ubiquitously expressed DGAT1 have reduced body fat, a lactation defect and are resistant to diet-induced obesity (10). Interestingly, they show normal TG levels in plasma and adipose tissue. DGAT2 is...

Phosphatidylserine exists lumenally in the ER, Golgi, and mitochondria but cytoplasmically in the trans-Golgi and at the plasma membrane, which suggests that functionally important flipping may occur during trafficking.

Microscopy of lipids in living cells is currently hampered by a lack of adequate fluorescent tags. The most frequently used tags, NBD and BODIPY, strongly influence the properties of lipids, yielding analogs with quite different characteristics. Here, we introduce polyene-lipids containing five conjugated double bonds as a new type of lipid tag. Polyene-lipids exhibit a unique structural similarity to natural lipids, which results in minimal effects on the lipid properties. Analyzing membrane phase partitioning, an important biophysical and biological property of lipids, we demonstrated the superiority of polyene-lipids to both NBD- and BODIPY-tagged lipids. Cells readily take up various polyene-lipid precursors and generate the expected end products with no apparent disturbance by the tag. Applying two-photon excitation microscopy, we imaged the distribution of polyene-lipids in living mammalian cells. For the first time, ether lipids, important for the function of the brain, were successfully visualized.