We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079 -1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2␤ and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain.Lipid droplets have been regarded as a depot of neutral lipids. They exist most abundantly in adipose cells and steroidproducing cells but can be found in virtually any kind of cell. The core of lipid droplets is occupied by triacylglycerol and cholesterol ester in various ratios depending on the cell type (1), but information on the lipid droplet surface has been scarce. Recently we as well as others showed that caveolins can exist in the lipid droplet surface (2-4). Caveolins, i.e. caveolin-1, 2, 3, are membrane proteins that are incorporated to the sphingolipid/cholesterol-enriched membrane microdomain and form the framework of caveolae (5). Furthermore, lipid droplets were reported to contain other microdomain proteins, i.e. Lyn and mitogen-activated protein kinase, as well as abundant free cholesterol (6 -8). These results suggest that the lipid droplet surface is a kind of membrane and that it might have some similarity to the microdomain.However, electron microscopy of conventional resin-embedded ultrathin sections cannot visualize any membranous structure around the lipid droplet. In the ultrathin section of specimens fixed by aldehydes and then by osmium tetroxide, the lipid droplet content appears vacant, and its periphery is usually seen as a thin intermittent line. In many diagrams, the lipid droplet surface has been depicted as a phospholipid ...

apposition of LDs to membrane cisternae connected to the rough ER. Two other procedures that decrease ADRP, i.e. RNA interference and brefeldin A treatment, induced the same morphological change, indicating that decrease in ADRP was the cause of the LD-ER apposition. In accordance with similar structures found between ER and other organelles, we propose that the ER membrane apposed to LDs should be named the LD-associated membrane, or LAM. The present results suggested that Rab18 regulates LAM formation, which is likely to be involved in mobilizing lipid esters stored in LDs.Key words: Lipid droplet, Mass spectrometry, Rab18, Endoplasmic reticulum, Membrane apposition SummaryRab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane

Fig. 1. Labeling of the liposome. (A)Outline of the method. Cells were rapidly frozen, freeze-fractured, and evaporated with carbon (C) and platinum/carbon (Pt/C) in vacuum. The replica of the split membrane was digested with SDS to remove noncast molecules and labeled by GST-PH. Both the cytoplasmic and exoplasmic halves of the membrane were examined. (B) Labeling of small unilamellar liposome replicas. Freeze-fracture replicas of liposomes containing 95 mol % of phosphatidylcholine (PC) and 5 mol % of phosphatidylinositol or a phosphoinositide were labeled. Only liposomes containing PI(4,5)P 2 were labeled intensely by GST-PH. A PH mutant, GST-PH (K30N, K32N), which does not bind PI(4,5)P 2, showed little labeling in the PI(4,5)P 2-containing liposome. (C) Quantification of the GST-PH labeling in the liposomes. The number of gold particles per 1 m 2 of the liposome surface is shown (blue). The labeling on the convex (green) and concave (yellow) surfaces showed equivalent results.

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