The principal lipids in animal cell lipid droplets are cholesterol, cholesterol ester, and triglyceride, but the protein composition of this compartment is largely unknown. Here we report on the proteomic analysis of lipid droplets. Using a combination of mass spectrometry and immunoblotting, we identify nearly 40 specifically associated proteins in droplets isolated from Chinese hamster ovary K2 cells grown in normal medium. The proteins fall in to five groups: structural molecules of the droplet-like adipose differentiation-related protein; multiple enzymes involved in the synthesis, storage, utilization, and degradation of cholesterol esters and triglycerides; multiple, different Rab GTPases known to be involved in regulating membrane traffic; signaling molecules such as p50RhoGAP; and a group of proteins that do not fit any classification but include proteins often found in caveolae/rafts such as caveolin-1 and 2 and flotillin-1. The proteome of droplets isolated from cells grown in the presence of oleate is largely the same except for an increase in the amount of adipose differentiation-related protein, caveolin-1, and a protein thought to be involved in phospholipid recycling called CGI-58. Based on the protein profile, the lipid droplet appears to be a complex, metabolically active organelle that is directly involved in membrane traffic and possibly phospholipid recycling. We propose the name adiposome for this organelle.Lipid droplets are generally regarded as simple storage depots for neutral lipids in animal and plant cells. Their morphologic appearance gives the impression they are inert cellular inclusions that derive metabolic sustenance solely from their association with smooth endoplasmic reticulum, mitochondria, or peroxisomes (1). This is especially true in professional fat storing cells of plant seeds and adipose tissue where they occupy much of the cytoplasmic space.Plant and animal lipid droplets are coated with proteins that may regulate their size. Plant oleosins, a large family of structurally related proteins, form a capsule around seed lipid bodies (2). By contrast, a family of four proteins (ADRP, 1 perilipin, S3-12, and TIP47) that share a 100-amino-acid-long region of homology at the N terminus called the PAT domain are associated with the periphery of animal lipid droplets (3). ADRP, perilipin, and S3-12 are expressed highly in adipocytes. Unlike perilipin and S3-12, ADRP is expressed ubiquitously. Both the oleosins and the PAT domain proteins may function as barriers that control the lipolysis of core lipids (4). Apparently oleosins and PAT domain proteins are not strictly required for the generation and stability of a lipid droplet because these proteins are not found in yeast (Saccharomyces cerevisiae) lipid droplets (5). Instead, the predominant proteins in the lipid droplet fraction of these cells are enzymes involved in sterol and triglyceride metabolism. The localization of these enzymes to yeast lipid droplets suggests that the droplet is a metabolic organelle with a central ...

Live cell, time-lapse microscopy was used to study trafficking of caveolin-1-GFP in stably expressing CHO cells. Multiple cytological and biochemical tests verified that caveolin-1-GFP was a reliable marker for endogenous caveolin-1. At steady state, most caveolin-1-GFP was either at the cell surface associated with invaginated caveolae or near the centrosome in caveosomes. Live cell fluorescence imaging indicated that while much of the caveolin-1-GFP in caveolae at the cell surface was relatively sessile, numerous, highly motile caveolin-1-GFP-positive vesicles were present within the cell interior. These vesicles moved at speeds ranging from 0.3-2 μm/second and movement was abolished when microtubules were depolymerized with nocodazole. In the absence of microtubules, cell surface invaginated caveolae increased more than twofold and they became organized into linear arrays. Complete depolymerization of the actin cytoskeleton with latrunculin A, by contrast, triggered rapid and massive movements of caveolin-positive structures towards the centrosomal region of the cell. The caveolar membrane system of CHO cells therefore appears to be comprised of three caveolin-1-containing compartments. These include caveolae that are confined to the cell surface by cortical actin filaments, the peri-centrosomal caveosomes and caveolar vesicles, which we call `cavicles', that move constitutively and bi-directionally along microtubules between the cell surface and caveosomes. The behavior of cavicles suggests that they function as transport intermediates between caveolae and caveosomes.

Caveolin-1 is a major structural component of raft structures within the plasma membrane and has been implicated as a regulator of cellular signal transduction with prominent expression in adipocytes. Here, we embarked on a comprehensive characterization of the metabolic pathways dysregulated in caveolin-1 null mice. We found that these mice display decreased circulating levels of total and high molecular weight adiponectin and a reduced ability to change substrate use in response to feeding/fasting conditions. Caveolin-1 null mice are extremely lean, but retain muscle mass despite lipodystrophy and massive metabolic dysfunction. Hepatic gluconeogenesis is chronically elevated, while hepatic steatosis is reduced. Our data suggest that the complex phenotype of the caveolin-1 null mouse is caused by altered metabolic and mitochondrial function in adipose tissue with a subsequent compensatory response driven mostly by the liver. This mouse model highlights the central contributions of adipose tissue for system-wide preservation of metabolic flexibility.

Caveolin-1 traffics to late endosomal/lysosomal membranes in response to manipulations of the cholesterol content of cells, suggesting that caveolin functions in the egress of cholesterol from this organelle. Cavicles associate with the periphery of the lysosome as they do with caveosomes, but these are separate organelles.

Abstract. Receptor-mediated