Lipid droplet proteins of the PAT (perilipin, adipophilin, and TIP47) family regulate cellular neutral lipid stores. We have studied a new member of this family, PAT-1, and found that it is expressed in highly oxidative tissues. We refer to this protein as "OXPAT.

Animals have evolved mechanisms to maintain circulating nutrient levels when energy demands exceed feeding opportunities. Mammals store most of their energy as triacylglycerol in the perilipin-coated lipid droplets of adipocytes. How newly synthesized triacylglycerol is delivered to perilipin-coated lipid droplets is poorly understood. Perilipin is a member of the evolutionarily related family of PAT proteins (Perilipin, Adipophilin, TIP47), which is defined by sequence similarity and association with lipid droplets. We previously showed that S3-12, which is also a member of this family, associates with a separate pool of lipid droplets that emerge when triacylglycerol storage is driven by adding oleate to the culture medium of adipocytes. Our current data extend these findings to demonstrate that nascent lipid droplets emerge with a coat composed of S3-12, TIP47, and adipophilin. After 100 min of oleate treatment, the nascent lipid droplets are more heterogeneous: S3-12 and TIP47 coat smaller, peripheral droplets and adipophilin coats a more medial population of droplets. Fractionation of untreated and oleate-treated adipocytes shows oleate-dependent redistribution of TIP47 and adipophilin from cytosolic fractions to the lipid droplet fraction. Inhibition of protein synthesis with cycloheximide does not block the oleate-induced formation of the nascent lipid droplets, nor does it prevent TAG accumulation. We suggest that the non-lipid droplet pools of S3-12, adipophilin, and TIP47 constitute a ready reservoir of coat proteins to permit rapid packaging of newly synthesized triacylglycerol and to maximize energy storage during nutrient excess.The ability to store energy as triacylglycerol (TAG) 1 and later mobilize energy from this reservoir as free fatty acids and glycerol allows animals to survive prolonged periods of fasting. Higher animals store TAG in specialized cells, adipocytes, which are able to efficiently synthesize TAG from fatty acid and glucose and package TAG into large storage droplets (10 -100-m diameter). Other cell types have a more limited capacity to store TAG and rarely accumulate large lipid droplets. The robust ability of the adipocyte to store TAG is not without limit. It has been hypothesized that, when the visceral adipose tissue depot fills up, excess lipid "overflows" into other tissues that are less able than adipocytes to package and store TAG. Fatty acids not esterified to glycerol are toxic even at low concentrations (1). In fact, failure to efficiently sequester lipid into the lipid storage droplets of adipocytes has been implicated in the pathogenesis of insulin resistance, nonalchoholic fatty liver disease, ␤-cell failure, and cardiomyopathy (2).Little is known of mechanisms by which cells, and adipocytes in particular, package newly synthesized TAG into storage droplets (3, 4). The structure of intracellular lipid droplets is similar to that of lipoproteins, a neutral lipid core is surrounded by a phospholipid monolayer and a protein coat. This structural organization is con...

Humans have evolved mechanisms of efficient fat storage to survive famine, but these mechanisms contribute to obesity in our current environment of plentiful food and reduced activity. Little is known about how animals package fat within cells. Five related structural proteins serve roles in packaging fat into lipid droplets. The proteins TIP47, S3-12, and OX-PAT/MLDP/PAT-1 move from the cytosol to coat nascent lipid droplets during rapid fat storage. In contrast, perilipin and adipophilin constitutively associate with lipid droplets and play roles in sustained fat storage and regulation of lipolysis. Different tissues express different complements of these lipid droplet proteins. Thus, the tissue-specific complement of these proteins determines how tissues manage lipid stores.

Most mammalian cells package neutral lipids into droplets that are surrounded by a monolayer of phospholipids and a specific set of proteins including the adipose differentiation-related protein (ADRP; also called adipophilin), which is found in a wide array of cell types, and the perilipins, which are restricted to adipocytes and steroidogenic cells. TIP47 was initially identified in a yeast two-hybrid screen for proteins that interact with the cytoplasmic tail of the mannose 6-phosphate receptor, yet its sequence is highly similar to the lipid droplet protein, ADRP, and more distantly related to perilipins. Hence, we hypothesized that TIP47 might be associated with lipid droplets. In HeLa cells grown in standard low lipid-containing culture media, immunofluorescence microscopy revealed that the cells had few lipid droplets; however, TIP47 and ADRP were found on the surfaces of the small lipid droplets present. When the cells were grown in media supplemented with physiological levels of fatty acids, the amount of neutral lipid stored in lipid droplets increased dramatically, as did the staining of TIP47 and ADRP surrounding these droplets. TIP47 was found primarily in the cytosolic fractions of HeLa cells and murine MA10 Leydig cells grown in low lipid-containing culture medium, while ADRP was undetectable in these fractionated cell homogenates. When HeLa and MA10 Leydig cells were lipidloaded, significant levels of ADRP were found in the floating lipid droplet fractions and TIP47 levels remained constant, but the distribution of a significant portion of TIP47 shifted from the cytosolic fractions to the lipid droplet fractions. Thus, we conclude that TIP47 associates with nascent lipid droplets and can be classified as a lipid droplet-associated protein.TIP47 was first identified by a yeast two-hybrid screen using the cytosolic domain of the cation-dependent mannose 6-phosphate receptor (M6PR) 1 as a bait to screen an expression library from human Jurkat cells (1). A glutathione S-transferase TIP47 fusion protein was subsequently shown to bind to glutathione S-transferase fusion proteins of the cytosolic tails of both the cation-dependent and cation-independent M6PRs in vitro and thus, this protein was named TIP47 (tail-interacting protein of 47 kDa). Diaz and Pfeffer (1) have proposed that TIP47 directs the retrieval of M6PRs from a prelysosomal compartment with delivery back to the trans-Golgi network through the interaction of TIP47 with the cytoplasmic tails of M6PRs. An essentially identical cDNA, PP17a 1 (2), and a truncated version of this cDNA, PP17a 2, have been obtained by screening a human placental expression library with an antibody raised against a 38-kDa protein purified from human placenta (3). Interestingly, the amino acid sequence of TIP47 (PP17a) is highly similar to sequences from the members of a growing family of lipid droplet-associated proteins that includes perilipins and the adipose differentiation-related protein (ADRP), also called adipophilin. The amino acid sequence of TIP47 (1) is...

Most animals store lipid intracellularly in proteincoated droplets. The protein coat usually contains at least one member of the PAT (perilipin, adipose differentiation-related protein, and TIP47) family. Evidence suggests that PAT proteins control access to the lipid they enclose. The protein S3-12, which has sequence similarity to the PAT proteins, was found in a screen for adipocyte-specific proteins. The adipocyte expression of S3-12 and its similarity to the PAT proteins suggest that S3-12 is involved in adipocyte lipid storage. To test this hypothesis, we supplemented 3T3-L1 adipocytes with fatty acids and assessed the distribution of S3-12 by immunofluorescence microscopy. Prior to fatty acid incubation, S3-12 was distributed diffusely throughout the cytoplasm on punctate structures of heterogeneous size. After 10 min of lipid loading, S3-12 localized to 500-nm structures concentrated at the adipocyte periphery. After longer incubations, S3-12 coated the surface of lipid droplets up to several micrometers in diameter. Initially, these droplets were distinct from those droplets surrounded by perilipin; but by 240 min, most perilipincoated droplets had some S3-12 on the surface as well. We additionally report that the formation of S3-12-coated droplets 1) required glucose and fatty acids that can be incorporated into triacylglycerol, 2) was blocked by an inhibitor of triacylglycerol synthesis, and 3) was insulin-dependent. This study reports for the first time the early morphological events in the genesis and maturation of adipocyte lipid droplets.