The perilipins are the most abundant proteins at the surfaces of lipid droplets in adipocytes and are also found in steroidogenic cells. To investigate perilipin function, perilipin A, the predominant isoform, was ectopically expressed in fibroblastic 3T3-L1 pre-adipocytes that normally lack the perilipins. In control cells, fluorescent staining of neutral lipids with Bodipy 493/ 503 showed a few minute and widely dispersed lipid droplets, while in cells stably expressing perilipin A, the lipid droplets were more numerous and tightly clustered in one or two regions of the cytoplasm. Immunofluorescence microscopy revealed that the ectopic perilipin A localized to the surfaces of the tiny clustered lipid droplets; subcellular fractionation of the cells using sucrose gradients confirmed that the perilipin A localized exclusively to lipid droplets. Cells expressing perilipin A stored 6 -30-fold more triacylglycerol than control cells due to reduced lipolysis of triacylglycerol stores. The lipolysis of stored triacylglycerol was 5 times slower in lipid-loaded cells expressing perilipin A than in lipid-loaded control cells, when triacylglycerol synthesis was blocked with 6 M triacsin C. This stabilization of triacylglycerol was not due to the suppression of triacylglycerol lipase activity by the expression of perilipin A. We conclude that perilipin A increases the triacylglycerol content of cells by forming a barrier that reduces the access of soluble lipases to stored lipids, thus inhibiting triacylglycerol hydrolysis. These studies suggest that perilipin A plays a major role in the regulation of triacylglycerol storage and lipolysis in adipocytes.Lipid droplets in adipocytes store the body's major energy reserves as triacylglycerols. These structures contain a large core of neutral lipid, primarily triacylglycerol, covered by a phospholipid monolayer. The intracellular mechanisms that control the storage and release of triacylglycerols are largely uncharacterized, yet are likely to be fundamental to understanding the regulation of energy metabolism in the body. Recent studies have shown that lipid droplets are covered with a proteinaceous coat; the functions and identities of the component proteins have not been fully elucidated. The first identified lipid droplet-specific proteins are the perilipins (1-7), a family of proteins coating the surfaces of lipid droplets of adipocytes and steroidogenic cells of adrenal cortex, testes, and ovaries, but lacking in other types of cells and in other cellular compartments. The perilipins are encoded by a single copy gene that gives rise to multiple mRNAs by alternative splicing mechanisms 1 ; these mRNAs are translated to yield the three described protein isoforms (2, 4). Perilipin A is the predominant isoform in both adipocytes and steroidogenic cells, perilipin B is found primarily in adipocytes, and perilipin C is unique to steroidogenic cells. Perilipin A is the most abundant protein on highly purified lipid droplets isolated from fully differentiated cultured 3T3-L1 adip...

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...

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