TIP47 associates with lipid droplets

Most mammalian cells package neutral lipids into droplets that are surround ed 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 i dentified in a yeast two-hybrid screen for proteins that interact with the cytoplasmic tail of the mannose B-phosphate receptor, yet its sequence is h ighly similar to the lipid droplet protein, ADRP, and more distantly relate d to perilipins. Hence, we hypothesized that TIP47 might be associated with lipid droplets. In HeLa cells grown in standard low lipid-containing cultu re media, immunofluorescence microscopy revealed that the cells had few lip id droplets; however, TIP47 and ADRP were found on the surfaces of the smal l lipid droplets present. When the cells were grown in media supplemented w ith 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 cytosoli c fractions of HeLa cells and murine MA10 Leydig cells grown in low lipid-c ontaining culture medium, while ADRP was undetectable in these fractionated cell homogenates, When HeLa and MA10 Leydig cells were lipid-loaded, signi ficant levels of ADRP were found in the floating lipid droplet fractions an d TIP47 levels remained constant, but the distribution of a significant por tion of TIP47 shifted from the cytosolic fractions to the lipid droplet fra ctions. Thus, we conclude that TIP47 associates with nascent lipid droplets and can be classified as a lipid droplet-associated protein.