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.