The expression of
apolipoprotein A-V (
apoA-V) in
hepatoma cells results in homing of this
protein to intracellular lipid droplets. When
hepatoma cells transfected with a full-length
apoA-V-
green fluorescent protein fusion
protein were cultured in medium that was not supplemented with
oleic acid (OA), intracellular lipid droplet size and number were reduced compared with those of cells supplemented with OA. Confocal microscopy studies revealed that
apoA-V associates with lipid droplets under both conditions. To define the structural requirements for
apoA-V lipid droplet association,
hepatoma cells were transfected with a series of C-terminal truncated
apoA-V variants. Confocal microscopy analysis revealed that, in a manner similar to mature full-length
apoA-V (343
amino acids), truncation variants apoA-V(1-292), apoA-V(1-237), and apoA-V(1-191) associated with lipid droplets, while apoA-V(1-146) did not. Western blot analysis of the relative abundance of
apoA-V in cell lysates versus
conditioned medium indicated that
apoA-V variants associated with lipid droplets were poorly secreted while apoA-V(1-146) was efficiently secreted. Ultracentrifugation of
conditioned medium revealed that, unlike full-length
apoA-V, which associates with
lipoproteins, apoA-V(1-146) was present solely in the
lipoprotein-deficient fraction. Deletion of the N-terminal
signal peptide from
apoA-V resulted in an inability of the
protein to be secreted into the medium, although it associated with lipid droplets. Taken together, these data suggest that the C terminus of
apoA-V is essential for lipid droplet association in transfected
hepatoma cells and
lipoprotein association in
conditioned medium while the
signal peptide is required for extracellular trafficking of this
protein.